CYTOGENETIC RELATIONSHIP BETWEEN CULTIVATED RICE AND FOUR

DIPLOID WILD RICE

RAJ KUMAR NIROULA

April 2003

CYTOGENETIC RELATIONSHIP BETWEEN

CULTIVATED AND NEPALESE WILD SPECIES OF RICE

RAJ KUMAR NIROULA

THESIS

SUBMITTED TO THE

TRIVHUVAN UNIVERSITY

INSTITUTE OF AGRICULTURE AND ANIMAL SCIENCE

RAMPUR, CHITWAN, NEPAL

IN PARTIAL FULFILMENT OF THE

REQUIREMENTS FOR THE

DEGREE OF

MASTER OF SCIENCE IN AGRICULTURE

(PLANT BREEDING)

APRIL 2003

II

III

ACKNOWLEDGEMENTS

The author wishes to express his sincere gratitude and appreciation to his major

advisor, Dr. Lakshmi Prasad Subedi, whose guidance was genuine and continuous from

germplasm collection to end of the laboratory experiment. He is grateful to Prof. Dr. R.C.

Sharma, Dr. Madhusudan Prasad Upadhyay (NARC) and Mr. Nav Raj Adhikari (members

of the advisory committee) for their constant guidance, inspirations, timely suggestions,

constructive criticisms, keen interest and continuous cooperation throughout the course of

this study.

The author is also greatly indebted to Prof. Dr. Ram Chandra Sharma, for making

financial support, relevant journals, assistantship, and some experimental materials beside

his role of committee member. Similarly, the effort made by Dr. M. P. Upadhyay to

provide fund from IPGRI (International Plant Genetics Resource Institute, Rome, Italy) is

highly appreciated.

The author is greatly indebted to Dr. S.D. Brar (Scientist, Plant Breeding, Genetics

and Biochemistry Division, IRRI) for his valuable suggestions, timely help, and providing

a volume of literatures. The author appreciates his great patience to handcarry the some of

the valuable and expensive chemicals from Philippines to Kathmandu (Nepal). The author

also extends his special thanks to Mr. Madhav Prasad Pandey for his valuable suggestions,

interest, sharing of his experience on the part of this study and his cooperation. He is very

grateful to Dr. Sarah Johnson (Cornell University) for her kindly help and suggestions to

improve the language of this manuscript.

The partial grant (C11G/6800-C11G) support from IPGRI is highly appreciated.

Similarly, the help and timely suggestions provided by Dr. Bhuwan Ratna Sthapit

IV

(Scientist, IPGRI), and Dr. Toby Hodgkin (Principal Scientist, IPGRI) are highly

appreciated. Without their cooperation the research would not be possible. He would also

like to express his gratitude to the following individuals for providing the constant

inspiration, timely suggestions, and co-ordination: Dr. G.L. Shrestha (Director,

GEM/Nepal), B.K. Joshi (Scientist, NARC), Dr. S.R. Shakya (Botany Division, TU). He

would also like to thank the library of IAAS, NARC, Central library TU, and IRRI,

Philippines.

The author also grateful to Prof. Dr. Tej Bahadur K. C. (Dean) and Prof. Dr. D.D.

Dhakal (Ex Dean), IAAS, Rampur, for providing the favorable environment that allowed

success of this study. Likewise, the author is grateful to all the department members and his

colleagues for their interest.

He would like to express his sincere appreciation to the following individuals: Mr.

Madan Adhikari, Mr. Bhanu Pokhrel (NARC), Anju. Baral, R. Upadhyay. S. Bhandari, Mr.

Bharat Khanal, Ram and Shyam Basnet, Benup Aryal, Ram Kumar B.K, Deepak and

Binod Ghimire, R. Burlakoti, S. Bhushal and Tulsi Photo Studio (Rampur) for their

financial support and research assistance during study.

At the last but not least the author extends his deep love to his parents Ram Bahadur

and Muna Maya, brothers Shesh Kumar and Prem Kumar (Rajesh) and all sisters and

brothers in law, without whose inspiration and moral support this piece of work would

have been left undone. To them, he has no words to thank.

V

DEDICATED TO

MY BELOVED PARENTS

RAM BAHADUR AND MUNA MAYA

VI

TABLE OF CONTENTS

Title

Acknowledgements III-IV

Table of contents VI-IX

List of tables XI

List of figures and plates XII

List of appendices XIII

Abbreviations XIV

Abstract 1-2

1. Introduction

1.1. Background 3-6

1.2. Objectives 7

2. Literature Review 8

2.1. Taxonomy and origin of rice 8-9

2.2. Rice biodiversity in Nepal 10

2.3. Description of Nepalese wild species of rices 10

2.3.1. Oryza granulata 10-11

2.3.2. O. officinalis 11

2.3.3. O. nivara and O. rufipogon 11

2.4. Genome and Genepool of rice 12

2.5. Importance of wild germplasm 12-15

2.6. Progress towards the utilization of wild relatives of rice 15-17

2.7. Wide hybridization 17-18

2.7.1. Barriers to wide hybridization 18

VII

2.7.1.1.Prefertilization barriers 18-19

2.7.1.2. Postfertilization barriers 19-20

2.8.1. Crossability 21

2.8.2. Causes of embryo abortion in wide crosses 21-22

2.8.3. Seed and embryo differentiation in hybrids 22-23

2.9. Embryo rescue in rice 23-24

2.10. Hardening of regenerated seedlings before field transfer 24

2.11. Hybrid embryo derived callus culture 25

2.12. In vitro fertilization 25-26

2.13. Media composition 26

2.14. Hybrid sterility 26-27

2.15. Chromosome pairing in haploid rice 27

2.16. Chromosome behavior between AA genome species hybrids 27

2.16.1. Meiosis in intervarietal crosses 27-28

2.16.2. Pachytene analysis 28-29

2.16.3. Diplotene, Diakinesis, and Metaphase I 29-30

2.16.4. Chiasma frequency 30-31

2.16.5. Anaphase bridge: cytological view 31-32

2.17. Meiotic behavior in intergenomic hybrids 33

2.18. Pachytene analysis in intergenomic crosses 33-34

2.19. Asynapsis and/or desynapsis 34-35

2.20. Auto and allosyndetic pairing 35-36

2.21. Nucleolus: number, type, and shape 36

2.22. Recent progress in rice chromosome research 37

VIII

3. Materials and Methods

3.1. Preparation of parental materials 38

3.1.1. Germplasm collection 38

3.1.2. Germination and green house rearing 38-41

3.2. Hybridization 41

3.2.1. Cross combination 41

3.2.2. Forced anthesis 41

3.2.3. Emasculation 42

3.2.4. Artificial pollination 42

3.3. Embryo rescue 42-43

3.3.1. Aseptic excision of immature hybrid embryo 43

3.3.2. Aseptic preparation of mature hybrid embryo 43

3.3.3. Inoculation of embryo 44

3.3.4. Media preparation 44

3.3.5. Media employed 44-45

3.3.6. Media efficiency determination 45

3.3.7. Incubation of culture\ 45

3.3.8. Seedlings transfer 45-46

3.4. Regeneration of plants from callus of hybrid embryos 46-47

3.5. In vitro pollination 47

3.6. Harvesting of F1 seeds 47-48

3.7. Determination of crossability 48

3.7.1. Crossability between O. sativa and common wild rice

species

48

IX

3.7.2. Crossability determination in intergenomics crosses 48-49

3.8. Morphological characterization 49

3.9. Chromosome preparation 49

3.9.1. Chemical preparation 49

3.9.1.1.Stain preparation 49-50

3.9.1.2.Fixative preparation 50

3.9.2. Standardization of suitable stages for meiotic study 50-51

3.9.3. Meiotic behavior study 51

3.9.3.1. Chromosome analysis 51-52

3.9.3.2. Pachytene analysis 52

3.9.3.3. Chiasma frequency determination 52

3.9.3.4. Detection of univalents, bivalents, trivalents,

and quadrivalents

53

3.9.4. Pollen fertility and sterility determination 53-54

3.9.5. Spikelet fertility and sterility determination 54-55

4. Results and Discussions

4.1. Description of Nepalese wild rice speices 56-57

4.2. In vitro manipulation 58

4.2.1. Media efficiency determination 58

4.2.2. Embryo rescue 59-62

4.2.3. Hybrid embryo derived callus culture 62-63

4.2.4. In vitro fertilization 63-66

4.3. Crossability 66

4.3.1. Crossability in intergenomic cross combination 66

X

4.3.2. Crossability between intragenomic species crosses 66-68

4.4. Morphology of the interspecific hybrids 68-69

4.5. Meiotic behavior in parents and their hybrids 69

4.5.1. Pachytene analysis in AA genome hybrids and their

parents and their parents

69-75

4.5.2. Pachytene analysis in intergenomic (A and C

genome)

77-79

4.5.3. Variation in nucleolus shape in parents and their

hybrids

79-81

4.5.4. Diakinesis and Metaphase I. 81-91

4.5.4.1.Chiasma frequency in parents and their

hybrids

91-97

4.5.4.2. Chromosome number and ploidy level 97-98

4.5.5. Meiotic behavior at Anaphase I and Telophase I in

parents and their hybrids

98-102

4.6. Hybrid fertility and sterility: cytological dissection 105-109

Summery and conclusion 110-112

Recommendation 113

Literature cited 114-143

Appendices 144-156

XI

LIST OF TABLES

2.1. Chromosome number, Genomic composition, geographical distribution

and useful traits of Oryza species.

13-14

2.2. Progress in gene introgression and transfer from wild Oryza species into

elite lines of cultivated rice

16

3.1. Description of germplasm collection, collection site, type of collection

and their species name.

39

3.2. Crossing scheme employed during experiment. 40

4.1. Morphological characters of four Nepalese wild species of rice recorded

during study period 2001/2002.

57

4.1a. Results of embryo rescue and crossability between O. sativa and two

distant relatives; O. granulata and O. officinalis.

65

4.1b. Seed set, and crossability between O. sativa L. and two common wild

rice species (O. nivara and O. rufipogon).

67

4.2.1. Chromosome behavior at pachynema in hybrids and their parents 76

4.2.2. Percent frequency of nucleolus shape variation in pachytene stage 80

4.2.3a. Meiotic configuration at diakinesis and metaphase I. from the

parents used in the hybridization

83-84

4.2.3b. Meiotic configuration at diakinesis and metaphase I from the

interspecific hybrids involving three different species.

88-89

4.2.3c. Percentage of normal and abnormal chromosome behavior at

diakinesis and metaphase I in the parents and F1’s

94-95

4.2.3d. Percentage of normal and abnormal chromosome behavior at

diakinesis and metaphase I in the hybrid involving O. sativa L. and O.

officinalis. Wall ex Watt.

96

4.2.4. Meiotic behavior at anaphase and telophase I in the parents and hybrids 103-104

4.2.5. Mean percentage of pollen and spikelet fertility in the parents and F1

hybrids

108

XII

LIST OF PLATES AND FIGURES

Fig. 2.1. Evolutionary pathway of the two cultivated species of rice. 9

Fig. 2.2. Schematic representation of stages in normal sexual reproduction and

related barriers to interspecific hybridization.

20

Plate1.Invitro manipulation: efforts made to regenerate intergenomic rice

hybrids, figure a-l.

64

Plate 2.Comparative morphology of parents and their hybrids. 60

Plate3. Morphology of hybrid plants, figure 1-8, and wild species O.

granulata.

61

Plate4. Figure showing meiotic behavior at pachynema in hybrids, figure 1-3. 71

Plate5. Figure showing meiotic behavior at pachynema in hybrids, figure 1-3. 72

Plate6. Figure showing meiotic behavior at pachynema in hybrids, figure 1-3. 73

Plate7.Figure showing meiotic behavior at pachynema in hybrids and parents,

figure1-5.

74

Plate8. Meiotic behaviour at diplotene and diakinesis in hybrids and parents, figure

1-11.

85

Plate9. Meiotic configuration at metaphaseI in different hybrid combinations, figure

1-12.

86

Plate10.Representative microphotograph of meiotic configuration at MI, AI, and

TI in hybrid plant, figure 1-9.

99

Plate11.Representative microphotograph of meiotic configuration at AI, and TI in

hybrid plants, figure 1-11 and figure 12, showing partial pollen sterility in

hybrids.

100

XIII

LIST OF APPENDICES

3.1. Nutritional components of different media employed for embryo

culture during study period.

144

3.2. Nutrient constitution for the preparation of hardening solution. 145

4.1. Comparative results of embryo culture on five different sterile media

(preliminary report).

146-147

4.2. Comparative results of different hardening techniques employed. 148

4.3a-4.3h. Phenotypic characters relationship between parents and their hybrids. 149-156

XIV

ABBREVIATION AND SYMBOL

AFLP

Ag.

AI

Dl

Dp

FISH

GISH

Hm

I

IAAS

II

III

In

IRRI

ISH

ISSR

IV

Lp

M2

MI

NARC

NB

RAPD

RFLP

TI

Tl

TU

VI

Amplified Fragment Length Polymorphism

Agriculture

Anaphase I

Deletion

Duplication

Fluorescence In situ hybridization

Genomic In situ hybridization

Heteromorphocity

Univalent

Institute of Agriculture and Animal Science

Bivalent

Trivalent

Inversion

International Rice Research Institute

In situ Hybridization

Inter Simple Sequence Repeat

Quadrivalent

Loose Pairing

Second generation mutant progeny

Metaphase I

Nepal Agriculture Research Council

Nucleolar Bodies

Randomly Amplified Polymorphic DNA

Restriction Fragment Length Polymorphism

Telophase I

Translocation

Tribhuvan University

Hexavalent

1

CYTOGENETIC RELATIONSHIP BETWEEN CULTIVATED AND NEPALESE

WILD SPECIES OF RICE

ABSTRACT

Name: Raj Kumar Niroula ID. No: R-2000-PB-21-M

Semester and Year of enrollment: Ist semester, 2000 Degree: M. Sc. (Ag.)

Major Subject: Plant Breeding Department: Plant Breeding

Major advisor: Dr. Lakshmi Prasad Subedi

Four wild species (O. rufipogon, O. nivara, O. officinalis, and O. granulata) and nine

cultivated varieties (IR 64, IR 72, Manshara, Jhinuwa, Pokhreli, Jethobudo, Kalanamak,

Ghaiya, and Masuli) were used to study the crossability between species, morphology,

pollen and spikelet fertility, and meiotic behavior of chromosomes in the parents and their

hybrids, during 2001-2002 at IAAS, Rampur. Embryo rescue, callus culture, and in vitro

fertilization were attempted to produce intergenomic hybrids and to overcome both pre and

post fertilization barriers through manipulating different media. Among these methods,

embryo rescue followed by new hardening technique was found to be capable of

regenerating the intergenomic hybrids. However, embryo rescue technique was not able to

overcome strong post fertilization barrier. Based on the embryo rescue results, the

crossability between O. sativa and O. officinalis ranged from 0-2.44%. Strong crossability

barrier was found between O. sativa and O. granulata, and hence no hybrids were

obtained. The crossability between O. sativa and common wild rice varied from 7.58-

51.05%, and on an average landraces had closer cross affinity than advanced cultivars.

Comparative study of morphology in parents and hybrids revealed that wild traits were

found dominant to cultivated forms. Percent mean pollen and spikelet fertility in parents

and hybrids varied from 33–78.16 and 0-49.93, and 30-91.46 and 0-86.49, respectively.

Although, Manshara/O. nivara had low pollen fertility, seed set was higher than its female

parent. No significant associations were observed between mean percentage of stainable

pollen and spikelet fertility. The meiotic behavior at different stages of meiosis in

intragenomic hybrid was more or less normal, and the frequency of aberrations was

comparable to their respective parents. Only six out of eleven hybrid combinations had

cryptic structural hybridity in 4-22.58% cells at pachytene stage. In these hybrids existence

of loose pairing, inversion, translocation, deletion, duplication, and heteromorphocity were

2

observed. Similarly, intergenomic hybrid (O. sativa/O. officinalis) showed high

abnormality in later stages of meiosis, although pachytene was normal in more than 50%

cells. Therefore, it is inferred that the abnormality in these hybrids was brought about by

desynapsis rather than absence of complete homology between their genome (A and C).

Most of the chromosomes in these hybrids appeared as univalents (24) at diakinesis and

metaphase I, and laggards and bridges at anaphase I and telophase I. Based on chiasma

frequency, O. granulata was found more primitive than other species. All the analyzed

wild species of Nepal were diploid (2n = 24). At diakinesis and Metaphase I, no sharp

reduction in chiasma frequency was observed except in O. sativa/O. officinalis derived

hybrids. The mean frequency of ring bivalent was always high and varied from 10.42-11.25

and had >21-<24 chiasmata/cell in most of the hybrids. Other minor irregularities such as

presence of univalents, quadrivalents, presence of nucleolar bodies, bridges and laggards,

laggards, bridges and fragments, and unequal segregation at diakinesis–telophase I were

observed and were comparable to their parents. However, cells with bridges + fragments

were highest in IR64/O. rufipogon (9.40%). On an average, no sharp structural

differentiation was found between common wild rice and landraces of Nepal. Thus the

partial sterility accounted in hybrids did not associate with chromosomal abnormalities

except in IR64/O. nivara, IR 64/O.rufipogon, Jethobudo/O. nivara, and O. sativa/O.

officinalis hybrids. Some of the landraces like Pokhreli, Jethobudo, and Kalanamak showed

high crossability, F1 fertility and close meiotic affinity with O. rufipogon, and those of

Manshara with O. nivara.

Dr. Lakshmi Prasad Subedi Raj Kumar Niroula

Major advisor Author

3

INTRODUCTION

1.1. Background

Rice is the principal lifeline food of more than half of the world’s population

(Mallik, 2002). Among cereals, rice is the major source of calories for most of the Asian

people where 90% of rice is produced and consumed. At present demand for rice is rapidly

increasing with the increase in population and will be increasing continuously, especially in

less developed countries (Virmani et al., 1997). In spite of several yield limiting factors, it

is expected, so much so, that by 2025, 800 million tones of rice will have to be produced

annually to feed ever increasing population (Swaminathan, 1998).

Similarly, Rice farming in Nepal is the foremost industry that contributes about

24% of the total gross agricultural products (Pokhrel, 1997), and the actual national

economy still depends on what happens to rice cultivation. It is widely grown in different

agro-ecological zones accounting for more than 50% of the total area and production

(Upadhyay, 1996). Although, rice (Oryza sativa. L.) in Nepal has been cultivated prior to

Vedic time (Upadhyay et al., 2002) and its cultivation ranges from the seasonal deep-water

river basin to steep sloppy upland field of over 2700 m. altitude, the increment in average

productivity during the past decays has been small. However, variations exist in its

landraces and wild relatives, from the floating rice of tropical climate to chill tolerant

upland rice varieties, indicating that Nepal has rich rice genetic resources (Shahi, 1999).

In spite of its low productivity, at present, it is estimated that Nepal has over 2000

local landraces (Shrestha and Shrestha, 1999), and four species namely O. rufipogon, O.

nivara, O. granulata, and O. officinalis out of 22 valid wild species of rice around the

world (Upadhayay et al., 2002; Shrestha and Vaughan, 1989) and these constitute the all

three basic gene pool of rice (Khush, 2000). Beside, Nepal also harbors weedy rice: Oryza

4

sativa f. spontanea, and Oryza related genera: Leersia hexandra, and Hygroryza aristata.

These landraces and wild relatives of cultivated crop plants constitute the excellent

insurance against the genetic vulnerability (Chang, 1976b). It is because of wild relatives

have been used as gene/s source, against the biotic and abiotic stresses, to provide

alternative cytoplasm and develop cytoplasmic sterility systems, to widen adaptation, to

improve stature, increase crossability between, and increased yield. (Harlan, 1976). It is

also note worthy that some crops could not maintain commercial status without genetic

support of their wild species (Harlan, 1976). Beside these wild species of rice can be used

in hybridization and varietal development and improvement programmes (Brar et al.,

2002), and to isolate the apomictic gene block/s for sustaining and the commercialization

of the hybrid rice production throughout the world through various biotechnological tools

(Li and Yuan, 2000).

Experiences at IRRI and in China and other countries have made remarkable

contributions to recognize the worth of wide hybridization and wild relatives of rice in rice

breeding and cultivar development. Considering the above mentioned facts, Nepal has still

great opportunity to exploit these wild relatives, however, their proper utilization and

exploration has not been carried out yet. Only very few landraces have been used in

varietal improvement and development programme. On the other hand, its tremendous

amount of genetic diversity is declining day by day (Lu, 1999a; Chang et al., 1982).

Therefore, to cope with such burning problems for present and future necessitates several

breeding strategies. Among them, varietal improvement and development through wide

hybridization including modern varieties, land races and wild relatives is one of the viable

options within country to extend the rice crop into new management regimes, new habitats,

or regions of marginal climate, edaphic adaptation and bridging the yield gap in its

production. However, their inclusion in the breeding program always necessitates the study

5

of crossability, genome relationship, and meiotic pairing between wild and cultivated

species (Murthy and Reddy, 1993; Bennett, 1966). Understanding of the evolutionary

change is essential to increase our efficiency as plant breeder engaged in promoting change

to meet human needs (Hutchinson, 1970). Not only, but also genome and evolutionary

relationship are crucial aspects for directing our effort to search for beneficial genes in wild

species of rice (Chen, 2002).

But several reproductive isolating mechanisms restrict the production of distant

hybrids and the transfer of useful genes (Khush and Brar, 1992; Sitch, 1990). They occur in

interspecific hybridization as a result of either sexual incompatibility or hybrid inviability

(Willims et al., 1987). From the viewpoint of breeding, abortion of hybrid embryos at

different stages of development is a more important and frequently encountered

postfertilization barrier in interspecific hybrids than others (Khush and Brar, 1992). It is,

however, possible to overcome this post fertilization barrier through rescue of immature

embryo (Jena and Khush, 1984; Laibach, 1929), and other in vitro techniques (Raghavan,

1985).

Once a distant hybrid was established, a carefully planned pre-breeding program

and repeated backcrossing through cytogenetical manipulation is necessary to transfer

useful genes from many wild species to an improved plant type (Singh et al., 1990). On the

other hand, knowledge of the cytogenetic relationships between cultivated species and their

allied wild relatives has proved to be essential in utilizing the desirable germplasm in the

most effective manner (Bernham, 1966; Hey and Henderson, 1962). Although in the past,

progress in crop production and productivity has been mainly due to conventional

approaches and techniques, we can’t expect to continue improving crop production

indefinitely by only conventional techniques. Several innovative approaches in breeding

crop plants have, therefore, been developed. One of these approaches in plant breeding

6

demands constant reference to the chromosomal status of the breeding materials. Such

approaches involve interspecific hybridization, alteration in ploidy, aneuploidy, structural

changes in chromosomes and regulation of recombination, and yield a wealth of

information about the genetic architecture of crop plants, which may become the basis for

future researches in plant breeding (Swaminanthan and Gupta, 1983).

Likewise, meiotic study is inevitable to generalize the background information on

evolutionary history, mode of speciation, cytological traits, species relationships etc. such

information’s have been greatly emphasized (McClintock, 1978; Hawkes, 1977; Morinaga,

1964; Li et al., 1961). That are essential for a conceptual approach to and design of a

breeding program and to introduce the alien variation from allied wild and weedy species

to the cultivated ones (Swaminanthan and Gupta, 1983).

Similarly, chromosome pairing study is a preliminary aspect, because it determines

the degree of variability, viability and stability of a variety and/or hybrid and progress of

breeding program when develop through wide hybridization. Brar and Khush (1997)

pointed out that there is urgent need to reinvestigate the chromosome pairing at earlier

stage, particularly at pachynema. It is because of strong desynapsis mechanism existed, at

later stages of meiosis, in the chromosome of cultivated and wild species having different

genome. Therefore, karyomorhological study, banding pattern, which is chromosome

specific (Griffiths et al., 2000), and meiotic behavior and its affinity studies at different

stages are the fundamental and preliminary aspect in rice cytogenetic study to utilize these

diverse germplasm in effective manner. This approach is one of the invaluable options to

all the plant breeders. Hence, keeping these facts about the value of wide hybridization

adopting various supplementary techniques of tissue culture and their cytogenetic study in

plant breeding, the present study was undertaken with the following objectives:

7

1.2. Objectives

i) To produce the intergenomic F1 hybrids through in vitro manipulation.

ii) To examine the crossability, pollen fertility and sterility, and morphological

characters in the parents and F1 hybrids.

iii) To study meiotic behavior, meiotic affinity, and chiasma frequency in parents and

their hybrids.

iv) To determine the chromosomal status of Nepalese wild rices.

8

2 LITERATURE REVIEW

2.1. Taxonomy and origin of rice

Rice is classified under the family Poaceae (gramineae), sub-family Oryzodeae,

tribe Oryzae. There are 12 genera under Oryzae tribe. Under the genus Oryza 24 spcecies

with ten different genomes including four valied section and three gene pools have been

reported so far (Khush, 2000). However, the number of species under genus Oryza delimit

the taxonomist to determine the actual number of species around the world. As a

consequences the number of species always vary with the taxonomist (Lu, 1999b) (Table

2.1).

The present day cultivated rice is probably originated in the humid tropics of

GONDWANALAND before that super continent began to fracture and drift apart about

135 million years ago to south and southeast Asia, Madagascar, Africa, Australia, South

America and Antartica (Chang, 1976c). Based on the molecular analysis several authors

suported this speculation (Joshi et al., 2000; Aggrawal et al., 1999). However, Cheng

(1993) considered the Asia is the place where the origin of the O. sativa occurred. Shrestha

and Upadhayay (1999), Shai (1999) also claimed that monsoonal swampy and tropical

south-faced foothills of Nepal are the most probable “original home land” of the Asian

rice. Khush (2000) also noted that most of the terai region of Nepal, and UP, Bihar of India

are the pimary centres for Asian aromatic rice.

Regarding the ancestral species of Asian cultivated rice, there are no single opinion:

some of the taxonomists, such as Chatterjee (1951), Ramiah and Ghose (1951), and Shastry

(1964) considered the annual taxa of common wild rice, O. nivara, must probably the

immmediate ancestor of Asian cultivated speceis. On the other hand, authors like

Morishima et al. (1961, 1963), Oka and Chang (1962), Oka and Morishima (1971), and

9

nivara (AA)

Nayar (1973) considered that the common perennial wild rice (O.rufipogon) is the direct

ancestor of Asian rice, and differentiaton in the cultivated rice resulted in part from the

geographical differentiation of the races of O. rufipogon (Second, 1982). However, Chang

(1976c) and Chang and Vaughan (1991) later proposed evolutionary trends of cultivated

rice, in which differentiation of perennial to annual first took palce and finally further

differentiation resulted into present cultivated Asian rice. However in contrary, Shao et al.

(1986 ) and Morishima (1986) pointed out that the intermediate of perennial-annual form

are the immediate prototype of rice.

Similar trends also took place in the Africa from other common wild rice (O.

longistaminata), resulted in to african cultivated O. glaberrima. Their proposed path way is

postulated in figure 2.1.

GONDWANALAND

South and South- East Asia Tropical Africa

Wild

perennial

Weedy annuals

Wild

Annual

Cultivated

annual

Figure 2.1. Evolutionary pathway of the two cultivated species of rice. Taxa boxed by solid

lines are wild perennials. Taxa boxed by broken lines are annuals. Arrow with solid

line indicates direct descent. Arrow with broken line indicates indirect descent.

Double arrows indicate introgression hybridization (adapted from Chang, 1976c).

Common

ancestor

rufipogon (AA) longistaminata (A

1A

1)

barathii (AgA

g)

sativa (AA)

indica javanica

japonica

glaberrima (AgA

g)

spontanea

stapfii

10

2.1. Rice biodiversity in Nepal

Nepal represents the unique variability in geographic pattern, and a similar variation

have been reported in its crop plants, particularly, in the case of rice (Shahi, 1999). Its

cultivation ranges from tropic to terperate regions and its allied related taxa pose

accrodingly a similar pattern in genetic variability. It has been reported that, Nepal

endowed with more than 2000 local rices and more than four wild related species namely

O. rufipogon, O. nivara, O. officinalis, and O. granulata (Shresthat, 2002; Shrestha and

Upadhayay, 1999; Shrestha and Vaughan, 1989). Within cultivated landraces differentation

extend from indica to japonica (Nayar, 1973).

Likewise intermediates form, O. sativa f.spontanea, have been reported throughout

the terai region of Nepal (Lu, 1999a). Beside, two Oryza related genera are also abundantly

found in wide range of altitude from160–1640 meter altitude (Shrestha and Vaughan,

1989). Based on molecular analysis, Sharma et al. (1999) reported that the landraces of

Nepal are highly diversed and most of the landraces posseessed one or more economic

traits.

2.3. Description of Nepalese wild rice species

2.3.1 Oryza granulata Griff.

O. granulata is one of the only two wild rice species in the genus Oryza that occurs

in the upland condition (Vaughan, 1994). In Nepal, this species is restricted to Chure hill of

Chitwan and Jhapa district (Shrestha and Vaughan, 1989). It is a small bamboo-like

miniature, perennial, and found under the dense vegetation. Morphology of O. granulata is

unique from other rice species by its small and dark green plants, tightly compact panicles,

and round-elliptical and awnless grains and flowers throughout the year (Vaughan, 1994).

11

The ploidy level of this species reported from other accessions (other countries) is diploid

with GG genome (Aggrawal et al., 1997)

2.3.2. O. officinalis Wall ex Watt.

This species was routinely observed in different parts of terai region of Nepal and

identified as O. officinalis just a decay ago (Shrestha and Upadhayay, 1999). It is usually

rhizomatous herb of variable height; basal panicle branches and is diploid with CC

genome. However, the Nepalese O. officinalis either diploid or teraploid, it is still

unknown. It is frequently grown in seasonally wet areas, ditches, swampy places, near

small water holes, and along lakeside, streams, or rivers and found under full sun or partial

shade (Vaughn, 1994).

2.3.3. O. nivara Shrama et Shastry, and O. rufipogon Griff.

O. nivara and O. rufipogon are widely distributed in Southeast Asia and other rice

growing countries (Vaughan, 1989). In the recent year, they are frequently known as

common wild rice and are recognized as the immediate prototypes of cultivated rice

(Khush, 2000; Chang, 1976a). Similarly, the large population of these two species has been

frequently reported throughout the terai region of Nepal (Shrestha, 2002; Upadhyay and

Gupta, 2000; Shrestha and Upadhyay, 1999). O. nivara is annual and it has short plant

stature with many basal tillers, compact panicles, broad and well filled grains with long and

tough awns and small anthers (Shrestha and Upadhyay, 1999).

On the other hand, O. rufipogon is perennial and It has distinct nature of tallness

and long awns along with black husk and long slender grain, larger anther and procumbent

type of growth habit and both the species are diploid (Vaughan, 1994).

12

2.4. Genome and genepool

Various approaches including morphological differentiation, meiotic chromosome

pairing in F1 hybrids, and molecular divergence analysis, have been used in genome

analysis, and in determining species relationships in Oryza (Khush and Brar, 2001). At

present, ten different genomes have been identified and assigned under the genus Oryza

based on meiotic pairing study and DNA based technology (Table 2.1). Based on ease of

gene transfer from wild related species to cultivated rice, Khush (2000) divided Oryza

species into primary, secondary and tertiary gene pool. According to him, Primary gene

pool consists of species having AA genome, secondary includes the O. officinalis complex

and tertiary gene pool comprises the O. meyeriana, O. ridleyi and O. schlechteri complex

(Table 2.1). Hybrid production and subsequent gene transfer from this complex are rare or

extremely difficult.

2.5. Importance of wild germplasm

Frankel and Brown (1984) stated that wild germplasm are donors of genetic

material rather than for direct use except some instances. They provide safe guard against

unpredictable future genetic vulnerability (Alcazar, 1993; Harlan and Starks, 1980; Chang,

1976b; Harlan, 1975). The wild relatives of the cultivated cereals form an important

reservoir of genetic variability for various economic characters such as disease and insect

resistance, tolerance for abiotic stresses, increased biomass and grain yield, and improved

quality characteristics (Harlan, 1976). The use of wild relatives is relatively demanding in

recent year as in some instances, the genetic variability for some of these traits are limited

or unavailable in the cultivated germplasm (Stalker, 1980). Not only, but also for example,

in case of rice biotic stress resistance seems to follow an approximately 1:50 rule i.e. wild

taxa offer 50 times more resistance genes than cultivated ones (Vaughan, 1989).

13

Table 2.1. Chromosome number, Genomic composition, geographical distribution and useful traits of Oryza species.

Species 2n Genome Distribution Useful or potentially useful traitsa

O. sativa complex

O. sativa L. 24 AA Worldwide Cultigen

O. nivara Sharma et Shastry 24 AA Tropical and subtropical Asia Resistance to grassy stunt virus

Blast , drought avoidance

O. rufipogon Griff. 24 AA Tropical and subtropical Elongation ability, source of

Asia, tropical Australia CMS, resistance to BB

O. breviligulata A. Chev. et Roehr. 24 AA Africa Resistance to GLH, BB,

drought avoidance

O. glaberrima Steud. 24 AA West Africa Cultigen

O. longistaminata A. Chev. et

Roehr.

24 AA Africa Resistance to BB, drought

avoidance, source of perennial

rice

O. meridionalis Ng 24 AA Tropical Australia Elongation ability and drought

avoidance

O. glumaepetula Steud. 24 AA South and Central Elongation ability and, source

America of CMS

O. officinalis complex

O. punctata Kotschy ex. Steud 24,48 BB,BBB

C

Africa Resistance to BPH, zigzag leaf

hopper

O. minuta J.S. Presl ex C.B. Presl 48 BBCC Philippines and Papua Resistance to sheath blight, BB,

New Guinea BPH, GLH

O. officinalis Wall ex Watt 24 CC Tropical and Subtropical Resistance to thrips, BPH, Asia,

Tropical Australia GLH, WBPH

O. rhizomatis Vaughan 24 CC Sri Lanka Drought avoidance,

rhizomatous

O. eichingeri A. Peter 24 CC South Asia and Resistance to yellow mottle

East Africa virus, BPH, WBPH, GLH

14

O. latifolia Desv.

48

CCDD

Table continues………

South and central America Resistance to BPH, high biomas

production

O. alata Swallen 48 CCDD South and central America Resistance to striped stemborer,

high biomass production

O. grandiglumis (Doell) Prod. 48 CCDD South and central America High biomass production

O. australiansis Domin. 24 EE Tropical Australia Drought avoidance, resistance

to BPH

O. meyeriana complex

O. granulata Nees et Arn. ex Watt 24 GG South and South East Asia Shade tolerance, adaptation to

aerobic soil

O. meyeriana (Zoll et Mor. ex

Steud.) Baill

24 GG Southeast Asia Shade tolerance, adaptation to

aerobic soil

O. ridleyi complex

O. longiglumis Jansen 48 HHJJ Irian Java (Indonesia) Resistance to blast, BB

and Popua New Guinea

O. ridleyi Hook. F. 48 HHJJ South Asia Resistance to stemborer, whorl

maggot, blast, BB

Unclassified

O. brachynatha A. Chev. et Roehr. 24 FF Africa Resistance to yellow stemborer,

leaf folder, whorl maggot,

tolerance to laterite soil

O. schlechletri Pilger 48 HHKK Papua New Guinea Stolonniferous

a BPH = brown plant hopper, GLH = green leafhopper, WBPH = white-backed planthopper, BB = bacterial blight, CMS = cytoplasmic male

sterility

Source: Khush and Brar (2001) and Khush (2000)

15

From time to time, the roles of wild relatives of cultivated forms have been emphasized

either theoretically or practically at greater extent in different crop species (Singh et al.;

1990; Moss, 1985; Frey et al., 1984; Swaminathan and Gupta, 1983; Stalker, 1980;

Hawkes, 1977; Harlan, 1976; Knott and Dvorak, 1976)

A few notable examples that raised the common concern in the utilization of wild

relatives are rust resistance in wheat (Sears, 1984; Knott, 1971), grassy stunt resistance in

rice (Khush, 1977) and transfer of CMS source for hybrid rice (Li and Yuan, 2000),

mildew and crown rust resistance in oats (Aung and Thomas, 1976; Browning and Frey,

1969), increased biomass and grain yield in oats, perlmillet, and sorghum (Frey et al.,

1984) and in rice (Xiao et al., 1998), and successful transfer of southern corn leaf blight

resistance gene into maize from a wild related species (Mann, 1997; Chang, 1976b;

Hooker, 1974). Recent advances in tissue culture and molecular marker technology and in-

situ hybridization technique have enabled to the utilization of wild species of crop plants to

fuller extent (Brar et al., 2002).

2.6. Progress towards the utilization of wild relatives of rice

Rice crop is grown world wide under a wide range of agroclimatic conditions

therefore its productivity is affected by several biotic and abiotic stresses. The genetic

variability for some of the traits, such as resistance to tungro, sheath blight, yellow stem

borer, tolerance to acid sulfate condition and drought tolerance, is limited in the germplasm

of cultivated rice. More over, continuous changes in insect biotypes and disease races are a

continuing threat to increase rice production. Under such conditions, wild species of rice

are a good source of useful variability (Brar et al., 1996). Thus there is urgent need to

broaden the rice gene pool through genes introgression for such traits from diverse rice

germplasm (Brar and Khush, 1995). However, in the past breeders did not utilize these

16

Table 2.2. Progress in gene introgression and transfer from wild Oryza species into elite lines of cultivated rice

Trait transferred to O. sativa (AA)

Grassy stunt resistance

Bacterial blight resistance

Blast resistance

Brown plant hopper resistance

Whitbacked planthopper resistance

Cytoplasmic male sterility

Tungro tolerance

Tolerance to acid sulfate soil

Yield improving trait

Earliness

Resistance to root–knot nematode

Name of gene/s donor

wild species

O. nivara

O. longistaminata

O. officinalis

O. minuta

O. latifolia

O. australiensis

O. brachyantha

O. minuta

O. officinalis

O. minuta

O. latifolia

O.australiensis

O. officinalis

O. sativa.f. spontanea

O. perennis

O. glumaepatula

O. nivara

O. rufipogon

O. rufipogon

O. officinalis

O. rufipogon

O. rufipogon

O.australiensis

O. longistaminata

Genome

symbol

AA

AA

CC

BBCC

CCDD

EE

FF

BBCC

CC

BBCC

CCDD

EE

CC

AA

AA

AA

AA

AA

AA

CC

AA

AA

EE

AA

References

Khush (1977)

Brar et al. (2002); Brar et al. (1996)

Brar et al. (2002)

Brar et al. (2002); Amante et al. ( 1992)

Brar and Khush (1997)

Multani et al. (1994)

Brar et al. (1996)

Amante et al. (1992)

Jena and Khush (1990)

Brar and Khush (1997)

Brar and Khush (1997)

Multani et al. (1994); Ishii et al. (1994)

Jena and Khush (1990)

Lin and Yuan (1980)

Dalmacio et al. (1995); Brar et al. (1998)

Dalmacio et al. (1996)

Brar et al. (1998)

Brar et al. (1998)

Khush et al. (2000); Khush et al. (1990)

Kobayashi et al. (1992).

Brar et al. (2002)

Martinez et al. (2002); Xiao et al. (1998)

Ishii et al. (1994)

Soriano et al. (1999)

17

potential germplasm due to great difficulties to deal with (Khush, 1994). These difficulties

mostly attributed to strong pre and post fertilization barrier and limited recombination

between homoeologous chromosome of two different genomes is the most problematic one

(Brar et al., 1996)

However, successful transfer of grassy stunt virus resistance genes and CMS source

from common wild rice are the two historical corner stone that rose the interest in the

utilization of wild species. Now, utilization of wild taxa of rice is a component research at

IRRI. At International Rice Research Institute (IRRI), a series of hybrids and monsomic

alien addition lines (MAALs) have been produced following hybridization and embryo

rescue technique. At present, utilization of wild species is highly advanced in rice breeding

program and become a routine tool. To date more than dozens of genes have been

transferred (Brar and Khush, 1997; Brar et al., 2002). Details about the gene transfer from

wild taxa to cultivated forms of rice were shown in Table 2.2.

2.7. Wide hybridization

The meaning of wide hybridization and distant hybridization is quite different.

Chang and Vaughan (1991) defined wide hybridization as hybridization between species

having the same genome and on the other hand hybridization between species having

different genomes, or unknown genome in the same genus is called distant hybridization.

Thomas Fairchild was the first who produced wide cross hybrids plants, between Carnation

and the Sweetwilliam, in 1717 (Allard, 1960). Similarly in rice, the first hybrids plants was

produced in 1898 by Takahashi in Japan (Morinaga, 1964). Since then several wide and

distant hybrids in rice have been produced to introgress the segment of particular genes

and/or chromosome in to cultivated ones following the simple tissue culture approach (Brar

18

et al., 2002).

Now, wide hybridization in rice has become a significant plant breeding tools for

the incorporation of genes of interest from wild to cultivated ones in current rice breeding

(Jena and Khush, 1984). Many breeders apparently have concluded that the interspecific

path is necessary for achieving their goals. A common justification for this choice is that

wild relative and progenitors of our crops can be tapped for “genes carrying special

attributes not apparently in the cultivated forms (Bates and Deyoe, 1973). Briggs and

Knowles (1967) have emphasized the interspecific path to transfer one or a few genes from

one species to another, achieve new character expression not found in either parent,

produce new alloploid species, and to determine the relationship of one species to another.

2.7.1. Barriers in species hybridization

Although a few species combinations are highly interfertile, hybridization between

the majority of related but distinct Oryza species is normally prevented in nature by

prefertilization and/or postfertilization barriers (Sitch et al., 1989a.b; Sitch and Romero,

1990). Williams et al. (1987) diagrammatically presented these barriers in his review paper

to illustrate the possible phases of incompatibility occurring during interspecific pollination

for pasture legumes. However, these barriers are similar to those seen in many other

grasses families (Harrison, 1982 in William et al., 1987). Therefore, this diagram is also

presented to cover better understanding (Figure 2.2).

2.7.1.1.Prefertilization barriers in interspecific cross

No so much research has been conducted regarding the prefertilization barrier in

species cross of rice. However, Sitch et al. (1989 a, b); Sitch (1990) and Sitch and Romero

(1990) made special treatment in this field and reported that prefertilization incompatibility

19

in interspecific and intergeneric crosses involving O. sativa. According to Sitch and

Romero (1990), pollen germination was normal in crosses of O. sativa with O.

brachyantha, O. eichingeri, O. officinalis, and O. ridleyi and slightly inhibited in crosses

with Rhynchoryza subulata. Pollen tubes of O. eichingeri and O. officinalis, and O. ridleyi

penetrated the stigma but growth was frequently inhibited in the style. Similar, stigma-

style incompatibility has been reported in intergeneric hybrids involving O. sativa and

Proteresia coaractata (Sitch et al., 1989a).

2.7.1.2.Post fertilization barrier in interspecific cross

Among post fertilization barriers hybrid embryo abortion at an early stage is the

characteristics feature of wide crossing in rice (Brar and Khush, 1994). However,

postfertilization barrier includes hybrid inviability, hybrid weakness and hybrid break

down and some times fertility distortion (Brar and Khush, 1986)

Hybridization among AA genome species generally gives rise to viable seeds and

plants, but the rate of successful crossing is very variable and embryo rescue has

sometimes been used (Nowick, 1986). In some cases, as reported by Chu and Oka (1970),

when O. sativa was pollinated by O. longistaminata, the F1embryos and endosperm begin.

to deteriorate about 6 days after fertilization, and when O. longistaminata is used as

maternal parent, hybrid embryos fail after 3 days due to presence of a barrier controlled by

a set of complementary dominant lethal genes. Interspecific hybrids involving AA group

are generally highly sterile but their chromosomes nevertheless homologous (Bouharmont,

1991). Their fertility can be easily restored and introgression is possible through back

crossing. On the other hand, distant hybridization among rice genomes necessitates the

embryo culture (Bouharmont, 1991). For example, as stated by Jena and Khush (1986)

production of interspecific hybrids between O. sativa and O. officinalis, embryo rescue is

essential as embryo abortion started after 10-14 days of fertilization.

20

Normal Reproduction Prefertilization Barriers

Postfertilization Barriers

Figure 2.2. Schematic representation of stages in normal sexual reproduction (left) and

related barriers to interspecific hybridization (right) Williams (1987) and Headly and

Openshaw (1980).

Pollen hydration and germination on stigma

Failure of pollen germination eg. by

osmotic mismatch of pollen and

stigmatic fluid

Pollen tube growth through stigma to reach style

Pollen tube growth through style to ovary

Pollen tube growth through ovary and into ovule

Pollen tube penetration of

embryo sac, and double fertilization

Embryo and endosperm

development to seed maturation and germination

Seedling growth

Vegetative growth

Reproductive success

Interspecific pollen / pistil

incompatibility or incongruity

expressed at a number of possible

levels in the pistil from stigma surface to penetrated embryo sac

Seed abortion

Seedling lethality

Poor vigor or abnormal growth

Hybrid sterility/ hybrid break down*

21

2.8.1. Crossability between intra and inter genomic species cross

Different investigators from time to time obtained differences in crossability

between species crosses depending upon the genotypes of female parent and geographical

races of wild species used. The crossability between O. sativa and O. rufipogon was greatly

varied from 10-30% depending upon the geographical race of wild species and genotype of

O. sativa used (Shastry, 1964; Henderson, 1964a.; Nezu et al., 1960; Ghose et al., 1960).

Sitch et al. (1989c) obtained 23% seed set in cross involving IR64/O. rufipogon and 42.2%

seed set in IR64/O. perennis. Not only, low crossability genes (Lcr) have been reported in

certain geographical races of common wild rice, O. rufipogon resulted in low seed set

(Sano, 1991).

Like wise, Jena and Khush (1986, 1984) reported that crossability between three

lines of O. sativa and O. officinalis varied from 1.0-2.3 %. However, Brar et al. (1991)

obtained quite low crossability which was ranged from (0-1.1%). Similarly, other workers

(Brar et al., 1991; Morinaga, 1964; Ghose et al., 1960) also obtained non or few hybrids in

this set of cross.

2.8.2. Causes of embryo abortion in wide cross

Successful development of an embryo depends upon the accompanying

development of endosperm tissue capable of nourishing the embryo and a harmonious

interaction of related embryo, endosperm, and maternal tissues (Singh et al., 1990). Based

on the comparative study regarding the endosperm development and cytokinin biosynthesis

in the normal and hybrid endosperm, Singh et al. (1990) suggested that the abortion of the

embryo is attributed to embryo and endosperm incompatibility and reduction in the

cytokinenis biosynthesis in hybrids endosperm. Similarly, Hadley and Openshaw (1980)

22

suggested that the differences in the dose effects of the genes as they act in the hybrid

tissue of the resultant genomic combination could also lead endosperm disintegration.

Hadley and Openshaw (1980) mentioned the three probable cause of hybrid

weakness or inviability: disharmonies between genes of the parental species, disharmonies

between genome of one species and the cytoplasm of the other, and disharmonies between

the genotype of the F1 zygote and the genotypes of endosperm or the maternal tissue with

which the developing F1 embryo is associated or due to the physiological upset (Skrim,

1942). Kobayashi and Sano (1996) reported Lcr in wild rice O. rufipogon in chromosome 6

that cause unidirectional cross incompatibility when crossed with japonica strains. In such

situation Lcr acts as postfertilization barrier at which hybrid embryo undergo to degenerate

within 4-5 days after fertilization (Sano and Kobayashi, 1996). Copper and Brink (1940 in

Hadley and Openshaw, 1980) purposed somatoplastic sterility in wide cross hybrid. In this

case the excessive growth of maternal tissues impairs the capacity for endosperm

development and leads to starvation and collapse of the embryo.

2.8.3. Seed and embryo differentiation in wide hybrids

In rice and other grass families fertilization and growth of the embryo are rapid and

provides the useful feature for the application of embryo culture and for the rescue of

hybrid progenies from incompatible crosses (Bouharmont, 1991). In case of normal

pathway, rice embryo is completely organized in less than two weeks. On the other hand

when widely related species are crossed, fertilization takes place in some spikelets, the

differentiation of the embryo can be normal for several days and the lower part of the ovary

is swollen. In contrast, the rest of the ovary remains slender and collapses sooner or later

due to some disturbances occurring during endosperm development (Ragahvan, 1985).

23

In some cases, an embryo can develop for several days in the absence of endosperm

but the differentiation of the organs is disturbed and delayed as compared to normal

(Zhang, 1978). In this situation malformation is prominent feature particularly in the

scutelum (Bouharmont, 1991). Zhang (1978) reported that irregular cell division in O.

sativa and O. officinalis cross and found endosperm degeneration started 5th

day after

pollination and considerable deformity was found in 7-10th

day of pollination. Similarly,

Kobayashi and Sano (1996) and Sano and Kobayashi (1996) found three cross

incompatible genes, which causes embryo abortion in unidirectional cross.

2.9. Embryo rescue in rice

Embryo culture in plant breeding refers to the culture of excised immature embryos

on artificial culture medium to obtain the normal viable plants with different objectives.

The credit of embryo culture goes to the Laibach (1929) and Skirm (1942) for

demonstrating the important application of the embryo culture technique to overcome

interspecific incompatibility in Linum and Prunus, respectively. In vitro culture of hybrid

embryo is useful in wide crosses where fertilization occurs and embryo begins to develop

but aborts before reaching full maturity (Nandan, 1997). This technique has considerable

application as a method of obtaining novel gene combination from interspecific or

intergeneric hybridization.

In the Genus Oryza, Niles (1951) has firstly been adopted the method of culture of

intraspecific hybrids seeds on artificial medium. However, detail events of rice embryo

culture have been noticed by Amemiya et al. (1956a.b). They studied the culture condition

and first germinative stage of immature rice embryos. Nakajina and Morishima (1958)

have obtained various interspecific hybrids by using the techniques designed by the

24

Amemiya et al. (1956a.b). Since then several workers have been obtained F1 hybrids from

otherwise unsuccessful interspecific cross in rice through embryo culture in a number of

cross combinations (Bouharmont, 1991; Iyer and Govilla, 1964; Li et al., 1961). On the

other hand authors like Katayama (1966a.b), Gopalkrishinan and Shastry (1966), Nowick

(1986), and Mariam et al. (1996) have also been adopted the embryo rescue to obtain the

interspecific rice hybrids for their cytogenenetic study by using different nutrients media.

Likewise, Yie and Liaw (1975) studied the growth and development aspects of

excised embryos of rice under in vitro and Ko et al. (1983) described a simplified fast and

efficient method of rice embryo culture particularly excision of the embryo. Similarly Jena

and Khush (1984), and de Guzman (1983) advanced the embryo culture techniques at

IRRI. Following these technique wide arrays of intersectional hybrids have been produced

at IRRI and in other countries (Abdullah, and Somantri, 1995; Brar and Khush, 1995; Brar

et al., 1991; Sitch et al., 1989c).

2.10. Hardening of regenerated seedling before field transfer

No much report have been found to raise the embryo rescue regenerated seedling of

rice in field condition despite of its great importance to establish the plants in the field

conditions. Only two reports paid special attention in the concerned subject (Iyer and

Govilla, 1964, Bouharmont, 1961). Transferring cultural seedlings direct to potted soil can

pose a serious survival problem. Bouharmont (1961) hardened seedlings by keeping their

roots in petridishes containing distilled water and growing under shade. On the other hand,

Iyer and Govilla (1964) improved survival rates by growing cultured seedlings in a nutrient

solution as suggested by Karim and Vlamis (1962) before transferring them into soil.

25

2.11. Hybrid embryo derived callus culture

This technique was increasingly utilized in a number of crosses involving distant

wild relatives of different crop species and a number of successes have been reported other

wise embryo culture technique failed to give hybrid plants (Brar and Khush, 1994).

Embryo derived callus culture in rice was suggested by Bajaj and Bidani (1980) and

reported wide range of genetic variability was recovered in the regenerants. Likewise,

Nowick (1986) successfully employed this technique in O. sativa/O. latifolia hybrids and

reported similar results (1980).

2.12. In vitro fertilization

Any manipulation of excised maternal and paternal tissue to accomplish pollen tube

penetration to the embryo sac for affecting fertilization is referred to as in vitro

fertilization. It is an important technique for overcoming the barrier inhibiting the pollen

tube growth and very early stage embryo abortion. To overcome these difficulties, in vitro

fertilization followed by culturing of fertilized ovules to maturity is a promising approach

and may be a viable alternative even to parasexual hybridization (Brar and Khush, 1986,

1994). Similarly, the method has been widely used in other crop species such as soybean

(Tilton and Russel, 1982), and maize (Gengenbach, 1982). This is one of the viable options

to attempt wide hybridization and alternation of cytoplasmically controlled traits. As stated

by Zhang (1985) fertilization of ovules in test tube was first time reported by Kanta et al.

(1962) in Papaver somniferum .

Yeh et al. (1980) was also made in vitro fertilization in distantly related species

using this technique in tobacco and wheat. However no much research have been made yet

regarding the cereals. Dhaliwal and King (1978) obtained 5% seed set when Zea mays

ovules fertilized by Z. mexicana pollen through this technique. Zhang (1985) suggested

26

that sterilization of parental flowers in rice for in vitro fertilization was difficult, despite of

that he obtained 5.8% success in inter varietal cross.

2.13. Media composition

A primary consideration for transplant survival is seedling vigor, which can be

determined by the growth medium and other culture conditions (de Guzman, 1983).

Several attempts have been made to standardize the media composition for in vitro rice

embryo culture, however, most of the reports shown that any media can successfully be

employed by slight modification (Ko et al., 1983; Yiew and law, 1975; Bouharmont, 1961;

Nakajima and Morishima, 1958; Amemiya, 1956a.b).

2.14. Hybrid sterility

Hybrid sterility in general normal phenomenon in wide crosses, and is attributed to

either chromosomal differences or genetic, and genetic-cytoplasmic interaction (Allard,

1960, Stebbins, 1958). In wide cross hybrids most of the cause of sterility is mainly

attributed to structural differences and limited pairing of the parental chromosome (Allard,

1960). In wide cross rice hybrids too sterility was brought about by similar phenomenon

(Henderson, 1961; Shastry and Misra 1961a.b; Yao et al., 1958). However, under normal

chromosome pairing conditions, sterility is attributed to genetic, and genetic and

cytoplasmic factors of the parents (Sage, 1976; Meyer, 1969; Plamer and Hadley, 1968).

But third one can be easily recognized through reciprocal crossing and no much important

(Hadley and Openshaw, 1980).

The sterility; the spikelet sterilities in the hybrids are mainly caused by the pollen

sterility (Guiquen et al., 1994). The sterility genes determined by allelic interaction seem to

be of wide occurrence (Sano, 1990) between distantly related taxa and serve as one of the

27

genetic mechanism for hybrid sterility (Sano, 1983). Three different genes causing F1

hybrids sterility were extracted and analyzed between the two species O. sativa and O.

glaberrima (Sano, 1983). Of them, two genes acted as gamete eliminator and the other

acted as pollen killer, which suggests that gametic abortion due to allelic interaction

frequently occurred between them.

2.15. Chromosome pairing in Haploid rice

Based on chromosome pairing study in haploid rice of O. sativa. L, Hu (1957)

reported existence of intragenome pairing and this was due to true pairing. Again Hu

(1960) analyzed the meiotic behavior in haploid rice variety belonging to O. glaberrima.

Stued. and reported that about 34.2% of the cells at diakinesis, metaphase I and anaphase, I

showed pairing association having 1II is common. However, he inferred later that the intra-

genome pairing in later was due to secondary association.

Rao (1984) reported similar results in haploid indica rice variety T1242 and

observed 90.6% cells showed chromosome association of 8I+2II, followed by 10I+1II in

9.4% of the cells studied by him. Based on his observation and previous observation of Hu

(1957, 1960), he concluded that consistent synapsis in haploid rice indicates partial

homology or residual homology between the chromosomes.

2.16. Chromosome behavior between AA genome species hybrids

2.16.1. Meiosis in inter-varietal crosses

Hsieh and Oka (1958, in Nayar, 1973) found also no disturbance in chromosome

pairing in several hybrids. Occasionally, univalents, stretched chromosomes, and anaphase

bridges were found. The authors then attributed to precocious separation or reunion of

sister chromatids after a break at the diplotene stage. Sampath and Mohanty (1954) found

28

low frequencies of bridges and fragments indicating the presence of paracentric inversions.

Venkataswamy (1963) found quadrivalent in certain crosses, which showed the presence of

reciprocal translocation. They observed univalents, quadrivalents, and anaphase bridges in

low frequencies in some crosses. Meiotic analyses in the pachytene stage was done by Yao

et al. (1958) for the first time they observed loops, which were interpreted as resulting from

inversions, in less than 10% of the cells examined in 5 out of 7 crosses. Diakinesis and MI

were normal. In further studies, Henderson et al. (1959) obtained bridges with fragments in

0.28% of AI cells in 9 out of 12 combinations. Anaphase bridges without fragments were

noticed in both parents and hybrids. They concluded that a genetic basis for the cause of

sterility was improbable, and that instead it could be attributed to cryptic structural

differences caused by inversion of an included type (Henderson, 1964b).

Shastry and Misra (1961a.b) repeated pachytene analyses and reported that meiosis

was highly abnormal, showing the presence of inversions, translocations, deletions, and

differential segments. In three semisterile hybrids, up to 31% of the chromatin length was

unpaired. MI and AI stages were normal. They proposed that the main cause of sterility

was cryptic structural differences of the chromosomes caused mostly by translocation

(Shastry, 1964). In contrast to these findings, Wu et al. (1964) found pairing abnormalities

only once during pachytene analyses of four hybrids. Likewise, there are number of

investigator who reported considerable paring anomalies in hybrids involving indica and

javanica varieties (Nayar, 1973).

2.16.2. Pachytene analysis

Yao et al. (1958) was the first who initiated the pachytene analysis however,

detailed analysis was made by Shastry and Misra (1961b) in indica-japonica rice hybrids.

Based on the pachytene analysis Yao et al. (1958) observed inversion loops in the five inter

29

varietal crosses and reported that cells having loops did not exceed the 10 % of the cells

examined. Similarly, Shastry and Misra (1961a.b) able to pin point the deletion,

duplication, inversion and translocation in the pachytene chromosome in indica -japonica

hybrids. They reported that about 31% of the chromatin length was unpaired in three

semisterile hybrids. During meiotic division translocated chromosomes appeared as crossed

shape quadruple at pachytene stage (Schulz, 1985) or small interstitial gap (Shastry and

Misra, 1961a.b). Dolores et al. (1979) reported that normal chromosome pairing, in hybrids

of O. sativa/O. nivara, on the basis of complete chromosome pairing at pachytene stage

with low frequency of structural difference.

Misra and Shastry (1969) also studied the chromosome association in reciprocal

hybrids and showed that in one of the hybrid combination (O. glaberrima/O. sativa),

normal pairing was restricted only to pachytene stage and showed univalents and trivalents

at low frequency. Based on the results they reported that the both species are identical but

differentiated by few chromosome structural changes. On the other hand its reciprocal

combination showed normal pairing at all stage.

2.16.3. Diplotene, diakinesis, and Metaphase I

Based on meiotic behavior study, Yeh and Henderson (1961) reported that meiosis

in all hybrids, involving cultivated rice O. sativa and five diploid wild rice (O. sativa var.

fatuwa, O. sativa.var. formosana, O. balunga, O. perennis. subspp. cubensis and O.

perennis, was basically normal. They observed no more (0-11% and 0-4% irregular

divisions at diakinesis and metaphase I (MI), and anaphase I (AI), respectively) irregular

cells than observed in inter-varietal hybrids. Yao et al. (1958) reported higher frequency of

rod shaped bivalents in five of 7 inter-varietal hybrids than homozygous controls and found

8% of the cells contained univalent at MI. The mean chiasmata frequency per bivalent was

30

2.7 with range 1.8-2.9. In 1954, Sampath and Mohanty made large scale cross between

indica and japonica varieties in which sterility was found 25-99 %. Meiotic behavior in all

hybrids was normal except stretched chromosome, and bridges with fragments at AI.

Shastry and Misra (1961b) reported no reduction in chiasma frequency having 1-3

chiasmata/bivalent at diakinesis and MI in four indica and japonica hybrids. Similarly,

Shastry and Misra (1961b) also observed quadrivalents association in two of the hybrids

and suggested that quadrivalent association is an indisputable indication of translocation

heterozygosity. During these stages the shape of the structure depends on the frequency of

crossover, size of the interchange fragments and location of chiasmata and most frequently

appeared as 8, ring or rod shaped at diakinesis (Schulz, 1985).

Brar et al. (1996) reported that meiotic behavior in O. sativa cv. IR64/O. rufipogon

hybrids was normal. They observed 11.97 average bivalent per PMC at diakinesis and MI.

Similarly, Dolores et al. (1979) studied the chromosome pairing in the F1 hybrids between

O. sativa including series of landraces and improved varieties, and O. nivara, and

suggested that normal chromosome pairing with certain degree of abnormality. They

reported only few quadrivalents and univalents at diakinesis and Metaphase I in some of

the PMCs. Lu et al. (1998) also observed normal meiotic behavior, at Diakinesis and MI, in

hybrids involving four AA genome species from different geographical origin. The mean

bivalent observed by them was ranged from 11.51-12.

2.16.4. Chiasma frequency

During 1910, Kuwada first studied meiosis in rice and reported that, the paired

chromosomes were being ring or x shaped, later becoming square or dumbbell shaped

(Nayar, 1973). These observations later lay that they had either two or one

chiasmata/bivalent depending upon the shape of the chromosome. Soriano (1961) obtained

31

14-22 chiasmata/cell in MI of five indica varieties. Similarly, Jena and Misra (1984)

studied chiasmata frequency in connection with evolutionary trends at diplotene and

metaphase I of meiosis in eleven diploid species of Oryza and found that the evolutionary

trend in that series was towards self-pollination and annual growth habit. In the recent

studied, Lu et al. (1998) reported that both intra and interspecific hybrids between AA

genome species from New world, Asia, and Australia showed > 23 average

chiasmata/PMC except for one hybrid (O. glumaepatula/O. nivara) which had 22.26/PMC.

2.16.5. Anaphase bridges: cytological view

Two types of inversions have been reported to date, paracentric and pericentric

inversions. Among them paracentric inversions are more frequently occurred in plant

kingdom (Schulz, 1985). It has been suggested that the occurrence of anaphase bridges and

fragments during meiosis be brought about by inversion in species chromosome, mostly

paracentric inversion while pericentric inversions lead karyotypic differences, if single

chiasma occurs within inverted regions. However, formation of bridge and acentric

fragments don’t always indicate the occurrence of paracentric inversion as these can be

brought about by spontaneous breakage and fusion of chromosomes during meiosis (Haga,

1953). Rees and Thompson (1955) reported that for about 27 generation, inbred rye

showed bridges and fragments and concluded that such structural change can also be

brought about by chromosome breakage and sister chromatid reunion. A heterozygous

inversion may lead to bridges at anaphase I, however, if the chromosomes involved in the

bridge fail to separate completely in the first meiotic division, the bridge may persist until

the second division. Therefore, diagonal type bridge notice in second division may be the

remnants of a bridge at anaphase I (Ahmad et al., 1977). On the other hand direct bridges at

anaphase II could originate in two different ways: a) the aberrant bivalent may pass intact

32

to one pole in the first meiotic division, so that separation at anaphase II would give rise to

the bridge; or b) if one chiasma occurred in the inversion lop and another in the region

between the centromere and the inversion loop, then a monocentric loop chromatid appears

at anaphase I which will give rise to a direct type anaphase II bridge (Ahmad et al., 1977).

Likewise Schulz (1985) also suggested that formation of bridge at anaphase II can be due

to four strand double crossover combined with two strand single cross over in the region

between the centromere and the inversion loop, which results in bridges in each of the two

AII.

A bridge will appear during meiosis only if there was at least one crossover in the

inverted segment. Such bridges and fragments were commonly observed in different type

wide hybrids of crop plants. Henderson et al. (1959) reported that 0-0.8% cells having

anaphase bridges with small fragments in inter-varietal crosses of rice. Anaphase bridges

without fragments and fragments and laggards were also noticed in both parents and

hybrids involving O. sativa and O. rufipogon hybrids and their hybrid swarm (Majumder et

al., 1997). Similarly, Sampath and Mohanty (1954) found Anaphase bridges and fragments

in 11 of the 85 intra-specific hybrids, and suggested that these were indication of

inversions. Similar trend of results has been reported in inter-varietal rice hybrids by

Demeterio et al. (1965). Likewise, Dolores et al. (1979) reported 0.5-7.4 % percent PMCs

had bridges and fragments in F1 hybrids of O. sativa and O. nivara cross. Beside rice,

anaphase bridges have been frequently reported in interspecific hybrids of soybean

involving wild and cultivated forms (Palmer et al., 2000; Ahmad et al., 1977) and

suggested that wild and cultivated forms were differentiated mainly due to structural

differences caused by paracentric inversion.

33

2.17. Meiotic behavior in intergenomic hybrids

Several authors reported large amount of irregularities in the intergenomic hybrids

between O. sativa and other wild species having genomes other than AA, and showed that

limited or if at all no pairing based on late stage of meiotic analyses, particularly diakinesis

and MI stage (Mahapatra et al., 2002; Brar et al., 1996; Jena and Khush; 1986;

Ranganadhacharyulu and Yesoda Raj, 1974; Wuu et al., 1963). Jena and Khush (1986)

reported that absence of complete chromosome pairing in O. sativa and O. officinalis

hybrids. They observed only occasional bivalents with mean ranged from 0-4 and

frequently 20-24 univalents/cell. However, Shastry et al. (1961) based on pachytene

analysis reported that complete chromosome pairing in satival-officinalis hybrids followed

by desynapsis in later stage and also found completely 24 univalents at Metaphase I.

Based on the meiotic observation (MI), Brar et al. (1996) also reported the similar

results in O. sativa/O. brachynatha and O. sativa/O. granulata hybrids. They observed

only 0.06, and 0.29 bivalent/PMC, respectively and suggested that limited chromosome

pairing between these taxa. Likewise, limited chromosome pairing (0.05 bivalents/cell and

0.03 bivalents/cell) has been observed by Abbasi et al. (1998) and Wuu et al. (1963)

respectively in O. sativa and O. brachyantha hybrids. Similarly, Shastry and Rao (1961)

reported that timing imbalance existed between O. sativa and O. australiensis genomes.

2.18. Pachytene analysis in intergenomic hybrids

At pachytene stage in the F1 hybrid of the cross, O. sativa/O. officinalis, Shastry et

al. (1961) observed complete pairing between the chromosome complement of the O.

sativa and O. officinalis and they concluded that the A genome was homologous to the C

genome. However, Li et al. (1964) questioned the observation of Shastry et al. (1961) and

reported that O. sativa/O. officinalis hybrids had 24 univalents at pachytene stage as well.

34

However, Katayama (1965) observed normal pairing in the F1 hybrids of O. sativa/O.

officinalis at pachytene and speculated that A genome was partially homologous to the C

genome. Similarly he postulated the similar phenomenon in his series of paper (Katayama,

1966a.b.). He also observed that the chromosome behavior in later stages was very variable

and showed that 3-12 II and 1-11 I in interspecific hybrid with the ABC, and ADC genome.

Shastry (1966) again concluded that failure of pairing in intergenomic O. sativa/O.

officinalis hybrids was due to desynapsis. However, he pointed out that it needs to be

investigation whether this phenomenon common in all known stocks or varietal variation

exists in the manifestation of this character. On the other hand, at pachytene stage

Ranganadhacharyulu and Yesoda Raj (1974) observed irregular meiotic behavior and

subsequently in later stage in hybrids involving O. Punctata and O. eichingeri.

2.19. Asynapsis and/or Desynapsis

Plants having reduced amounts of chromosome pairing during meiosis have been

reported in number of crop plants such as, in wheat (Li et al., 1945), in rice (Ramanujam

and Parthasarathy (1935, cited by Nayar, 1973); Chao et al. (1960); Chao and Hu (1961),

Misra and Shastry (1969), and Wang et al. (1965). Asynaptic refers to the condition during

which pairing event falls apart particularly pachytene stage of meiosis. On the other hand

desynaptic refers to the condition during which pairing event is normal but in the later

stages chromosome fail to pair. Both conditions favor the production of non-functional

gametes and hence caused higher sterility.

Chao et al. (1960) obtained seven sterile plants in a M2 progeny of 34 plants after

the treatment of neutron irradiation. They set less than 1% seeds while their sib plants set

about 80% seeds. Their chromosome pairing was normal at pachytene, but they showed 10

I at diakinesis and 7 I at MI. Later, they concluded that the sterile plant arose as a mutation,

35

which was monogenetically controlled, for desynaptic behavior and its expression mainly

depended on the certain temperature regime (Chao and Hu, 1961;Wang et al., 1965).

Similar observation was found by Li et al. (1945) in desynaptic mutant wheat

obtained through the varietal cross, and reported that some modifier genes act upon, and

showed stable and unstable type. In wheat, Li et al. (1945) found that higher temperature

favors the bivalent formation and low temperature acts opposite. Similar, phenomenon was

made by (Misra and Shastry, 1969) in O. glaberrima/O. sativa hybrids, as well, and the

fertility of desynaptic and asynaptic mutant had significantly lower. But the occurrence of

asynaptic phenomenon is less frequent than desynaptic (Nayar, 1973). Similarly, Brar and

Khush (1997) reported that chromosomes of cultivated and wild species of rice showed

strong desynapsis at later stages of meiosis.

2.20. Autosyndetic and allosyndetic pairing

According to Stebbins (1950 in Li et al., 1961) autosyndesis refers to the pairing of

chromosomes derived from the same parental gametes of a particular plant, regardless of

the similarity or difference from each other, while alllosyndesis refers similarity to pairing

between chromosomes derived from different parental gametes. Li et al. (1961) explained

this phenomenon in interspecific hybrids between O. praguaiensis/O. australiensis and O.

australiensis/O. alata, and showed that frequency of alllosyndesis was predominant type of

chromosome pairing.

Similarly, Shastry and Rao (1961) observed 9.87% PMCs having autosyndetic

pairing within sativa chromosomes in O. sativa/O.australiensis hybrids. Based on the

chromosome size differences between O. australiensis and O. sativa Li (1964), and Li et

al. (1961) also observed both auto and alllosyndesis phenomenon. It has been frequently

36

reported that O. australiensis and O. officinalis have larger chromosome than others

(Morinaga, 1964; Bouharmont, 1962; Ghose et al., 1960). However, Kurata (1986) and

Kurata and Omura (1982) did not observed any significant karyotype differences between

O. sativa and O. officinalis. However, Abbasi et al. (1999) detect auto and allosyndetic

pairing among A and E genomes through, molecular cytogenetical techniques, genomic in

situ hybridization.

2.21. Nucleolus: number, type, and shape

Kuwada (1910) found one nucleolus in the majority of PMCs. Selim (1930)

reported two nucleoli in PMCs of indica varieties and one in japonica varieties. Since then

different authors found differences in number, shape and size of nucleoli during the course

of mitosis in several varieties of rice (Nayar, 1973). Misra and Shastry (1967) studied

nucleolus variation in five indica varieties, two javanica varieties and two japonica

varieties and found 1-2 big nucleolus and 2-5 smaller ones, even up to 18 some times and

stain similarly as the nucleolus. Latter these were identified as supernumerary nucleoli

(Misra and Shastry, 1967). They are either free or attached to pachytene chromosome.

However, most of them lay free in cytoplasm.

They compared them to the nuclear bodies observed by Walters (1963) in several

Bromus species. About a third of these were attached to chromosomes and the rest lay free.

They proposed that evolutionarily advanced species might show more of these bodies

consequent to increase competition in nucleoar activity in them as a result of chromosomal

structural changes undergone by them. Walters (1963) found usually only one nucleolar

body in a cell, and they were not present in somatic cells.

37

2.22. Recent progress in rice chromosome study

Rice karyotype analysis is the basis of cytogenetics (Wu and Chung, 1986).

Unfortunately, smallness of the rice chromosome, it was found very difficult in the past to

karyotype it. Shastry et al. (1960), and Shastry Misra (1961a.b.) initiated rice karyotyping

based on the pachytene analysis in rice. Since, then improved techniques of rice

chromosome preparation have been emerged (Hamoud et al., 1991; Faridi and Sitch, 1989;

Quy and Phai, 1985; Wu and Chung, 1986; Kurata and Omura, 1982; Kurata et al., 1981a;

Kurata et al., 1978; Khan, 1975; Hu, 1964; Sen, 1963). These technique have been

employed to study the karyotypic variation in Oryza sativa and chromosome pairing at

pachytene stage to analysis of interspecific hybrids (Dolores et al., 1979; Reddi and Reddi,

1977; Katayama, 1966a.b.; Ranganadhacharyulu and Raj, 1974) and to identified

translocation and trisomics (Kurata, 1986;Kurata et al., 1981b; Sato et al., 1980). Similarly,

these earlier developed techniques have been extended to construct the linkage maps in rice

(Iwata et al., 1984; Sato et al., 1980). In mid 1980’s, Fuki developed analytical method to

characterize the all chromosomes of rice by image analyzing system and further utilized in

developing somatic rice chromosome map (Fukui, 1996, Fukui and Ijijima, 1991; Fukui et

al., 1988; Fukui, 1986).

In the recent years, cytogenetic techniques in rice have been highly developed and

still growing up. Now techniques to visualize the genes and nucleotide sequence within

chromosomes at different stages have been reported (Fukui and Ohimido, 2000, de Jong et

al., 1999; Singh et al., 1996; Gustafson and Dille, 1992). However, the principles of these

techniques have already been exploited in rice such as GISH, FISH and ISH to characterize

the parental genomes in interspecific hybrids including different taxa with O. sativa (Fukui

and Ohimido, 2000; Asghar et al., 1998; Abbasi et al., 1999, 1998; Fukui et al., 1994).

38

3 MATERIAL AND METHODS

The whole experiment was laboratory based and therefore, all the study was

conducted at green house and at Central laboratory of Institute of Agriculture and Animal

Science (IAAS), Rampur, during the academic year of 2001-2002. IAAS is located at 840

29’ E and 270 37' N and, 244 m above sea level.

3.1.Preparation of parental materials

3.1.1. Germplasm collection

Male parents used in the hybridization consisted of four wild species, namely O.

rufipogon, O. nivara, O. granulata, and O. officinalis. Similarly, female parents consisted

of six local land races belonging to O. sativa viz. Kalanamak, Jethobudo, Pokhreli,

Manshara, Jhinuwa, and Ghaiya, two improved IRRI varieties viz. IR 64 and IR 72 and one

commercial varieties; Masuli. Three of the four wild species of rice and five landraces

were collected from different parts of Nepal. Seeds of one wild species, O. officinalis, was

kindly provided by Dr. M.P. Upadhaya, (Botany division, NARC, Khumaltar, Kathmandu).

The species name, site of collection, type of collection, and collection date were

summarized in Table 3.1.

3.1.2. Germination and greenhouse rearing

All the collected live male parent plants were planted in plastic buckets (buckets

were provided by Prof. Dr. R.C. Sharma) filled with soil sterilized by formaldehyde. Each

species was planted in ten buckets. On the other hand, collected seeds of O. officinalis and

O. nivara were germinated by keeping dehulled treated seeds in incubator at 330 C for 7-10

days. Dehulled seeds were kept in sodium hypochlorite @ 1% (v/v) for 15 minutes

39

Table 3.1. Description of germplasm collection during study period of 2001

Sn. Name of species Site of collection Type of collection Season

a ,* improved varieties and Landraces of Nepal, respectively

1

2

3

4

5

a

c

d

O. rufipogon

O. nivara

O. granulata

O. officinalis

O. sativa. cv.

IR 64 a

and IR 72 a Masuli

a

Ghaiya*

Pokhreli * Jethobudo*

Manshara* and Jhinuwa*

Kalanamak*

Ajighara swampy area of Rupendehi district (Plain) and Bulbule

park of Surkhet (Canal) Valley

Canal, road side ditches , swampy area, and farmers field

(Nepalgunj, Banke district), Latikoili VDC (2) of Surkhet district

(Valley) and Khumber forest ponds (Bardiya district)

River bank forest (Chure hill)of Piple-6, Chitwan district (Valley)

NARC, Botany division, Khumaltar, Kathmandu

IAAS, Rampur campus, Chitwan Nepal

Batule Chaour, Pokhara Municipality 16, Kaski (Valley)

Lamjung (Hill)

Siddhartha Adarsha VDC, Rupendehi (Plain)

Plants

Plants and Seeds

Plants and rhizome

Seeds

Seeds

Seeds

Seeds

seeds

August 24-25

August 22-24

May 19-21

April 15-18

May 24-25

May 24-25

May 26-27

May 26-27

40

Table 3.2. Crossing scheme employed during the study period 2001-2002

Female parent

O. sativa cv.

Manshara Jhinuwa Jethobudo Pokhreli Kalanamak Masuli Ghaiya IR 64 IR 72

Male parent

Wild species

Oryza nivara x x x x x - - x x

O. rufipogon x x x x x - - x x

O. officinalis x x x x x - - x x

O. granulata x x x x x x x x x

–crossing did not make

41

following the procedure of Vaughan (1994). Then germinated seeds were planted into ten

buckets filled with same material. Staggered planting of all female parents (including six

landraces, commercial variety Masuli and two IRRIs’ varieties) was made continuously for

two months at four days of interval, to synchronize the flowering time of parents. Each

female seed was planted in two replication; one bucket/replication, and two seeds/bucket at

a time. Usual agronomical practices were adopted as needed. All these mentioned activities

were done in normal rice growing season of 2001 at IAAS green house.

3.2. Hybridization

3.2.1. Cross combination

Each female parent was crossed with all four wild species except Ghaiya and

Masuli, which were crossed only with O. granulata in the second season of 2002. In

general twenty-eight hybrid combinations were expected at the end of the crossing.

However, due to non- synchronization of flowering, crossing of Manshara and IR 72 with

O. rufipogon was not carried out. The one way cross schemes employed during the

crossing were shown in the Table 3.2.

3.2.2. Forced anthesis

Out of four wild species, two species O. nivara and O. rufipogon, often bloomed

after 1.30 p.m. but the entire female parents bloomed after 9.30 a.m. and completely ceased

by 1.30 p.m. at Rampur environment during October-November 2001. Therefore, forced

anthesis was done by keeping these two male parents in well-illuminated and heated room

(36-380C) by fluorescent light and electric heater for an hour. Water was also sprayed to

facilitate the blooming and maintain constant temperature by hand sprayer.

42

3.2.3. Emasculation

Hand emasculation of female parents to be crossed was done without clipping the

flag leaf prior to 1-2 days of crossing continuously for one and half months. The number of

emasculated spikelets in each panicle ranged from 10–93 depending upon the availability

of pollen source. The emasculated panicles were bagged by Glassine bag and pollination

was done in the next day.

3.2.4. Artificial pollination

Both approaches and hand pollination methods were adopted for pollination.

During hand pollination, fresh anthers ready to dehisce were collected and immediately

crushed in the petridish and then pollens were shaded on the stigma with the aid of brush

repeatedly, without disturbing stigmatic surfaces particularly, for low amount of pollen

sources, for 2-3 days. In approach method, emasculated spikelets of female and male

panicles were bagged together within a single glassine bag, and occasional shaking was

provided for 2-3 days mostly at 9 -11 o’clock. On the other hand, pollinated spikelets, of

distant crosses involving O. officinalis and O. granulata, were sprayed by GA3 and NAA

@ 75 ppm (1:1) once a day regularly up to five days of pollination.

3.3. Embryo rescue

At the beginning of the experiment, embryo rescue was partly practiced for all

hybrid caryopsis. However, it was later found that hybrid between intragenomic (AA type)

cross, (crosses of O. sativa with O. rufipogon and O. nivara) set good amount of mature

seeds without noticing abortion of embryo. Due to inviability of zygotic embryo, later

rescue work was extensively practiced for intergenomic cross hybrids only. When hybrid

43

embryos came to age of 7-10 days, they were cultured in sterile nutrient medium. During

embryo rescue work, following sequential activities were followed:

3.3.1 Aseptic excision of immature hybrid embryos

Excision of the embryo was carried out by adopting the procedure suggested by Ko

et al. (1983) and Bouharmont (1991). The method consisted of removing the envelope

(husk) from the cut made at the top of the spikelets at the time of emasculation with the aid

sterile forceps. Then immature ovaries were excised without disturbing their structural

integrity. Excised ovaries were then surface sterilized in freshly prepared solution of

sodium hypochlorite (1%). The soft green ovaries were shaken up in this solution for 15

minutes. Then isolated ovaries were thoroughly washed thrice in sterile water and the lower

part of the ovary was cut and pressed out with the help of sterile forceps without touching

by hand. The excised embryos were washed thrice with sterile distilled water to free them

from chlorine. After that, embryos were soaked in the same sterile water for two to five

hours in an aseptic inoculation chamber i.e. under Laminar Bench.

3.3.2. Aseptic preparation of mature hybrid embryo

Mature seeds of hybrid obtained from crosses involving O. rufipogon and O.

nivara, as male parents with other female parents were also cultured. Similarly, the mature

embryos of the wild species were also cultured in the nutrient medium. Such mature

caryopsis were first surface sterilized after removing the husk in freshly prepared solution

of sodium hypochlorite (1%) for 35 minutes. Then either a part or the whole endosperm

with embryo was inoculated, after peeling off a portion of the pericarp opposite to the

embryo.

44

3.3.3. Inoculation of embryo

After providing the proper time for imbibition of excised embryos, embryos were

then placed on the sterilized filter paper for 30-45 seconds to remove the excess surface

water from embryos. Then isolated embryos were aseptically inoculated into the tube

containing respective culture medium by the help of a needle with a loop.

3.3.4. Media preparation

At the beginning of the experiment all the media were prepared without making

stock solution. However, later, Bouharmont, (1991) and ¼ th

MS media (Murashige and

Skoog, 1962) were prepared by making stock solution. Each ingredient was first divided

into four groups and stock solution of them was prepared following the procedure

mentioned by Razdan (2001). Stock solution, consisting of major salts (20X concentrated),

minor salts (200X concentrated), organic nutrients (200X), and iron (200X concentrated),

were made by weighing the respective nutrient composition as given in the protocol

(appendix.3.1). Each stock solution was then stored in refrigerator at 50 C until used.

3.3.5. Media employed

In the beginning of this study, five different Media namely Nistch’s (1969), White’s

(1953), ¼ MS (Murashige and Skoog, 1962), SR (Ko et al., 1980) and Bouharmont (1991)

were employed to determine the media efficiency for hybrid embryo regeneration at IAAS

tissue culture laboratory environment. Each B, W, N, ¼ MS and SR medium was

supplemented with 10% coconut milk. All the media were gelled by 0.7% Agar Agar, after

adjusting the appropriate pH (appendix 3.1). Finally prepared medium was then boiled in

the micro-oven and dispensed into culture tubes, 20-25 ml. in each, stoppered with cap or

45

closed by aluminum foil and finally tubes were autoclaved at 15 psi. for 20 minutes. Then

media were kept in laminar hood for two hours and allowed for setting.

3.3.6. Media efficiency determination

Media efficiency was determined by repeating each experiment twice at IAAS,

tissue culture laboratory. The preliminary results were recorded (appendix 4.2) and best

media was choosen for further embryo rescue work. After findings the best media for

complex hybrid embryo germination and subsequent development, the final embryo rescue

study was conducted mostly on Bouharmont (1991) and partly on ¼ Murashige and Skoog

(1962) media supplemented with 10% coconut milk as an organic supplement.

3.3.7. Incubation of culture

The cultures were maintained in a temperature controlled chamber at 2510C under

dark until germination and then continuous light (~110-foot candles) up to 2-4 weeks.

Regenerated seedlings were removed out from the culture tubes after three-leaf stage. The

roots of the seedlings were thoroughly washed to remove agar in the tap water and finally

washed with sterilized distilled water.

3.3.8. Seedlings transfer

Hardening of seedlings was done following the four methods: 1) direct transfer, 2)

petridish method, 3) Iyer and Govill (1964) method, and 4) new method to enhance rooting

and to make plants hardened in external environment. First one is the direct transfer of

seedling obtained after embryo rescue. Second approach was hardening of seedlings in the

moist filter paper over the petridish as suggested by Bouharmont (1961). Third method

46

consisted of hardening in the nutrient solution at temperature controlled chamber as

reported by Iyer and Govilla (1964).

The fourth and last method was the latest one that was developed during

experimentation of this study. The newly developed hardening medium and procedures

consist of sterilized 2:1 sand and soil (300 gm. in each 13 x 10cm2

plastic bag) floated with

a nutrient solution (Karim and Vlamis, 1962) up to 1.5 cm above the mixture surface in the

bags. In which the molybdenum source (H2MoO4) was modified as sodium molybdate

(Na2Mo4.2H2O). Stock solution of each of the chemical was made and finally the complete

nutrient solution was prepared (appendix 3.2)

The whole small plastic bags with fragile seedlings were kept in plastic bucket, then

covered by large white plastic bag with the aid of stakes to maintain humidity. The covered

plastic bags were removed every day for 1-2 hours after 6-7 days of hardening up to three

days and finally whole plastic bags were removed and seedlings left for two more days in

the same medium. This hardening activity was undertaken in a well illuminated room and

even on open field. Then finally hardened seedlings were planted to plastic buckets filled

with well fertilized sterilized soil, and grown in the glass house of IAAS during winter

season in 2001/2002.

3.4. Regenerating plants from callus of hybrid embryos

In most distant crosses, hybrid embryos (7-14 days old) are manipulated to produce

F1 plants directly. In some distant crosses, however, the hybrid embryos fail to differentiate

in to plants and embryo abortion started earlier. To over come this barrier, and to maximize

the chances of getting hybrids plants, the undifferentiated embryos were induced to

proliferate as callus on a culture medium at an early stage and hybrid plant were

regenerated from the callus following the technique of (Nowick, 1986). Immature embryo

47

to be cultured were aseptically excised and placed on the hybrid medium as proposed by

Chen et al. (1991). The hybrid medium consisted of Murashige and Skoog (1962) organic

salts and N6 mineral salts and supplied with 6% sucrose, 4mg/l NAA, and 2mg/l kinetin.

Culture was maintained with 12 h light –12 h dark at 25±10C. After 35-40 days of

inoculation seedlings were removed following the same procedure as practiced for the

embryo culture. Other further activities were also similar to the method as method

described in embryo culture.

3.5. In vitro fertilization

In vitro fertilization was also carried out for O. granulata and O. sativa cross

combination following the procedure of Zhang (1985). The spikelet of the female parents

O. sativa cv. Jhinuwa was emasculated by the hot water treatment method (450) for five

minutes, and the spikelets ready to bloom were sterilized by wiping with a piece of ethanol-

moistened gauge several times, soaked in 70 % ethanol for 5-10 seconds, and were washed

with sterile water three times. A spikelet with a piece of peduncle was then placed upright

on solid medium of type N6 containing 4% sucrose and pH 5.8.

The spikelets of male parent O. granulata was surface sterilized by presoaking in

70% ethanol for 7 seconds, and finally soaking in 1% sodium hypochlorite for five minutes

and thorough washing was provided. After this treatment, anthers were drawn out of the

spikelet with a set of sterilized forceps and each was inserted in the maternal spikelet in a

test tube. Cultures were maintained under darkness at 250 for 15days and then transferred

to the Bouharmont (1991) media for regeneration following the embryo rescue technique.

3.6. Harvesting of F1 seeds

Mature and perfectly set hybrid seed was harvested separately at 24 - 28 days after

48

fertilization. Harvested seeds were immediately kept in dessicator at 300 for 3 days after

proper tagging and labeling. Then these seeds were again germinated and sown as

mentioned previously (3.1.2).

3.7. Determination crossability

3.7.1. Crossability between O. sativa and common wild rice

Crossability percent of common wild species (O. nivara, and O. rufipogon) with O.

sativa was determined by counting the total number of true F1 hybrids in proportion to the

total number of florets pollinated during pollination (Wuu et al., 1963). However, in this

study, all F1 plants were not reared at first due to lack of space and hence only 5 F1 seeds

were planted for cytological analysis and characterization purpose. All F1 hybrids exhibited

the purple basal leaf sheath color at an early stage of seedling. Since then, the

morphological marker was used to identify the F1 hybrids for those crossed combinations

involving O. sativa/O. nivara and O. sativa/O. rufipogon. On the next season, all the seeds

harvested from these cross combinations were germinated and reared up to 20 days in the

laboratory condition and looked for the presence of basal leaf sheath color as purple.

It is because of all the cultivars belonging to O. sativa used here had green basal

leaf sheath color. Therefore, hybrids were ascertained by observing the presence and

absence of purple basal leaf sheath color at an early stage of seedlings. In this connection

earlier finding was used to identify the hybrids, some of them again reared up to maturity

and finally confirmed that all were hybrids. Non germinated seeds, and albino plants which

were later died, were not considered in the calculation of crossability percent.

3.7.2 Crossability determination in intergenomic crosses

The crossability between intergenomic species were also determined, based on the

49

hybrid plant establishment in the field condition after embryo rescue, as method reported

by (Li, 1964).

3.8. Morphological characterization

For the purpose of morphological characterization, each different female parent and

their hybrid combinations were planted to five plastic buckets and wild species were

planted in ten plastic buckets during winter season of year 2001/2002. A number of

different observations regarding the morphology of included study materials were done

from early vegetative stage to until harvest. About > 30 observable characters were

characterized during study period. Each observation was taken from random sample of size

five i.e. one sample from each bucket. The observation of each character was taken based

on the standard characterization procedure (IBPGRI- IRRI, 1980; Sharma and Shastry,

1965).

3.9. Chromosome preparation

Chromosome, of the 15 F1 hybrids, 7 cultivars and four wild species of rice, was

prepared from immature anthers following the usual squashing technique as mentioned by

(Wu and Chung, 1986; Khan, 1975). In some of the study enzymatic maceration was also

employed as described by Kurata et al. (1982). Most of the observations were made from

temporary slide.

3.9.1. Chemical preparation

3.9.1.1. Stain

Most of the chromosomes were stained by 1% acetocarmine solution and this

solution was prepared by following the procedure of Evans and Reed (1981). Their

50

procedure involves: One gram of acetocarmine powder weighted in electronic balance was

dissolved in 100 ml glacial acetic acid solution having strength 45%. Before dissolving,

45% solution of glacial acetic acid was heated up to boil. For dissolving the powder

continuous heating and stirring was provided for 5-10 minutes under hot Plate with

magnetic stirrer. Prepared solution was allowed to cool and finally one drop of 45% GAA

and trace amount of ferric chloride was supplied as mordant That prepared solution was

then stored at 50C in refrigerator until used.

3.9.1.2. Fixative

During study, two kinds of fixative were used: 1. Caryony’s fluid, and 2. Acetic-

alcohol solution. Caryony’s fixative was prepared by mixing Glacial acetic acid,

Chloroform, and 95% Ethanol in the proportion of 1:3:6, respectively. Then finally

prepared solution was provided with trace amount of ferric chloride. On the other hand,

Acetic-alcohol fixative was prepared following the procedure of Khan (1975). This was

prepared by mixing one part of Glacial acetic acid with 3 parts of 95% ethanol. This

prepared solution was also supplied with trace amount of ferric chloride to ensure the better

stain during staining. Every time these fixatives were freshly prepared, just before fixing

the materials.

3.9.2. Standardization of suitable stages

The suitable stage for studying the meiotic behavior were first standardized by

harvesting the young panicles from early booting, varying in length below the junctura of

the flag leaf (Khan, 1975). These harvested young panicles were fixed for 24 hours at 140-

250C in freshly prepared fixative and then tested under microscope by staining freshly

prepared 1% acetocaramine solution. Since then different stages of meiotic behavior were

51

studied by harvesting the young panicles (the distance between boot and junctura of the

flag leaf was 0-6.5 cm depending upon the species and their F1 derivatives) based on the

results of standardization of suitable stage for meiotic study.

3.9.3. Meiotic behavior study

Young spikelets at suitable stage were fixed in acetic alcohol (1:3 v/v) to which

traces of ferric chloride was added to intensify the staining of the chromosomes. The

materials were kept in the fixative for 24 hours at low temperature (140-25

0C) and then

transferred to 70 % ethanol until used for smearing. Fixed anthers were removed from the

spikelets and smeared in one or two drops of 1 % acetocarmine. Slight warming and gentle

tapping were provided to promote the excellent spreading and differentiation of the

chromosomes.

Data were recorded based on the analyses of 10 randomly selected immature

spikelets of parents, including five F1 plants of each cross for each stages of studied. Data

concerning the meiotic behavior included only from those PMC’s that yield well spread

and distinct configuration after screening a large number of dividing cells. Microscopic

photographs, and drawings, if necessary, were made from temporary slides with the aid of

microscopic camera and Camera Lucida at table level with the mirror at 450 inclination and

using 100X oil immersion objective and 10X eye piece of Olympus Microscope,

respectively.

3.9.3.1. Chromosomes analysis

Meiotic chromosomes associations were mostly studied at, diakinesis, metaphase,

anaphase, and telophase I. Similarly, meiotic associations at pachytene stage were also

52

studied in a selected number of the cells. Diplotene stage was also occasionally analyzed in

some cells of the few hybrids.

3.9.3.2. Pachytene analysis

Detailed karyotypic analysis (individual chromosome length, arm ratio etc.) of

pachytene stage chromosomes was not carried out due to stickiness and poor resolution of

the staining. The pattern of pairing in this stage was determined as normal and abnormal

only from those cells having well spread and distinct pairing pattern. Percent normal cells

and cells showing cryptic structural hybridity (Shastry and Misra, 1961a.b) were calculated

based on the microscopic observation. Microscopic photograph and necessary drawings

were made in limited PMCs after screening the large number.

3.9.3.3. Chiasma frequency determination

Chiasma frequency in the parents including wild rice and hybrids was determined

based on the meiotic association at the diakinesis and metaphase I following the procedure

of Lu et al. (1998). During diakinesis and metaphase I chromosome were highly condensed

and easily recognized as rod and ring shaped and proposed 1 and 2 chiasmata/bivalent,

respectively. Similarly, two (Bothmer et al., 1988) and four chiasmata/bivalent were

considered for each trivalent and quadrivalent association, respectively.

The chiasma frequency/PMC and per bivalent were determined only from those

cells that showed normal complete set of chromosome. Observed cells under microscope

were numbered randomly as first observed cell numbered 1, second observed 2 and so on.

Each cell was characterized by observing meiotic association at particular stages. Observed

bivalents were further characterized as rod or ring.

53

3.9.3.4. Detection of univalents, bivalents, trivalents, and quadrivalents

No rigid techniques was employed to detect the chromosome association at

diakinesis and metaphase I. However, chromosome, simple rule as described in every

photographs and drawings in the rice cytogenetics paper were thoroughly concerned and

based on these observed photographs, all most all the univalents, bivalents, trivalents and

quadrivalents were detected during microscopic observation. When chromosome number

exceeds 12 and some of the chromosomes found comparatively smaller in size than

remaining within the same PMC, were considered as univalents.

Similarly, bivalents were uniform and comparatively larger than univalents and

showed 12 in numbers. Bivalents were mostly either ring or rod shaped but univalents were

almost ring shaped and small. On the other hand trivalents were found to be larger than

bivalents and overall counts should meet 24 univalents (for example 9II + 2III). Similarly,

quadrivalents were large enough to detect and doubled in size of bivalents.

3.9.4. Pollen fertility and sterility

Pollen fertility and sterility of included materials were determined following the

method described by Virmani et al. (1997). The stain used in this method was I-KI, which

was prepared by dissolving one gm each of potassium iodide and iodine in 100 ml of

deionized water. Randomly selected 15-20 spikelets were taken from the just emerged

panicles of selected tillers of five plants in a vial containing 70% ethanol. All the anthers

from at least 6 spikelets were taken and placed on the fresh slide and anthers were then

crushed with 1% iodine potassium iodide (I-KI) stain. After removing the debris, a cover

slip was placed and observed under the light microscope (10X). The numbers of pollens

were counted in four random microscopic fields and observed pollen grains were classified

54

based on their shape, size, and extent of staining (Young et al., 1983; Chaudhary et al.,

1981) as follows:

Categories of pollen and their features

Categories of pollen Shape and staining behavior Classification

Unstained withered sterile (UWS) Withered and undeveloped Sterile

unstained

Unstained spherical sterile (USS) Spherical and smaller Sterile

unstained

Stained round sterile (SRS) Round and small, lightly Sterile

or incompletely stained,

rough surface

Stained round fertile (SRF) Round and Large, darkly stained, Fertile

smooth surface

Based on the classification, mean percent pollen fertility and sterility were

calculated. Then plants were classified on the basis of pollen fertility as fertile (61-100%

pollen stained), partially fertile (31-60%), partially sterile (1-30%) and sterile (<1%)

(Virmani et al., 1997; Subedi, 1982).

3.9.5. Spikelet sterility

Spikelet sterility in the respective parents and their hybrids were determined by

selecting and counting the filled and unfilled grains in the main tillers of selected five

plants of each. The spikelets of the selected plants of all the parents and their F1 hybrids

55

were bagged before flowering up to maturity. At maturity, the bagged panicles were

examined for seed set. The mean percent spikelet sterility was determined by counting the

total number of seed set in proportion to the total number of spikelets/plant. Then the

percentage sterility from each panicle was added and divided by five to calculate the mean

sterility percent. Then parent and hybrids were classified as fertile (81-100% seed set),

partially fertile (31-80%), partially sterile (1-30%) and sterile (<1%) (Virmani et al., 1997;

Ikehashi and Araki, 1984).

56

4 RESULT AND DISCUSSION

4.1. Description of Nepalese wild species of rice

Out of four species, three species, namely O. rufipogon, O. officinalis, and O.

granulata had perennial growth type and profuse tillering (Table 4.1). All the species

exhibit specific morphological features depending on the species, however, most of them

had spreading growth habit, high stigma exertion rate, high spikelet shattering, photoperiod

sensitive to insensitive, fully awned to awnless; dark green leaves with basal leaf sheath

coloration. The flowering period of these wild species was quite longer than cultivated

species and two species, O. granulata and O. officinalis, mostly bloomed at early in the

morning, and ceased by 8-9 o’clock. Similarly, two common wild species, O. nivara and O.

rufipogon bloomed at noon and often later than that cultivated forms.

The plant height was varied depending upon the species and was shortest in O.

granulata and the tallest in O. rufipogon. O. granulata and O. officinalis did better under

partial shade, where as O. nivara and O. rufipogon showed well in open sun shine. O.

granulata, in its natural state, was mostly found in upland field under the shade of dense

vegetation. Other diagnostic features of these wild species are presented in Table 4.1. and

illustrated in photograph (Plate 2 and Plate 3.). Similar observation were made by earlier

researchers (Jackson et al., 2000; Vaughan, 1994; Sharma, 1986; Sharma and Sampath,

1985; Shrestha and Vaughan, 1989, Sharma and Shastry, 1965, Gopalkrishinan, 1962).

In some of the species, marked ecotype specific morphological variation was

observed for example, in O. rufipogon, collection from Surkhet had open panicle with

creeping nature of culm, but from Kapilbastu, it had compact panicle with procumbent type

of culm. Vaughan (1994) and Sharma and Shastry (1965) reported similar observation with

minor geographic differentiation within this taxa.

57

Table 4.1.Morphological characters, of four Nepalese wild species of rice, observed during study

period 2001/002

Characters

O. nivara O. rufipogon O. officinalis O. granulata

Apiculus color black black brownish grey brownish white 200 grain wt 4.9 gm 3.89 gm 1.35gm 2.63 gm Anther color yellow deep yellow Pale-white white Auricle color pale green pale green light green light green Awn color brown brown brown -

Awn density long & fully long & fully short & partly Awnless Awn length 5.21cm 5.3cm 0.82cm - BLSC purple lines purple lines brownish green brownish white Blade pubescence intermediate pubescent pubescent pubescent Branching of culm present(2-3) present(2-3) present (3-4) present (2-3) Culm angle intermediate procumbent intermediate open <600c culm diameter 0.34cm 0.3cm 0.33cm 0.21cm

Culm length 46.1cm 73.25cm 89.11cm 44.55cm Culm number >25 >40 >35 >45 Flag leaf angle descending horizontal horizontal horizontal Habit annual perennial perennial perennial Heading days 80 135 109 50-70 (continuous) Height 57cm 92.3cm 107.14cm 54.85 Internode length 19cm 16.0cm 30.0cm 13.0cm

Internode color purple lines purple lines DBPL dark green Leaf color DGLPT DGLPT dark green dark green Leaf angle dropping dropping dropping horizontal Leaf length, 50.5cm 37.2cm 32.5cm 17.67 Leaf width 1.23cm 1.2cm 1.47cm 1.72cm Ligule color white papery white papery white whitish green Ligule length 1.15cm 2.15cm 0.35cm - Ligule shape 2-cleft 2-cleft truncated small truncated

MNBLNP 2 1 3 1 or (sessile) Panicle exertion just exerted well exerted well exerted well exerted Panicle length 15.3cm 19.04cm 27.16cm 10.3cm Panicle shattering high high high high Panicle type open compact - open open compact Pedicel length 3.4mm 3mm 3.8mm minute Rachilla minute prominent minute minute

SB heavy absent heavy absent Seed coat color black black brownish grey brownish white SLC brown brown brownish green blackish white Stigma color dark purple dark purple DPWT white and large TNPB 6 7 8 absent Underground stem Photoperiod reaction

absent insensitive

absent sensitive

present sensitive

present insensitive

TNPB = total number of panicle branches, DPWT = dark purple with white tip

DGLPT = dark green with light purple tip, SB = secondary branching, TNPB = total

number of panicle branches, SLC = sterile lemma color, MNBLNP = maximum number of

branches at the lowest node of panicle

58

4.2. In vitro manipulation

4.2.1. Media efficiency

In the preliminary study, hybrid as well as two selected parental embryos subjected

to different cultural media showed quite variation in germination and other observations

depending on the composition of media employed (Appendix 3.1). Although, the parental

embryos germinated within two days irrespective of the media used, significant variation

was observed in intergenomic hybrids depending on the media composition (5-13 days).

The effect of media composition was apparently seen in the case of intergenomic hybrid

embryos, and some of the seedlings died even after germination (Appendix 4.1). The

frequency of dead seedlings after germination was highest in Whites, Nistch and SR

Medium.

The observed variation in the germination and subsequent growth in the media

employed, suggest that complex hybrid embryo needs complete media as the normal

endosperm supplied (Singh et al., 1990; de Guzman, 1983; Hadley and Openshaw, 1980;

Murashige, 1979). Therefore, it was found that successful culture of embryo needs

complete medium and success will be depend on the choice of media (Brar and Khush,

1994; Yeung and Thorpe, 1981). In other words, the more complex the hybrid, complex

media will be needed to foster the better germination and vigorous growth (de Guzman,

1983; Yiew and Law 1983). However, the cultural conditions may also affect the

germination, and subsequent growth and development of the seedlings (Amemiya,

19656a.b; Ko et al., 1983), and it may also be varied from laboratory to laboratory and

expertise in handling of the materials (Razdan, 2001).

59

4.2.2. Embryo rescue.

Embryo rescue was extensively practiced for crosses involving O. officinalis and O.

granulata. Embryo degeneration after fertilization was varied with remoteness of the wild

species used. It was quite earlier in O. sativa/O. granulata (5-10 days) than O. sativa/O.

officinalis hybrid (9-17days) depending on the female genotype used. The germination of

hybrid embryo was varied from 0-66.67% (Plate 1 Fig. e-h.). In some of the combinations

the germination of hybrid embryos obtained from O. sativa/O. granulata, was also found

good, however most of them were died after germination. Therefore, hybrid between O.

sativa and O. granulata were not obtained. Most of the caryopsis were without embryo or

only filled with watery endosperm (Table 4.1a) however, very few embryos were obtained

in the year 2002. They were cultured, but most of the embryos did not respond. About 127

embryos were cultured and only 34 hybrids were germinated, however, all were died after

germination. Only one hybrid seedling was obtained, but that was also died during

hardening process indicating that there should be strong pre and post crossability barriers,

or mostly handicapped by post germination barrier.

Similar prefertilization barrier was found by Sitch et al. (1989a.b), Sitch (1990) in

the genus Oryza and reported in other taxa like O. brachyantha, O. minuta. O. ridleyi,

when crossed with O. sativa, they all showed strong prefertilization barriers. On the other

hand hybrid involving O. sativa and O. officinalis gave 16 hybrids plants.however, field

establishment of regenerated seedlings was found very difficult. At initial stage 150

interspecific hybrids died due to lack of appropriate hardening technique. However, later

proposed method as described in materials and methods significantly improved the field

establishment and gave nearly 100% success (Plate 1. fig. j-l) (appendix 4.2).

Similar results were presented by several researchers (Brar et al., 1991, Sitch et al.,

1989c; Jena and Khush, 1986, 1984; Iyer and Govilla, 1964) regarding the hybrid caryopsis

60

1

2

3

Plate 2. Figure 1–3. Morphology of parents and their hybrids. 1. Female parent (Manshara), F1 (Manshara/O.

officinalis), male parent (O. officinalis), from left to right respectively. 2. Jethobudo, F1, and O. rufipogon,

respectively 3. Pokhreli, F1, and O. nivara, respectively.

61

1 2

5 4

7 8

6

9

3

Plate 3. Figure 1– 8. Morphology of interspecific hybrids. 1. IR 64/O. nivara., 2. Pokhreli/O. rufipogon.,

3. Jhinuwa/O officinalis,, 4. Kalanamak/O. nivara., 5.Pokhreli/O.officinalis., 6. Kalanamak/O. officinalis.

7. Jethobuo/O. nivara., 8. IR 72/O. nivara, respectively., 9. Wild species O. granulata.

62

formation and their in vitro germination, however, except Iyer and Govilla (1964) others

did not mention any details about the hardening process. Jena and Khush (1986) obtained

variation in germination and their range were 56.57-70.05% in 1/4th

MS medium.

The successful hybrids obtained in O. sativa and O. officinalis suggests that there

might be less crossability barrier than that found in O. sativa/O. granulata. They mostly

showed post fertilization barrier and can be overcome by embryo culture. Several

investigators have successfully been overcome such barriers by adopting embryo culture in

different species hybrids (Philips1992; Sitch et al., 1989c; Williams, et al., 1987; Jena and

Khush 1986, 1984; de Guzman, 1983; Keim, 1953). However, Brar et al. (1991) obtain 3

hybrid plants from O. sativa/O. granulata, in one combination out of three following this

usual technique. This suggested that degree of incompatibility is varying with genotypes

used in the crossing program. During study, O. granulata was crossed with nine genotypes

(Table 3.2) up to two seasons and several hormonal manipulations were also made.

However, most died after germination at single leaf stage. Therefore, after several attempts,

it is concluded that there should be strong crossability barrier between cultivars. of O.

sativa studied and O. granulata. There is no proof towards such slight discrepancy

obtained in this study and that results found by earlier investigators, except to say

differences in strains used in crossing. This over all result suggest that production of hybrid

plants from O. sativa/O. granulata cross is very difficult (Khush, 2000; Ghose et al. 1960).

4.2.3. Hybrid embryo derived callus culture

Out of 35 hybrid embryos (Pokhreli/O. officinalis) cultured, 15 embryos

immediately differentiated into callus. Most of the callus was whitish and friable (Plate 1.

fig.a-b). Responsive embryos induced callus within 8-15 days of culture. However, 5 test

tubes containing callus did not differentiate into plantlets. Only 5 tube gave plantlets and

63

remaining 5 test tube were heavily contaminated with fungal and bacterial origin. Within

25-30 days of culture, these went to rhizogenesis at first and then caulogenesis (Plate 1. fig.

c and d). Bajaj and Bidani (1980), and Nowick (1986) obtained similar results in rice.

Nowick (1986) was able to isolate hybrid plants from O. sativa/O. latifolia and grown up to

maturity. However, he did not report detail result in his paper about the success rate.

Slight discrepancy in the result obtained might be due to cultural condition,

laboratory environment and genotypic factors, as they are crucial to regenerate plants

(Misso et al., 1989; Ko et al., 1983). Bajaj and Bidani (1980) reported that most of the

callus went rhizogenesis after 5-15 days, however, in this experiment most of the callus

formed shoot, but rhizogenesis was slightly inhibited. These slight discrepancies in the

mode of regeneration might be attributed to hormonal difference in medium and genotypic

differences. Other than genotypic difference, age of embryo also plays crucial role from

formation of callus to whole plant regeneration. Similar type of research in rice was also

conducted by Bouharmont and Dekeyser (1985) in African rice and reported that only

embryo derive calli gave successful plant regeneration Thus it was found potential tool to

regenerate hybrid plants when embryo abortion started quite earlier.

4.2.4. In vitro fertilization

Out of 65 spikelets pollinated only 20 spikelets showed swelling of ovules after 5

days of pollination (Plate 1.fig. i). At ten days after of pollination, 35 tubes were heavily

infected by fungal and bacterial pathogens. Nine ovaries were excised after 12 days of

pollination and found that only 3 had embryo and endosperm. Remaining ovaries were full

of watery endosperm and some were empty. No seedlings were obtained after culturing

these three embryos on Bouharmont (1991) medium. However, after 5 days of incubation,

64

i l

e f g h

a b c d

Plate 1. Figure a–c. Efforts employed to regenerate intergenomic rice hybrids. a-d. very young hybrid embryo

subjected to induce callusing and subsequent regeneration i.e. regeneration of hybrid plants through callus

culture, Pokhreli/O. officinalis., (direct regeneration without subculture on medium proposed by Chen et al.,

1991)., c. regeneration of the shoot, and d. figure showing profuse rooting in the callus derived seedlings some

of the seedlings are albino in origin which were died after transplanting in the pot., e-h embryo rescue

Manshara/O. officinalis, Kalanamak/O. Officinalis, and Jhinuwa/O. officinalis, respectively., g. In vitro

pollination O. sativa/O. granulata, showing swelling of ovary after 10 days of pollination., j-l new hardening

technique employed to reduce the mortality in field transplantation.

k j

65

Table 4.1a. Results of embryo rescue and crossability between O. sativa, and O. officinalis, and O. granulata

parental combination

No. of floret

pollinated

Seed set

%

Seeds

without

embryo

Total no.

of embryo

culture

Germinati

on %

Died aft

germin-

ation

No. of seedling

well grown

Harde-

ning

done

No of F1

plant

obtained

Crossab

ility %

Ghaiya/O. granulata1 508 21.46 91 18 27.78 5 - - - 0

Jhinuwa/O. granulata 411 34.06 116 24 20.83 5 - - - 0

Masuli/O. granulata2 513 32.94 143 26 26.92 7 - - - 0

IR 72/O. granulata 413 19.37 73 7 14.29 1 - - - 0

IR 64/O. granulata 389 21.85 72 13 23.08 3 - - - 0

Manshara/O. granulata 211 11.85 21 4 - - - - - 0

Pokhreli/O. granulata 221 16.29 26 10 30.00 3 - - - 0

Jethobudo/O. granulata 341 21.99 60 15 53.33 8 1 1 - 0

Kalanamak/O. granulata 147 15.65 15 10 20.00 2 - - - 0

Jhinuwa /O. officinalis 205 20.00 23 18 50.00 2 5 5 5 2.44

IR 72 /O. officinalis 115 31.30 21 15 20.00 3 - - - 0

IR 64/O. officinalis 163 6.75 8 3 66.67 2 - - - 0

Manshara/O. officinalis 203 21.67 24 20 55.00 7 5 5 4 1.97

Pokhreli/O. officinalis 174 14.94 13 13 61.54 4 4 4 3 1.72

Jethobudo/O. officinalis 125 8.00 2 8 37.50 3 - - - 0

Kalanamak/O. officinalis 201 29.85 42 18 33.33 4 4 4 4 1.99

1, 2 crossing were made only with O. granulata in the second year of 2002

66

only one embryo was germinated and that was also finally collapsed at single leaf stage.

This result still suggested that existence of a strong crossability barrier, between O. sativa

and O. granulata.

4.3.Crossability

4.3.1. Crossability in intergenomic combinations

Regardless of the other factors, based on the embryo rescue worked the crossability

between O. sativa/O. officinalis was ranged from 0-2.44% and average pooled mean was

1.35 % among all crosses with cultivars (Table 4.1a.). Similarly, it was zero for cv. of O.

sativa and O. granulata, although a large number of florets pollinated and a lot of in vitro

efforts were carried out up to 2 years.

However, the present result was comparable to report of Jena and Khush (1986,

1984). They found the crossability between three lines of O. sativa and O. officinalis varied

from 1.0-2.3 % and pooled mean was 1.7% (Jena and Khush, 1984). However, Brar et al.

(1991) obtained quite low crossability which was ranged from (0-1.1%) and the pooled

mean was 0.15% in five varieties of O. sativa. They got only two hybrid plants from two

varieties, pollinating 1301 spikelets of five cultivars. Similarly, other workers (Brar et al.,

1991; Morinaga, 1964; Ghose et al., 1960) also obtained non or few hybrids in this set of

cross.

4.3.2. Crossability between intragenomic cross

The seed set and crossability percentage between O. sativa and two common wild

species of rices were shown in Table 4.1b. Among combination seed set and crossability

was varied from 7.58-53.15and 7.58-51.05 percent, respectively. Among O. sativa/O.

rufipogon hybrids, highest crossability was scored for Jethobudo/O. rufipogon and was

67

Table.4.1b. Seed set, and crossability between O. sativa L. and two common wild rice

species (O. nivara and O. rufipogon).

Cross combination

Female/ Male

Total

spikelets

pollinated

Seed

set %

No. of F1 plants

obtained

Crossability

%

Kalanamak/O. nivara

Kalanamak/O. rufipogon

Jhinuwa/O. nivara

Jhinuwa/O. rufipogon

Manshara/O. nivara

Jethobudo/O .rufipogon

Jethobudo/O. nivara

Pokhreli/O. rufipogon

Pokhreli/O. nivara

IR 64/O.nivara

IR 64/O. rufipogon

IR 72/O.nivara

120

133

108

132

181

143

208

250

320

140

138

101

40

49.63

45.37

7.58

45.86

53.15

32.4

48.80

30.00

35.00

50.00

16.83

48

65

49 (2a)

10

81 (3a)

73 (3b)

67 (4a , 3

b)

122 (5a, 2

b)

96 (15a)

49

69 (6a, 2

b )

14 (3a)

40.00

48.87

43.51

7.58

44.75

51.05

28.84

46.00

25.31

35.00

44.20

13.86

a and b for non germinated seeds, and non viable albino plant, respectively

68

least in Jhinuwa/O. rufipogon. Similarly, among O. sativa and O. nivara hybrids,

crossability was highest in Manshara/O. nivara (44.75%) and was least in IR 72/O.nivara

(13.86%).

Based on crossability result, it was clear that the landraces like Pokhreli, Jethobudo,

and Kalanamak showed close cross affinity with O. rufipogon and Manshara with O.

nivara. In most of the combinations landraces of Nepal showed good comparable

crossability than the improved varieties (IR64 and IR72). However, degree of cross affinity

varied with genetic makeup of the female genotypes (Table 4.2.1b.) suggest that different

cultivars have different cross affinity with their ancestral wild species. Similar such

variation in crossability have been reported in literatures (Brar et al., 1991; Sitch et al.,

1989c; Chu et al., 1969; Shastry, 1964; Henderson, 1964a.; Nezu et al., 1960; Ghose et al.,

1960). They all reported that the crossability between O. sativa and O. rufipogon was

greatly varied from 10-30% depending upon the geographical race of wild species and

genotype of O. sativa used. Therefore, this slight discrepancy obtained in this study in seed

set might be attributed to differences in geographical race of wild species used as male

parent.

4.4. Morphology of the interspecific hybrids

All the hybrid plants, obtained from the crosses involving O. sativa with O. nivara

and O. rufipogon, were intermediate between the two parents in some traits but showed a

preponderance of the characters of wild species, such as basal leaf sheath coloration,

spikelet related traits, apiculus colour, seed coat colour, awning character, leaf color,

panicle exertion, panicle shattering, panicle type, culm number, internode colour branching

of culm and spreading growth habit, stigma color, size of anther, etc. (Appendix 4.3a-3h,

and Plate 2-3. fig. 1,2,3, and 1-8, respectively). On the other hand, F1 plants from four

69

landraces of O. sativa and O. officinalis, obtained through embryo rescue showed

intermediate traits in some cases, but wild traits of the species were highly dominant as

mentioned above. They were perennial and had rhizomes in their roots. They were robust

and highly vigorous than the either parent (Appendix 4.3a-3h, and Plate 2. fig.1-3). Many

workers, in several intragenomic and intergenomic hybrids, (Jena and Khush, 1986; Li,

1964; Morinaga, 1964; Hey and Henderson, 1961; Morinaga and Kuriyama, 1957) reported

similar dominance of most of the wild type traits.

4.5. Meiotic behavior in parents and their hybrids

4.5.1. Pachytene analysis in AA genome species hybrids and their parents

The meiotic behavior at pachytene stage is presented in Table 4.2.1. Except IR 64

all female parents including wild forms showed complete pairing (Plate.4, fig. 1) IR 64

showed lose pairing at two of the bivalents in one PMC out of 25 studied. Five hybrids

showed complete pairing in all bivalents out of eleven studied in this series. Other

remaining hybrids showed absence of complete homology, and the frequency was quite

high in Jethobudo/O. nivara, Kalanamak/O. nivara, IR 72/O. nivara and IR

64/O.rufipogon. However, it was not too large as reported by Shastry and Misra (1961a.b)

in interracial hybrid between Japonica/Indica. They reported that 31.04% of the hybrid did

not show normal pairing and most of them had terminal deficiency, heteromorphocity,

duplication (mostly reverse repeat), interstitial differential segment (small translocation),

inversion, translocation and lose pairing, even though later stages were normal. In this

study, the frequency of such structural hybridity was ranged from 4.0-22.58% of the cells

observed (Plate.4-7. except figure 2-5 in Plate 7). In IR64/O. rufipogon hybrid seven

PMC’s showed structural hybridity in 2-6 bivalents mostly with terminal unpaired region

70

and small interstitial translocation, frequently appeared with unpaired gap (Plate 4. figure.

2)

On the other hand, inversion was observed in one of the bivalent of IR72/O. nivara

hybrid (Plate 4. fig.1), but the frequency was quite low and only two PMCs showed

inverted region out of 24 studied. One of the PMC’s had only heteromorphocity in more

than one bivalents. Similarly, Pokhreli/O. nivara hybrid showed interstitial gap with

heteromorphocity and duplication in three bivalents in one PMC out of 19 observed (Plate

4, fig.3). On the other hand, Kalanamak/O. nivara hybrid showed structural abnormality in

13.04% cells out of 23 studied and included abnormalities were interstitial small

translocation and mostly heteromorphocity (Plate. 5,.fig.2). Similar irregularities were

recorded for Jethobudo/O. nivara hybrid in 14.29% of the PMCs out of 14 studied. Among

irregularities, the heteromorphocity, lose pairing and small interstitial translocation was

modal class (Plate.5, fig. 3). But it was unable to resolve the causes of heteromorphocity,

however, such differences might also be occurred due to deletion (deficiency) or tandem

repeats in along the length (Shastry and Misra, 1961b) or sometimes condensation

differences between species chromatin (Misra and Shastry, 1967). But what ever the causes

of such chromatin differences, on an average pairing was essentially normal along

chromomere to chromomere in hybrids between O. sativa and two common wild species of

rice.

Other hybrid combination in this series showed complete pairing and did not record

any of the irregularities, indicates that very close relationship between their chromosomes,

even the number of cells was increased. Such structural hybridity have been frequently

observed in intragenomic hybrids involving O. sativa and O. nivara (Dolores et al., 1979).

They observed 32.4% structural hybridity including loose pairing (23.5%), reciprocal

71

i

q

d

3 3

d d

Plate 4. Figure 1-3. Representative photomicrograph of chromosomes behaviour during meiosis at pachynema and

Camera Lucida drawings (right side). 1. Showing inversion (i, appeared as Ω), IR 72/O. nivara 2. Showing

heteromorphocity and terminally unpaired bivalent (single arrow), quadrivalent association (q), small translocation

(double arrow), reverse repeat (d), and loose pairing (l), respectively. IR 64/O. rufipogon ., 3. Showing interstitial translocation (double arrow), reverse repeat (duplication) (d), terminally unpaired (single arrow) Pokhreli/O.

nivara.

i 1 i 1

l

d

2

l

d

2

d

3 3

72

Plate 5. Figure 1-3. Representative photomicrograph (left) and drawings (right) of chromosome behaviour

during meiosis at pachynema. 1. 12 II, showing small inversion (inverted segment mostly appeared as Я) and reverse repeat (d), IR 64/O.nivara., 2. 12 II, showing small interstitial translocation and terminally

unpaired segments in two bivalents (single and double arrow, respectively), Kalanamak/O. nivara., 3. Showing interstitial translocation (double arrow), terminally unpaired (single arrow) and loose pairing (l)

along length in two of the bivalents, Jethobudo/O. nivara.

.

i

d

i d

i

i

1 1

2 2

l

l

3 l

l

3

73

Plate 6. Figure 1-3. Representative photomicrograph (left) and drawings (right) of

chromosome behaviour during meiosis at pachynema. 1. Showing loose pairing in one of the

12 bivalent in IR 64 parent., 2. Showing translocation (double arrow) in two of the 12

bivalents of Jhinuwa/O. officinalis, intergenomic hybrids., 3. 12 normal bivalents in

Manshara/ O. officinalis intergenomic hybrids. In this series of hybrids most of the normal bivalents had complex networking.

3 3

l l

1 1

2 2

74

Plate 7. Figure. 1-5. Showing heteromorphocity in 2 of the 12 bivalents (single arrow), Manshara/O.

officinalis., 2. 12 II Manshara parent., 3. 12 II, O. rufipogon., 4. Showing well differentiated

heterochromatic region of chromosome, O. granulata., 5. 12 II, O. nivara.

1.014µ 5

4 3

2 1

75

translocation (3.9%), and trivalent association (2.9%) out of 34 cells studied in IR8/O.

nivara.

Many investigators studied the pachytene chromosomes in interracial and

intervarietal rice hybrids, and reported the similar results (Shastry and Misra, 1961a.b; Yao

et al., 1958). Yao et al. (1958) observed inversion loops, mostly pericentric in five of the

intervarietal rice hybrids in < 10 % of the PMCs. Further more, Shastry and Misra

(1961a.b) reported about 11.9-31.04% of the chromatin length had such structural hybridity

and suggested that such structural hybridities are the sole cause of sterility rather than other

factors. Therefore, summing up the present results the structural hybridity observed in this

study was very low or comparable to interracial and intervarietal differention. Based on this

stage it is concluded that Nepalese common wild rices are not cytologically so

differentiated as much differentiation found in the Japonica/indica rice hybrids. Thus

pairing was essentially normal.

76

Table 4.2.1. Chromosome behavior at pachynema in the parents and hybrids

Number cells observed

Hybrid studied

Total PMC’s

observed

Normal

pairing

Cryptic structural hybridity in one or more

than one chromosome ( a, b, c, d, e, f)*

IR64/O. nivara

25

24 (96 %)

1 (4%) e and d

IR64/O. rufipogon 31 24 (77.42%) 7 (22.58%) a, b, c, and f

Pokhreli/O. rufipogon 35 35 (100%) 0 (0%)

Pokhreli/O. nivara 19 18 (94.74 %) 1 (5.26 %) b, d, and f

Kalanamak/O. nivara 23 20 (86.96%) 3 (13.04%) a, b, and f

Kalanamak/O. rufipogon 38 38 (100%) 0(0%)

IR 72/O. nivara 24 21 (87.50) 3 (12.50 %) a, and e

Jhinuwa/O. nivara 39 39 (100 %) 0 (0%)

Manshara/O. nivara 32 32 (100 %) 0 (0%)

Jethobudo/O. rufipogon 53 53 (100 %) 0 (0%)

Jethobudo/O. nivara 14 12 (85.71%) 2 (14.29%) a, b, and f

Manshara/O. officinalis 11 6 (55%) 5 (45%) a, b, and f

Jhinuwa/O. officinalis 15 9 (60%) 6 (40%) b, and f

IR72 21 21 (100%) 0 (0%)

IR64 25 24 (96%) 1 (4%) a

Kalanamak 31 31 (100%) 0 (0%)

Jhinuwa 9 9 (100%) 0 (0%)

Manshara 36 36 (100%) 0 (0%)

Pokhreli 31 31 (100%) 0 (0%)

Jethobudo

O. nivara

O. rufipogon

O. officinalis

17

23

21

19

17 (100 %)

23 (100 %)

21 (100 %)

19 (100 %)

0 (0%)

0 (0%)

0 (0%)

0 (0%)

* Loose pairing (a), deletion (c), duplication (d), inversions (e), translocation (f), and heteromorphocity (b).

77

4.5.2. Pachytene analysis in intergenomic hybrids (A and C genomes)

The pairing pattern of chromosome between O. sativa and O. officinalis is shown in

Table 4.2.1. Out of four combinations, only two hybrids were analyzed at this stage. Both

hybrid combination showed partial homology between A and C genomes. The complete

normal pairing was found in 15 cells out of 26 observed. It was 54.54% in O. sativa. cv.

Manshara/O. officinalis and 60.0% in Jhinuwa/O. officinalis. However, the degree of

homology varied from cell to cell and most cells possessed 2-3 cryptic differential

bivalents in 11 of the cells (Plate. 6, fig. 2 and Plate 7, fig. 1). Similar ranges of results

have reported by several researchers (Shastry et al., 1961; Katayama, 1966a. b, 1964).

Shastry et al. (1961) obtained complete pairing, however, Katayama (1966a. b., 1965)

observed partial homology. On the other hand, Li et al. (1964) was only one who reported

24 univalents at this stage in O. sativa/O. officinalis hybrids. However, Shastry (1966)

again claimed that the pairing was normal between these species hybrid. In this study too

there was no evidence to report the abnormal pairing, instead that nearly normal pairing

was found. Pairing was normal in more than 50 percent cells in both sativa-officinalis

hybrids studied, however, in this study most of the PMCs showed complex networking of

the chromosome and very few of the cells were observable. No any univalents were

detected, although, 11 cells had large unpaired interstitial differential segment in 2-3

(frequently 1-2) bivalents (Plate 6,.fig.2).

From this observation it is concluded that certain segment of the bivalents are

subjected to evolutionary change, most probably due to small translocation. Remaining

hybrid PMC’s showed complete 12 bivalent. Thus the normal pairing shown by more than

50 percent cells in sativa-officinalis hybrids clearly indicates that A and C genomes should

be partially homologous. Neither 100 percent complete pairing nor presence of univalents

found in this study suggests that definitely certain structural change between these taxa

78

might be operated with the trends of evolution. This result was closely associated with

findings of Shin and Katayama (1979). They reported that two of the hybrids, which have

an additional chromosome of O. officinalis, formed univalent, other six hybrids studied by

them did not show univalent and frequently formed trivalents at diakinesis and metaphase

I. Based on the occurrence of many univalents in the PMCs of two alien addition lines of

O. officinalis, Shin and Katayama (1979) suggested that two chromosomes of O. officinalis

(W0065) have genes which are responsible to desynapsis of the pairing between species

homologous chromosomes. However, they did not observe the pachytene chromosome,

therefore they could not deduced the genetic control of univalent formation in the two types

of alien addition lines. Such asynaptic and desynaptic genes controlling chromosome

pairing at MI of PMCs have been reported in Zea (Beadle, 1933), Triticum (Li et al., 1945;

Riley and Law, 1965), and in rice (Misra and Shastry, 1969). Beside, desynaptic mutant in

rice has been artificially induced (Yasui et al., 1993). The asynapsis or desynapsis was in

most cases controlled by a single recessive gene (Shin and Katayama, 1979) and such

genes are more active particularly at low temperature (Li, 1945).

In this study, also two of the twelve bivalents mostly did not show complete pairing

in O. sativa. cv. Manshara/O. officinalis hybrid. Five of the PMC’s in this hybrid showed

such cryptic behavior. This differential region observed in this study indicates that certain

evolutionary change might have evolved and the desynapsis mutant further erecting the

strong reproductive barrier between these two complex. Not only at pachytene, even at

diplotene, 3-6 bivalents and trivalents and rarely quadrivalents were observed (Plate. 8,.fig.

1-3). Among these bivalents some were loosely paired with a single chiasmata (Plate.8,.

fig. 2). This latter stage analysis also supported the results of pachytene analysis. Such

frequent bivalent formation at this stage have been reported by Katayama (1966a., b). He

79

also frequently observed bivalents in O. sativa (AA)/O. minuta (BBCC) and O. sativa

(AA)/O. latifolia (CCDD) hybrids. Based on this analysis he concluded that such bivalent

mostly due to partial homology between A and C genomes; not due to pairing between A

and B or D genomes. Thus the data from the present study, also clearly shows that three is

partial homology between A and C genomes and did not support the earlier generalization

as either very limited pairing or pairing is completely failure made by Li (1964), Morinaga

(1964), (Jena and Khush, 1986), Nezu et al. (1960). However, all of them drew their

conclusion based on data from diakinesis and Metaphase I. except Li et al. (1964). On the

other hand these events might not be always true, as pairing event is largely effected by

plant growing environmental condition (Palmer et al., 2000; Ahmad et al., 1977;

Henderson, 1964a.; Dowrick, 1957; Li, 1945) or geographical differentiation in the race

and genotype of the female parent used (Shin and Katayama, 1979; Shastry, 1966).

Therefore, The discrepancy found in the pairing of pachytene chromosomes among these

workers might be due to their use of different strains of O. officinalis.

However, whatever the findings in the past had, the included materials in the

present study showed partial homology between A and C genomes. The result obtained

herein also clearly indicates that the failure of bivalent formation at later stage in distant

hybrids always not a proof of lack of homology. Such failure of synapsis can possibly be

brought by many external and internal factors (Misra and Shastry, 1969). Thus the present

study revealed that, A and C genomes, more or less completely pair but differentiated by

few chromosome structural changes, although these species are morphologically highly

differentiated.

4.5.3. Variation in nucleolus shape in parents and their hybrids

Shapes of nucleolus at pachytene were studied in all the parental forms and their

possible hybrid derivatives (Table 4.2.2). Most of the landraces had inconsistent shape and

80

Table 4.2.2. Percent frequency of nucleolus shape variation in parents and hybrids

Parents/ hybrids

Kalanamak

Total PMCs

observed

205

Single large

nucleolus

43.41

Semi-

doubled

10.24

Single with

one Bud

45.37

Single with two

bud

0.98

Single with

three bud

-

Two Nucleolus /cell

-

Manshara 214 36.45 44.86 18.69 - - -

Jethobudo 242 19.42 19.83 60.74 - - -

Pokhreli 214 47.66 11.68 40.65 - - -

Jhinuwa 190 52.63 7.89 39.47 - - -

IR64 148 67.57 2.03 28.38 - - 2.03

IR72 142 95.77 1.41 2.82 - - -

Manshara / O. nivara 132 38.64 1.52 59.09 0.76 - -

Jethobudo/ O. rufipogon 246 73.98 0.81 24.39 0.81 - -

Jethobudo/O. nivara 261 70.88 1.92 27.20 - - -

Pokhreli/O. nivara 206 49.03 9.71 41.26 - - -

Pokhreli/ O. rufipogon 96 98.96 - 1.04 - - -

Jhinuwa/ O. rufipogon 171 45.61 14.62 38.01 1.75 - -

Jhinuwa/ O. nivara 225 44.44 17.78 37.78 - - -

Jhinuwa/ O. officinalis 180 11.11 38.89 44.44 5.56 - - Pokhreli /O. officinalis 170 47.06 38.24 8.82 5.88 - -

Kalanamak/O. nivara 181 13.81 4.97 81.22 - - -

Kalanamak/O. rufipogon 211 61.61 1.42 36.97 - - -

Manshara/ O. officinalis 202 39.60 35.64 19.80 3.96 0.99 -

IR64/O.nivara 234 51.28 10.68 38.03 - - -

IR64/O. rufipogon 273 99.27 - 0.37 - - 0.37

IR72/ O. nivara 269 14.13 9.29 76.21 - - 0.37

O. nivara 280 25.00 0.00 74.64 - - 0.36

O. rufipogon 183 87.98 7.10 4.92 - - -

O. granulata 339 100.00 - - - - -

O. officinalis 201 97.51 1.49 1.00 - - -

81

mostly represent single, semi-doubled and single with small one bud. However, the single

with large and single with one bud type were commonly observed in most of the parents

except Manshara, which had semi-doubled type in 44.86% cells. On the other hand IR 72

only showed single large nucleolus in 95.77% cells. Among wild species: O. granulata and

O. officinalis contained single large nucleolus, however, small frequency of cells with other

type of shape in O. officinalis was also observed. It was found interesting that O. granulata

had only one single large nucleolus than all the species studied, even after screening large

number of cells. On the other hand high frequency of single nucleolus with one bud type

was observed in O. nivara in 74.64% cells.

Most of the hybrid showed similar variable structure with preponderance of single

type except O. sativa/O. nivara hybrid, which showed quite dominance over O. sativa

nucleolus. Only two of the hybrid combinations showed two nucleolus/cell. No sharp effect

of hybridization stimuli was detected in this object. Misra and Shastry (1967) carried out

similar study. They observed variation in pachytene nucleolus in the shape within sativa

varieties. However causes of such structural changes during this stage was still mysterious.

But these variations can be treated as marker to determine the diversity among wild and

cultivated forms.

4.5.4. Diakinesis and Metaphase I

The chromosome behaviour at these stages in parents and hybrids are shown in

Table 4.2.3a, 3b, 3c, and 4.2.3d and illustrated in photomicrograph (Plate 8 and 9). Among

wild species, O. officinalis scored highest percentage (96.64%) of normal bivalent (12II)

and O. nivara showed least one (89.24%) (Table 4.2.3c). Similarly, among cultivated forms

IR 72 had highest normal cells (97.65%) followed by Pokhreli was least (80.39%). On an

average all the parental forms showed normal cells having 12II and were ranged in 80.39-

82

97.65% of the PMC’s. On the other hand, highest univalents (15.03%) was scored in

Pokhreli among parents and least in IR 64 (0.35%). Similarly, almost parents, except, O.

rufipogon, O. officinalis, IR72, and Kalanamak had quadrivalent association, and these

were scored in 0.63-5.72% cells. The highest (5.72%) quadrivalent association was

observed in Jethobudo among parents, although pachytene stage was normal. Similarly,

Jethobudo and Kalanamak showed VI association at low frequency in 0.44 -1.91% cells.

On the other hand, it was found quite interesting that Jhinuwa parents scored highest 11-

12II with nucleolar bodies (7.88% cells). All other parents except IR 64 had such cells and

its occurrence was varied in 0.65 -7.88% cells. Aneuploids were also observed at low

frequency in Jethobudo and Pokhreli parent (0.44% and 0.65% cells, respectively) (Table

4.2.3b and 3c.).

83

Table 4.2.3a. Meiotic configuration at Diakinesis and Metaphase I from the parents used in

the hybridization

84

85

Plate 8. Figure 1-11. Representative photomicrograph of meiotic behavior at diplotene and

diakinesis in interspecific rice hybrids. 1-3 Diplotene. 1. 1 III (double arrows, fork shaped) + 4 II +

13 I, Kalanamak/O. officinalis., 2. 1 IV (shown by single arrow, cross shaped, indicator of typical

case of translocation) + 2 III (double arrows, frying pan shaped) + 3 II + 8I, Pokhreli/O. officinalis., 3. 1 IV (single arrow) + 2 III (double arrows) + 2 II +10 I, Manshara/O. officinalis. 4-11 diakinesis.

4. 12 II (early diakinesis) appeared as α (three chiasmata), ∞ or 8 (2 chiasmata) and V or Χ (mono

chiasmatic bivalent), and ring (dichiasmatic bivalent), O. granulata., 5. 12 II O. officinalis., 6. 12 II

+ nucleolar bodies, Pokhreli/O. nivara., 7. 11 II only, IR 64/ O. nivara., 8. 11 II + 2 I (single

arrow), Jhinuwa/O. nivara., 9. 24 I, Kalanamak/O. officinalis., 10. 24 I, Manshara/O. officinalis., 11.

24 I, Jhinuwa/O. officinalis.

1

4 5

6 7 8

9 10 11

3

2 2

86

5

Plate 9. Figure 1-12. Representative photomicrograph of chromosome behavior during

meiosis at MI in interspecific rice hybrids. 1. Showing 12 ring II, Kalanamak/O.

rufipogon.., 2. Showing 12 II, four of the II’s are rod shaped, Manshara/O. nivara., 3. 12

ring II, Jhinuwa/O. nivara., 4. 11 II + 2 I (single arrow), IR 72/O. nivara., 5. 11 II + 2 I

(single arrow), precocious separation of one bivalents resulted into univalent like structure,

Pokhreli/O. rufipogon., 6. 11 II + 2I (single arrow), IR 64/ O. rufipogon., 7. 11 II + 2 I

(single arrow) Kalanamak/O. nivara., 8. 11 II + 2 I (single arrow), Kalanamak/O.

rufipogon., 9. 24I, Pokhreli/O. officinalis, 10. 2 II (1 rod + 1 pseudo-bivalents) + 20 II,

Manshara/O. officinalis., 11. 51 chromosomes, showing hybridization stimuli, Pokhreli/O.

officinalis., 12. 25 I, Pokhreli/O. officinalis.

1 2 3 4

6 7 8

9 10 11 12

87

Similar, such abnormalities in true parental forms were reported by Nandi (1936), Sasaki

(1935). They frequently observed one quadrivalents and 2-4 univalents. However, in this

study too such irregularities were more or less similar, the frequency of univalents

observed in Pokhreli were quite high (0-8) (Table.4.2.3b). This event might be either

attributed to heterogeneity of its population or to winter effect, because, the analysis of this

material was carried out on the second week of December 2002. Such effect of

environment particularly low temperature was greatly emphasized by Li (1945), Ahmad et

al. (1977).

Among hybrids, involving O. sativa and common wild rice (O. nivara and O.

rufipogon), chromosome association was essentially normal except Pokhreli/O. nivara and

IR72/O. nivara; they showed mark reduction in normal bivalent association. More than

17% of the cells were with aberration in these hybrids. In other hybrids, reduction in the

normal bivalent formation was found in < 11% cells except IR 64/O. nivara. IR 64/O.

nivara had 16.26% cells without normal bivalent. On the other hand, it was found

interesting that normal bivalent formation was slightly increased by 0.57%, 6.71% and

2.34% in hybrid forms of Jethobudo, Pokhreli, and Kalanamak with O. rufipogon,

respectively. As compared to female parent (IR 64), IR 64/O. rufipogon showed reduction

in normal bivalent in 12.07% cells. Similarly, highest univalents were observed in

Pokhreli/O. nivara, and 18.18% cells had univalents with ranged from 2-6 (Table 4.2.3c)

and was least in Kalanamak/O. rufipogon (4.56%). Almost all hybrids showed quadrivalent

association except Kalanamak/O. rufipogon and Manshara/O. nivara, and it was highest in

Pokhreli/O. nivara in 6.82% cells. Other abnormalities includes were, formation of

hexavalent except Jhinuwa/O. nivara, Pokhreli/O. nivara, Pokhreli/O. rufipogon, and

Kalanamak/O. rufipogon, presence of nucleolar bodies (0.91-12.27%), (Plate. 8,. fig. 6),

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Table 4.2.3b. Meiotic configuration at Diakinesis and Metaphase I from the interspecific

hybrids involving three different species

89

Table 4.2.3b. Meiotic configuration at Diakinesis and Metaphase I from the interspecific

hybrids involving three different species

90

and occurrence of aneuploids (0.40-7.17%) in all hybrid combinations (Plate. 8, fig. 7).

However, in Pokhreli/O. rufipogon, the aneuploidy found in parental forms was slightly

reduced. The abnormalities observed at diakinesis and MI were also partly illustrated in

microphotograph (Plate. 8, fig.1-11, Plate.9,. fig. 1-12, and Plate.10,. fig. 1-2).

Similar, observations were reported by Dolores et al. (1979) in cross involving O.

sativa and O. nivara. They reported that a series of abnormality including univalents

trivalents, quadrivalents, nucleolar bodies and aneuploidy, but their frequencies were

comparable to their respective parents. Although, no trivalents were observed in this study.

Not only these small one, high frequency of abnormalities such as frequent 0-3

quadrivalents formation, univalents and chromosomal elimination have been reported in O.

sativa and O. rufipogon hybrids (Majumder et al., 1977; Majumder and Ram, 1992) and

even in true lines of partially sterile rice variety (Ram and Majumder, 1996). The frequent

occurrence of nuclear bodies in this study at diakinesis in both parents and hybrids,

however, necessitates that the new speculation needs to be made to generalize the causes of

such abnormalities. There are several proposition about the occurrence of such bodies, like

structural changes (Walter, 1963) or due to fragmentation of chromosome by translocation

(McClintock, 1934 in Parthasarathy, 1938). However, the nature of the data observed here

showed that there might be other causes too. It is because of the frequency of such bodies is

quite high even in parent like Jhinuwa (7.88%), although pachytene pairing was normal.

Similarly, univalents, and quadrivalents were frequently observed not only in interspecific

hybrids, but also commonly reported for interracial and intervarietal hybrids (Sampath and

Mohanty, 1954; Yao et al., 1958). Similar reports have been put forward in interspecific

hybrid involving intragenomic cross hybrids (Yeh and Henderson 1961, 1962, Morinaga,

1964, Misra and Shastry, 1969).

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Likewise hybrid involving O. sativa and O. officinalis showed pronounced

irregularities at these stages. Nearly 100% cells had irregularities and the mean frequency

of bivalent was ranged from 0.98-1.59 in most of the crosses (Table 4.2.3d). The

irregularities were characterized as formation (14-24 I) univalent with modal class 24 I

(Plate. 8., fig.9, 10,11, and Plate. 9,. fig. 9 and 10). Besides, at low frequency, some of the

PMC’s had haploid-polyploid chromosomes complement. There are voluminous literatures

that deal about such abnormality at these stages, although pachytene was normal in O.

sativa and O. officinalis hybrids (Jena and Khush, 1986, Nayar; 1973; Hu and Chang,

1966, Katayama, 1966a. b; Morinaga, 1964; Li 1964; Ghose et al., 1960, Nezu et al.,

1960). Similar irregularities have been reported in other intersectional species hybrids (Brar

et al., 1996; Morinaga, 1964; Li 1964; Shastry and Rao, 1961; Ghose et al., 1960; Nezu et

al., 1960). Most of them observed 24 univalents and occasionally 1-5 bivalents. On the

other hand, hyperploidy and hypoplody was reported by Hu and Chang (1966), Dolores et

al. (1979). Hu and Chang (1966) observed more than 48 chromosomes in two of the

geographical races of diploid O. officinalis hybrids and Nayar (1973) concluded that such

phenomenon is due to the hybridization stimuli (Plate. 9, .fig. 11 and 12 and Plate 10,. fig.

2). Therefore, the over all result and discussion regarding these two stages showed that

except intergenomic hybrids, pairing events obtained in the hybrids was comparable to

their parents in one or more respects thus pairing was essentially normal.

4.5.4.1. Chiasma frequency

Chiasma frequencies among parents included in the hybridization are shown in

Table 4.2.3a and representative illustrations were shown in Plate. 8., for diakinesis and 9.

for MI. Among wild species O. granulata showed least number of chiasmata/cell

(20.96±2.03, 22.16±1.95) and per bivalent (1.75±0.18,1.85±0.17) at diakinesis and

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metaphase I, respectively with pooled mean 21.56/cell and 1.8/bivalent. Similarly, O.

rufipogon and O. officinalis scored second and third position interms of chiasmata,

respectively. On the other hand, annual wild species O. nivara showed highest

chiasmata/cell and per bivalent, which is comparable to cultivated Oryza sativa forms (>23

chiasmata/cell and >1.9/bivalent). However, in all forms, ring bivalent was predominantly

found (8.94-11.17) and rod bivalent varied from 0-8, and was highest in O. granulata.

Some bivalents were visibly larger than others, they had 2 or 3 chiasmata and their shape

was ∞ or α. Some others had only 1 or 2 chiasmata and structure formed like V, X, or ring

shape (Plate. 8,. fig.4.). Similar trends in chiasma frequencies was reported by Jena and

Misra (1984) and they suggested that perennial and cross pollinated species in general

showed lower chiasma frequency than their related annual self pollinated species. This

generalization was also found evident in this study as well, particularly, in the case of O.

rufipogon, which is perennial and occurrence of certain degree of cross-pollination due to

its high stigma exertion for long duration than O. nivara.

It is frequently reported that the out crossing rate in this species was diverged and

quite high (30-50%) (Rao et al., 1997; Morishima and Oka, 1995). O. granulata and O.

officinalis are perennial, however, based on chiasma frequency, it was appeared that O.

granulata and O. rufipogon were more primitive than O. officinalis. Similar proposition is

made by Shastry and Sharma (1985) based on high amount of heterochromatic segment at

pachytene stage. In this study also O. granulata had highest amount of heterochromatic

regions than other species (Plate 7, fig.4). On the other hand annual species O. nivara

scored higher chiasma frequency (>23), which was the indication of its highly evolutionary

advanced. Thus it is concluded that evolutionary changes would be going on in the genus

Oryza and general trends seemed to perennial to annual and cross pollinated to self

93

pollinated through effective genetic recombination (higher chiasma frequency) (Jena and

Misra, 1984).

Similarly, all the cultivated forms of Oryza sativa showed more or less same level

of chiasmata frequency (>23/cell), except Pokhreli landraces, which had (22.59

chiasmata/cell), indicate that the landraces are so advanced as much advanced found in

improved ones. However, highest chiasma frequency was found in IR72 (23.36/cell).

Among landraces Kalanamak was more advanced than others. However, the chiasma

frequency/ bivalent was found in Pokhreli also showed same level of evolutionary advance

and low chiasmata frequency/cell was mainly attributed to more univalent formation at

these stages. The mean frequency of rod bivalents in these series varied from 0-9 however,

0-3 was the mode (Table 4.2.3a.). Similar ranged of results were presented by several

authors (Soriano, 1961; Chu et al., 1969). On the other hand, among hybrids, involving

common wild rice (O. rufipogon and O. nivara) and O. sativa did not showed sharp

reduction in chiasma frequencies as expected and were comparable to their either parent

indicated that significant association between their genomes Table 4.2.3a.

The highest pooled chiasmata frequency/cell and per bivalent was found in

Jethobudo/O. nivara (22.965 and 1.945, respectively). Another interesting event was that,

the chiasma frequency of hybrids (Pokhreli/O. rufipogon) slightly exceeds the frequency of

female parent, this suggests that chromosome pairing can be improved by genes from wild

species (Table 4.2.3b). The mean number of rod shaped bivalent in these series of hybrids

varied from 0.53-1.80 Similar result of increased in meiotic pairing from diploid wild

relatives of Triticum have been reported by Sears (1976). Not only in the wheat Nowick

(1986) also suggested that some of the AA genome cultivated rices have pairing promoter

genes.

94

95

96

97

Similar results were reported by other several investigators in hybrid among AA genome

species (Yao et al., 1958; Yeh and Henderson, 1962, 1961; Shastry and Misra, 1961a.b.).

However, Chu et al. (1969) obtained slight reduction in the chiasmata/bivalent in AA

genome hybrids. According to him the mean number of chiasmata in the sativa variety was

1.98±0.65 and was 1.91±0.61 in O., rufipogon but chiasmata among twenty hybrids was

varied from 1.08±0.62 to 1.83±0.59. Although, marked reduction in chiasma frequency

was observed in O. sativa and O. officinalis hybrids, this does not imply the lack of pairing

it was due to desynapsis and was varied from1.33-2.57/cell and 0.72-1/bivalent,

respectively. The average mean ring bivalent was varied from 0.53-0.89. Such sharp

reduction in the chiasma frequency and there by frequently formation of rod shaped

bivalents was reported by Sarkar et al. (1994) in Maize/Teosinte hybrids. This implies that

very limited pairing between these genomes at diakinesis and Metaphase I stages due to

desynapsis. Jena and Khush, (1986), Morinaga (1964) Lu (1964) observed similar pattern

of pairing in these species hybrids. Similar, events were reported in number of distant

crosses of rice (Mahapatra et al., 2002, Brar et al., 1996, Li, 1964; Morinaga, 1964),

however, they did not calculate chiasma frequency.

4.5.4.2. Chromosome number and ploidy level

From the cytological observation it was confirmed that all the wild species from

Nepal discovered so far, had a consistent chromosome number of 2n = 2x = 24 in both

meiotic PMCs and pollen mitosis (Table.4.2.3a.). Therefore, it is reported that all the wild

species, O. nivara, O. rufipogon, O. granulata, and O. officinalis, from Nepal are diploid

(Plate. 8, and 9.). This observation showed the similarity with the results of Bouharmont

(1962), and Vaughan (1994). Similar result was presented by Lu et al. (1997) and reported

98

that O. nivara, O. rufipogon, and O. granulata had 2n=24. On the other hand, 2 Indian, out

of 138 accession of O. officinalis showed tetraploidy (2n = 4x = 48), and known as O.

malampuzhaensis (Vaughan, 1994). In this study too minor meiotic irregularities was

observed and meiotic abnormalities includes were few univalent and PMCs with abnormal

nuclear bodies in all the species, however, the frequency was too small. Similarly, Lu et al.

(1998) suggested that four AA genome species, O. nivara, O. rufipogon, O. glumaepatula

and O. meridionalis, from different geographical origin of a world also showed consisted

number of chromosome, 2n = 24.

4.5.5. Meiotic behavior at Anaphase and telophase I

The chromosome behaviors at these stages are shown in Table 4.2.4. Among

parents the percentage of normal cells varied from 87.88-98.54, which was highest in IR 72

and lowest in Pokhreli. All parents showed a minimum degree of all arrays of

abnormalities except bridges and fragments and bridges and laggards Observed

abnormalities includes were unequal segregation, bridges, laggards, late disjunction and

early division of chromosomes. Their existence was observed in 0.69-3.93, 0.72-3.03,0.69-

2.96, 0.85-4.24 and 0.85-1.95 percent cells, respectively (Table 4.2.4, Plate. 10,. fig. 6-9

and Plate 11,. fig.1-11). All types of anomalies were not recorded within single parent

however more than two abnormalities were commonly observed. Among hybrids between

O. sativa and common wild rices, Jethobudo/O. rufipogon scored highest percentage

normal cells and was lowest in IR64/O.nivara. In this series of crosses, percentage of

normal cells varied from 70 -95%. Other abnormalities include were more or less

comparable to their respective parents except bridges + fragments and bridges + laggards

(Plate.11,.fig.8). Four (Kalanamak/O. nivara, Jethobudo/O. nivara, Pokhreli/O. nivara and

IR64/O.rufipogon) of the 11 hybrids had bridges + fragments. The percentage cells with

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3

Plate 10. Figure 1-9. Representative photomicrographs of chromosome behavior during meiosis at

metaphase I, and anaphase I in interspecific rice hybrids. 1. Prometaphase, 24 I, Kalanamak/O.

officinalis., 2. Prometaphase, 33 I, 9 II + 15 I, 4 are pseudobivalents due to e-e and s-s contact,

Jhinuwa/O. officinalis., 3. AI, showing normal disjunction, Jethobudo/O. rufipogon., 4. AI, showing

normal disjunction, 12 chromosomes are ready to go one pole, Jethobudo/O. rufipogon., 5. AI, normal

disjunction, IR 72/O. nivara., 6. AI, showing two lagging fragmented univalent and single univalent,

Kalanamak/O. nivara., 7. Two pairs of chromosome have formed bridges, apparently the results of

delayed terminalization of chiasmata (sticky bridges), Kalanamak/O. nivara., 8. AI, delayed disjunction, Pokhreli/O. rufipogon., 9. AI, unequal segregation, Manshara/O. officinalis.

1 2

4 5 6

7 8 9

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Plate 11. Figure 1-11. Representative photomicrographs of chromosome behavior during meiosis at

anaphase I, and telophase I in interspecific rice hybrids.1. MI-AI, showing sticky bridges and laggards,

Kalanamak/O. officinalis., 2. AI, showing laggards Manshara/O. officinalis., 3. MI-AI, showing laggards

Jhinuwa/O. officinalis., 4. MI-AI, 24 I randomly distributed along cell plate, Kalanamak/O. officinalis., 5.

AI, sticky bivalent showing possibility of unequal segregation, IR 64/O. rufipogon., 6. MI-AI, 18 I were appeared in metaphase plate, Manshara/O. officinalis., 7. AI, showing bridges + fragments and laggards,

Pokhreli/O. officinalis., 8. Showing typical anaphase bridges + fragments, indication of paracentric

inversion, IR 64/O. rufipogon., 9. TI, 2 lagging, and 1 laggards, Pokhreli/O. nivara., 10. TI, showing

laggards, Kalanamak/O. officinalis., 11. AI, showing anaphase false bridge due to delayed

terminalization of chiasmata, Manshara/O. nivara., 12. Showing partial pollen sterility, IR 64/O.

rufipogon.

1 2 3

4 5 6

7 8 9

10 11 12

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bridges + fragments were ranged from 0.44-9.40, which was quite higher in IR64/O.

rufipogon (9.40%).

On the other hand, bridges + laggards frequently observed in all hybrids except

Jethobudo/O. nivara, IR64/O.nivara, Jethobudo/O. rufipogon and Pokhreli/O. rufipogon

and their occurrence varied in 0.71-2.76% PMC’s. Highest percentage of cells with late

disjunction was found in IR 64/O. nivara (9.09%). On the other hand, hybrid involving O.

sativa/O. officinalis showed high abnormalities at these stages and most of them had high

frequency of laggards and it was observed in 33 -49% PMC’s studied. All hybrids in this

series showed bridges with fragments and its existence varied in 6.70-9.23% cells

(Plate.11, fig. 7). Cells with laggard +fragments were observed in 16.50-21.86 % cells

(Plate.11,. fig.9-11). Similar results were reported in many investigators in intragenomic

cross hybrids (Majumder et al., 1997; Majumder and Ram, 1992; Dolores et al., 1979

Misra and Shastry, 1969; Shastry, 1964; Hey and Henderson, 1961, 1962; Nezu et al.,

1960; Ghose et al., 1960), and in intervarietal and interracial rice hybrids (Ram and

Majumder, 1996; Demeterio et al., 1965; Henderson et al., 1959, Yao et al., 1958; Sampath

and Mohanty, 1954).

Dolores et al. (1979) observed bridges and fragments in five of the 11 crosses

involving O. sativa and O. nivara. However, they observed at low frequency in 0.3-0.7%

PMCs. According to them, the maximum abnormalities at these stages were bridges, late

disjunction and early division of chromosome. On the other hand, Sampath and Mohanty

(1954) found bridges and fragments in 11 of the interracial hybrids out of 33 studied by

them. Such irregularities was most frequently observed in <10% cells, and they concluded

that the occurrence of anaphase bridges and fragments mostly due to presence of inversion

in some of the chromosomes. Henderson et al. (1959) also observed 0.28% cells with

bridges and fragments in interracial rice hybrids. Other several investigators reported

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occurrence of anaphase bridges in interspecific crosses without mentioning their

frequencies (Hey and Henderson, 1961, 1962; Misra and Shastry, 1969).

However, in this study a quite high bridges and fragments formation at these stage

might be due to genotypic differences in the female parents used or probable explanation

for such abnormalities would be the induced inversions. High frequency of bridges

formation at anaphase mostly attributed to delay terminalization of chiasmata or sticky

bivalent formation or breakage and randomly reunion of the chromatids at earlier stages

regardless of homology as reported by Walter (1963) (Plate.10, fig. 7 and Plate.11, .fig.11).

Likewise, the quite high frequency of laggard formation at these stages undoubtedly the

results of univalents at diakinesis and metaphase I. Besides, these aberrations, unequal

separation, early disjunction, and late disjunction observed in this study also confirmed the

results of Dolores et al. (1979) (Plate. 10, fig. 8-9 and Plate. 11, fig.5). These commonly

occurred anomalies might be precocious separation of bivalent at metaphase (Plate.9,.

fig.5) or anaphase or due to timing imbalance (Shastry and Rao, 1961) to separate the

bivalent (Plate.10,.fig.5) or might be due to effect of hybridity. Besides these anomalies

some of the hybrids showed improvement in pairing in these stages than their parental

forms indicating that genes from wild rice improved the pairing efficiency as reported by

Sears (1976) in diploids wild wheat. On the other hand high abnormalities in later stages of

meiosis in O. sativa and O. officinalis hybrids have been frequently reported by several

researchers (Jena and Khush, 1986; Katayama, 1966a.b., 1965; Morinaga, 1964; Li, 1964).

However, they did not report bridges and fragments, as their study only confined to

diakinesis and metaphase I except Katayama (1966a.b). He only demonstrated laggards and

bridges in his figure but not reported the fragments (acentric) and bridges.

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104

In this study also most of the cells showed laggards (Plate.11,.fig. 1-4) and bridges as a result of same phenomenon as described above

(Plate.11, fig.7). Formation bridges and fragments at these stages was quite different from the previous study, indicates that inversion would

be certainly occurred during evolution in one – three chromosome or these might be due to effect of hybridity. Probably inversion should be

true as even in the pachytene frequently 2 of the bivalents did not completely paired. Most of the hybrids showed univalents either at the

equatorial Plate as laggards or distributed randomly throughout the nucleus (Plate.11,.fig.1, 2, 3, 4, and 6). Very few of the bivalents separated

and go to opposite poles (fig.6). Thus in these stages was pairing was normal, except for O. sativa and O. officinalis hybrids, as the aberration

found in this stages were similar to previous results of other investigators, and even low as reported in interracial hybrids having similar

genome. Not only, but also the frequencies of irregularities were more or less comparable to their parents as well.

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4.6. Hybrid fertility and sterility: cytological dissection

Pollen and spikelet fertility in F1 hybrids and their parents are shown in Table 4.2.5.

The F1 hybrids, among O. sativa and O. nivara, Manshara/O. nivara had highest pollen and

spikelet fertility (25 and 86.49%), respectively. Pokhreli had the lowest pollen fertility

(45.22%) among female parents. O. officinalis showed lowest pollen fertility (33%), and it

was the highest Kalanamak (71.30%) among male and female parents, respectively. IR

72/O.nivara, on the other hand, showed lowest pollen fertility among hybrids in this series.

Similarly, the F1 Pokhreli/O. rufipogon had the highest mean percentage for both

pollen and spikelet fertility (49.53 and 71.69%), and it was least for IR64/O.rufipogon

(23.0 and 2%, Fig. 12, Plate. 11), respectively, in this series of hybrids. It was found quite

interesting that two of the hybrid Kalanamak/O. rufipogon and Jethobudo/O. rufipogon did

better interms of spikelet fertility, although, the pollen fertility had lower than their

respective female parents.

On the other hand, among O. sativa and O. officinalis hybrids, most of the hybrids

had average pollen fertility <4.55% (0-4.55%). All the F1 hybrids had 100% spikelet

sterility however, partiality fertility was recorded for their female parents (Table 4.2.5).

Similar range of F1 sterility have been reported in intragenomic crosses, involving O. sativa

and different AA genome species hybrids (Dolores et al., 1979; Hey and Henderson,

1961,1962; Richharia, 1960; Morinaga and Kuriyama, 1957). Dolores et al. (1979)

observed 35-60% fertility as lowest in two of the O. sativa/O. nivara hybrids. Other hybrid

studied by them showed normal pollen fertility according their classification (>60%).

Morinaga and Kuriyama (1957) observed 4.3% averages pollen fertility and found 100%

sterility in winter grown O. sativa and O. glaberrima hybrids. On the other hand, Hey and

Henderson (1961) obtained 8.2-99.99% fertile pollen and 0-94.8% spikelet fertility in

hybrid involving O. sativa and five wild species of Oryza having AA genome. However in

106

this study, most of the hybrid involving O. sativa and common wild rice as well as parents

showed comparatively less pollen fertility (partial fertility). Such discrepancy might solely

due to large environmental effect, because pollen fertility and sterility was determined from

the winter grown plants. Such sterility have been largely reported not only for interspecific

crosses, but for intraspecific crosses as well (Shastry and Misra, 1961a.b; Henderson et al.,

1959; Sampath and Mohanty, 1954; Yao et al., 1958). Henderson et al. (1959) observed

28.7-98.4% sterility in intervarietal crosses. In some of the hybrid pollen fertility was quite

low as compared to spikelet fertility for example, Manshara/O. nivara. On the other hand,

the observation made by IKI staining technique did not reflect any significant correlation

between pollen and spikelet fertility and it was found dubious to generalize the results

based on this technique. Such dubious results have been frequently reported in literatures

(Song et al., 2001; Joshi, 2000)

There was a large deal about causes of pollen and spikelet sterility in many crop

species and many of them reported that chromosomal causes (cryptic structural hybridity)

(Yao et al., 1958 Shastry and Misra, 1961a.b.; Allard, 1960; Stebbins, 1958), or genic

(Sano, 1993; Ikehashi and Araki, 1986; Ikehashi, 1991, 1986; Oka, 1974) or cytolopalsmic

(Dolores et al., 1979) and or environmental (Palmer et al., 2000; Dolores et al., 1979;

Ahmad et al., 1977). Dolores et al. (1979) found significant differences in 3 of the 6

reciprocal crossed hybrids involving O. sativa and O. nivara, and demonstrated the

apparent effect of cytoplasm on varying degree of sterility. But, in this study reciprocal

crosses did not carry out and degree of sterility caused by cytoplasmic differentiation could

not be assessed.

However it was clear that the partial sterility obtained in O. sativa and common

wild species did not attribute to chromosomal hybridity except IR64/O. rufipogon,

Jethobudo/O. nivara, Kalanamak/O. nivara and IR 72/O. nivara. Although most of these

107

hybrids showed 12.50-22.58% cells with cryptic structural hybridity, the degree of

abnormality was quit far below than reported for interracial rice hybrids. Therefore,

comparative analysis of the present data and those from interracial hybrids, other causes of

sterility should certainly be occurred as reported by other investigators. However, Stebbins

(1958) speculated that small inversion involving 1-5 genes would be sufficient to cause

sterility. Similarly, many investigators obtained 100% sterility in O. sativa/O. officinalis

hybrids (Li, 1964; Morinaga, 1964; Jena and Khush, 1986) and reported that complete

sterility was due to limited or complete failure of chromosome pairing. However they never

analyzed the pachytene stage and their evidence mostly based on the diakinesis and

metaphase I. But from this present study, the 100% sterility for both pollen and spikelet in

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109

O. sativa/O. officinalis hybrids is not due to failure of chromosome pairing. It was solely

due to desynapsis of chromosome followed by timing imbalance (Shastry and Rao, 1961)

at later stages and a few part by cryptic structural hybridity rather than lack of homology.

Thus, the present study supported the results of Shin and Katayama (1979), Shastry et al.

(1961) and suggests that earlier generalization should be re-examined.

Now summing up the results in most of the intragenomic hybrids, pairing was

essentially normal, although, no significant association between normal pairing at

pachytene stage and latter stages of meiosis was found. Therefore, based on the present

result, sterility could not be accounted for by structural differentiation among AA genome

species. The partial sterility might be due to complex action of other causes such as

genetic, cytoplasmic or genetic-cytoplasmic, and or environmental.

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SUMMARY AND CONCLUSION

In vitro manipulation for the regeneration of intergenomic hybrids, crossability

between species, morphology, pollen and spikelet fertility, and meiotic behavior in three

distant hybrids and parental species were studied during 2001-2002 at IAAS, Rampur. For

this study four Nepalese wild species of rice (O. nivara, O. rufipogon, O. officinalis, and O.

granulata) and nine cultivars of O. sativa. L were used.

In vitro manipulations: embryo rescue, callusing, and in vitro fertilization was

attempted to overcome both pre and post fertilization barrier through manipulating

different media. Among them embryo rescue followed by latest hardening technique was

found potential to regenerate the intergenomic rice hybrids. However, embryo rescue

technique could not be able to overcome the strong post fertilization barrier. Based on the

embryo rescue results, the crossability between O. sativa/O. officinalis was ranged from 0-

2.44%. Strong crossability barrier was found between O. sativa/O. granulata, and hence no

hybrids were obtained. On the basis of direct crosses, the crossability of O. sativa with O.

nivara and O. rufipogon was varied from 7.58%-51.05%. However, on an average

landraces were showed close cross affinity than advanced cultivars.

Comparative study of morphology in parents and hybrids showed that wild traits

were dominant to cultivated forms. Among parents and hybrids, percent mean pollen and

spikelet fertility was varied from 33–78.16 and 0-49.93, and 30-91.46 and0-86.49,

respectively. However, Manshara/O. nivara hybrid set more seed than its female parents

despite of its low pollen fertility. No significant association between percentage of

stainable pollen and spikelet fertility was observed.

The meiotic behavior at different stages of meiosis in intragenomic hybrid was

more or less normal, and the frequency of aberrations were comparable to their respective

111

parents. Only four out of eleven hybrid combinations showed cryptic structural hybridity

including loose pairing, inversion, translocation, deletion, duplication, and

heteromorphocity at pachytene and their occurrence varied from 0-22.58% of the cells

observed. Similarly, intergenomic hybrid (O. sativa/O. officinalis) showed high

abnormality in later stages of meiosis due to desynaptic gene/s present in the genome of O.

officinalis, although pachytene was normal in more than 50% cells. Therefore it was

inferred that the abnormality in these brought about by desynapsis rather than lack of

complete homology between their genome (A and C). Most of the chromosome in these

hybrids appeared as univalents (24) at diakinesis and metaphaseI, and laggards and bridges

in anaphase I.

Based on chiasma study in wild species, O. granulata was found to be more

primitive than other species included in this study. It was also known that all the wild

species of Nepalese wild rices were diploid (2n = 24). At diakinesis and Metaphase I, no

sharp reduction in chiasma frequency was observed except O. sativa/O. officinalis derive

hybrids. The mean frequency of ring bivalent was always high and varied from 10.42-11.25

and had >21-<24 chiasmata/cell in most of the hybrids. Other minor irregularities such as

formation of univalents, quadrivalents, presence of nucleolar bodies, bridges and laggards,

laggards, bridges and fragments and unequal segregation at diakinesis–telophase I were

comparable to their parents. However, cells with bridges + fragments was quite high in

some of the intragenomic hybrids and the percentage of cells having such irregularity

varied from 0.44 – 9.40 and proved the existence of inversion. It was highest in IR64/O.

rufipogon (9.40). On an average, based on all the stages of observation, no sharp structural

differentiation was found between common wild rice and land races of Nepal. Therefore,

partial sterility accounted in hybrids did not associated with chromosomal abnormalities

112

except IR64/O. nivara, IR 64/O.rufipogon, Jethobudo/O. nivara, and O. sativa/O.

officinalis hybrids.

Beside some of the landraces like Pokhreli, Jethobudo, Kalanamak showed high

crossability, F1 fertility and meiotic affinity with O. rufipogon and Manshara with O.

nivara. Not only pairing efficiency, certain stages of meiosis were also improved and

indicated that pairing can be enhanced by genes from wild rice. Therefore, based on

crossability, F1 fertility, and meiotic affinity, Pokhreli, Jethobudo, Kalanamak leads the two

speculations: either these land races descended directly from this perennial taxa or large

amount of genes introgression should be occurred between these landraces and O.

rufipogon. Similar case should exist between O. sativa. cv. Manshara with O. nivara.

Therefore it was found evident that both annual and perennial forms of common wild rice

were the immediate prototype of these and they should originate at the swampy areas of

Pokhara valley and Kapilbastu of Nepal. Not only these evidences, these landraces were

still frequently co-exist with the population of these common wild rice in the farmers’ field.

Thus, it is better to generalize that both annual and perennial forms of common

wild rice, such as found in Nepal, are the prototype of cultivated species. However, this

speculation was mainly generalized based on the three mentioned affinity, further study

will be needed to generalize the results to full extent in the same materials through recent

developed molecular technology.

113

RECOMMENDATION

Thorough study should be conducted based on pachytene stage through traditional,

as well as molecular techniques in more number of cells as far as possible.

The original population of O. nivara and O. rufipogon found in Nepal, particularly

population from the foothills, should be studied in both natural as well as artificial

environment through better sampling strategies. Also include hybrid swarms between

cultivated and these common wild forms as far as possible.

Parasexual hybridization should be carried out to produce the hybrids between O.

sativa and O. granulata.

114

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Appendix 3.1. Nutritional components of different media employed for embryo culture

during study period 2001/002

Amount (mg l –1)

Components MgSO4.7H2O KH2PO4 NaH2PO4.H2O NH4NO3

CaCl2.2H2O (NH4)2. SO4 Ca(NO3 ) 2. 4H2 O KNO3

KCl MgSO4

CaHPO4. 2H2 O Micro components

FeSO4. 7H2 O H3BO3 MnSO4. 4H2O MnSO4. 5H2O MnSO4. H2O ZnSO4. 7H2O Na2MO4. 2H2O

CuSO4. 5H2O CoCl2. 6H2O KI Na2 EDTA. 2H2O Na2 EDTA Sucrose Agar

Vitamins Nicotinic acid Thiamine HCl Pyridoxine HCl Myo-inositol Folic acid Glycine Biotin

Growth regulators NAA Kinetin Casein hydrolysate pH

White’sa 750 - 19 -

- - - 80 - - -

- 1.5 5 - - 3 -

0.01 - 0.75 - - 2% 0.8%

0.05 0.01 0.01 - - 3 -

- - - 5.8

Nistch’sa 185 68 - 720

- - - 950 - - -

27.8 - 25 - - 10 0.25

0.025 0.025 - 37.3 - 2% 0.8%

5 0.5 0.5 100 0.5 2 0.05

- - - 5.8

SRb

296 179.52 - 720

352.8 - - 2730 - - -

27.8 3. - 10 - 2. 0.25

0.025 0.025 0.75 - 37.3 6% 0.9%

1 10 1 100 - - -

0.2 0.2 250 5.8

MSb

370 170 - 1650

440 - - 1900 - - -

27.8 6.2 22.2 - - 8.6 0.25

0.025 0.025 0.83 37.3 - 3% 0.8%

0.5 0.5 0.5 100 - 2 -

- - - 5.8

Bouharmontb

- - - -

- 3000 1000 800 500 120 25

- - - - - - -

- - - - 5% 0.7%

1 10 1 100 - - -

0.2 0.2 -

- 5.9

a [White’s (1953), Nistch’s (1969) ] adopted from Razdan (2001)

b SR (Ko et al.,1983), Murashige and Skoog (1962), and Bouharmont (1991) media, respectively.

145

Appendix 3.2. Composition of the nutrient solution (Karim and Vlamis, 1962) employed to

prepare hardening solution

Nutrients (molar concentration)

KNO3 ” ” … … … … 1 ml./ l.

Ca (NO3)2 ,, ,, … … … … 1 ml./ l.

KH2PO4 ,, ,, … … … … 2 ml./ l.

MgSO4 ,, ,, … … … … 4 ml./ l.

FeSO4 1.112 gm / l … … … … 1 ml./ l.

Johnson’s Micronutrient solution … … … … 1 ml./ l.

KCL … … 1.77 ppm

H3BO3 … … 0.27 ppm

MnSO4. H2O … … 0.274 ppm

ZnSO4. 7 H2O … … 0.131 ppm

CuSO4. 5 H2O … … 0.032 ppm

H2MoO41 … … 0.0096 ppm

1 H2MoO4 was modified by Na2MO4. 2H2O

146

Appendix 4.1. Comparative results of embryo culture on five different sterile media

Hybrid combination No. of No. of No. of No. of tube No. of No. of

embryo embryos embryos not attacked by seedlings seedlings

Culture germinated germinated Micro- died after well grown

organisms germination

Bouharmont (1991)

IR 64 / O. nivara 7 7 - 1 1 5

Manshara / O.officinalis 7 6 - 1 - 6

Kalanamak / O. officinalis 7 5 1 1 - 5

Kalanamak / O. nivara 7 6 1 - - 6

Kalanamak / O. rufipogon 7 5 - 2 1 4

IR 72 / O. granulata 7 4 3 1 3 -

IR 72 7 7 - 2 - 5

O. granulata 7 7 - 1 - 6

SR (Ko, et al., 1983)

IR 64 / O. nivara 7 5 - 2 2 3

Manshara / O.officinalis 7 3 2 1 3 -

Kalanamak / O. officinalis 7 4 3 - 2 2

Kalanamak / O. nivara 7 4 3 - 1 3

Kalanamak / O. rufipogon 7 6 1 - - 6

IR 72 / O. granulata 7 2 5 2 - -

IR 72 7 6 1 2 - 4

O. granulata 7 5 2 - 1 4

White’s (1953)

IR 64 / O. nivara 7 5 2 - 2 3

Appendix continued………..

147

Appendix continues………..

Manshara / O.officinalis 7 4 3 1 2 1

Kalanamak / O. officinalis 7 2 4 1 2 -

Kalanamak / O. nivara 7 4 3 - - 4

Kalanamak / O. rufipogon 7 3 4 - - 4

IR 72 / O. granulata 7 1 6 - 1 -

IR 72 7 7 - 2 2 3

O. granulata 7 7 - 3 1 3

Nistch’s (1969)

IR 64 / O. nivara 7 4 3 1 - 3

Manshara / O.officinalis 7 1 4 2 - 1

Kalanamak / O. officinalis 6 3 4 - 2 1

Kalanamak / O. nivara 7 6 1 - 1 5

Kalanamak / O. rufipogon 7 5 2 - - 5

IR 72 / O. granulata 7 3 4 2 2 -

IR 72 7 7 - 2 1 4

O. granulata 7 6 1 - 2 4

¼ MS (1962)

IR 64 / O. nivara 7 7 - 1 - 6

Manshara / O.officinalis 7 5 2 2 1 2

Kalanamak / O. officinalis 7 4 3 1 - 4

Kalanamak / O. nivara 7 7 - 2 - 5

Kalanamak / O. rufipogon 7 7 - - - 7

IR 72 / O. granulata 7 2 5 - 2 -

IR 72 7 7 - 1 - 6

O. granulata 7 7 1 1 - 5

148

Appendix 4. 2. Comparative results of different attempts employed to establish hybrids

plants in the field from embryo culture regenerated seedlings

Techniques employed

Cross combination

and success rate (%)

Total

seedlings

hardened

Direct

transfer

Bouharmont

(1961) method

Iyer and Govilla

(1964) method

Present

method

IR 64/O. nivara 19 7 4 5 3

Success rate 0.0 25.0 60.0 100.0

Kalanamak/O. officinalis 12 6 2 2 2

Success rate 0.0 0.0 100.0 100.0

Kalanamak/O .nivara 24 6 5 8 5

Success rate 0.0 20.0 50.0 80.0

Kalanamak/O. rufipogon 26 5 7 8 6

Success rate 0.0 0.0 75.0 100.0

Manshara/O. officinalis 11 3 3 3 2

Success rate 0.0 0.0 66.67 100.0

Total 92 27 21 26 18

Mean success rate 0.0 9.0 70.33 96.0

149

Appendix.4.3a. Phenotypic characters relationship between parents and their hybrids

Characters Male parent Female parent Female parent Dominance of

O. nivara (8) Manshara (1) Jhinuwa (2) F1 (1/8) F1(2/8) character of

F1(1/8) F1(2/8)

Apiculus color dark brown Brown weak red black dark brown Black 8 8

200 grain wt. 4.9gm 3.46gm 4.25gm 4.5gm 4.9gm 8 8

Awn density long and - - long and long and 8 8

Fully fully fully

Awn length 4.0cm - - 3.78cm 3.38cm 8 8

Branching culm present(2-3) present(1-2) absent present(2-3) present(2-3) 8 8

Heading days 80 96 102 74 99 8 I

Height 57cm 112.14cm 127.43cm 76.43cm 119.57cm 8 I

Internode color purple lines pale green pale green purple lines purple strips 8 8

Leaf color dark green green green dark green dark green 8 8

with light with light with light

purple tip purple tip purple tip

Panicle exertion just exerted moderately well exerted Just exerted just exerted 8 8

exerted

Panicle length 15.3cm 19cm 23.6 15.65cm 23.46 8 2

Panicle shattering high - - high high 8 8

Panicle type open intermediate intermediate open open 8 8

Seed coat color dark brown dark reddish dark brown dark brown dark brown 8 I

Yellow

Stigma color dark purple white white dark purple dark purple 8 8

BLSC purple green green purple purple 8 8

Culm number >30 25 15 >30 >35 8 8

BLSC = basal leaf sheath coloration

150

Appendix. 4.3b. Phenotypic characters relationship between parents and their hybrids

Characters Male parent Female parent Female parent Dominance of

O. nivara (8) R72(6) IR64(7) F1 (6 / 8) F1(7/8) character of

F1(6 / 8) F1(7/ 8)

Apiculus color dark brown light yellow light golden dark brown dark brown 8 8

200 grain wt. 4.9gm 4.36gm 3.91gm 4.5gm 4.9gm I 8

Awn density long and - minute long and long and 8 8

fully partly fully fully

Awn length 6.01cm - 0.5mm 3.19cm 3.8cm 8 8

Branching culm present(2-3) - - present (2-3) present(2-3) 8 8

flowering days 80 103 104 99 100 I I

Height 57cm 64.42cm 112.28cm 74.42 123cm 8 I

Internode color purple lines whitish green light green purple lines purple line 8 8

Leaf color dark green green green dark green dark green 8 8

with light with purple with purple

purple tip tip tip

Panicle exertion Just exerted just exerted well exerted Just exerted just exerted 8 2

Panicle length 15.3cm 21.14cm 23.84cm 17.36cm 16.95 I I

Panicle shattering high - - high high 8 8

Panicle type open compact intermediate open intermediate 8 2

Seed coat color brown light golden straw yellow dark brown dark brown 8 8

Stigma color dark purple white white dark purple dark purple 8 8

BLSC Purple green green purple purple 8 8

Culm number >25 21 22 >25 >25 8 8

BLSC = basal leaf sheath coloration

151

Appendix.4.3c. Phenotypic characters relationship between parents and their hybrids

Characters Male parent Female parent Female parent Dominance of

O. rufipogon (9) Pokhreli (5) Jethobudo (4) F1 (5/ 9) F1(4/9) character of

F1(1/8) F1(2/8)

Apiculus color black dark purple dark purple black black 9 9

Awn density long and - very short, long and long and

Fully - partly fully fully 9 9

Anther size large small small large large

Anther color deep yellow light yellow light yellow deep yellow deep yellow 9 9

Awn length 4.02. cm - 4.07cm 3.65cm 9 9

Branching culm present(2-3) - 0.48cm present (2-3) present (2-3) 9 9

Heading date 125 112 109 120 118 I I

Culm angle procumbent erect erect procumbent procumbent 9 9 Height 92.8cm 146.42cm 119cm 117.57cm 123cm I I

Internode color purple lines light green pale green purple lines purple lines 9 9

Leaf color dark green green green dark green dark green 9 9

with light purple tip purple tip 9 9

purple tip

Panicle exertion well exerted well exerted well exerted well exerted well exerted I I

Panicle length 19.04cm 22.8cm 23.6 18.78cm 22.61cm I I

Panicle shattering high - - high high 9 9

Panicle type compact open compact intermediate compact compact I I

Seed coat color brown GRB light golden dark brown dark brown 9 9

Stigma color dark purple white white dark purple dark purple 9 9

BLSC purple green green purple purple 9 9

Culm number >40 16 22 >25 .>30 I I

BSLC = basal leaf sheath coloration

GRB = golden reddish brown, I = intermediate, H = heterosis

152

Appendix. 4.3d. Phenotypic characters relationship between parents and their hybrids Characters Male parent Male parent Female parent Dominance of

O. nivara (8) O .rufipogon (9) Kalanamak (3) F1 (3/8) F1 (3/9) character of F1(3/8) F1(3/9) Apiculus color dark brown black black dark brown Black 8 I Awn density long and long and - long and long and 8 9 fully fully fully fully Anther size small large small small large I 9

Anther color yellow deep yellow yellow yellow deep yellow I 9 Awn length 6.01cm 4.02cm - 5.09cm 4.27cm 8 9 Branching of culm present (2-3) present (2-3) - present (2-3) present (2-3) 8 9 Heading days 80 125 109 101 113 I I Culm angle intermediate procumbent intermediate intermediate procumbent I 9 Height 57cm 110cm 132.72cm 82.6cm 122.57cm I I Internode color purple purple pale green purple lines light green 8 9 lines lines with with purple

green dot lines Leaf color dark green dark green green dark green dark green 8 9 with light with light with purple with purple purple tip purple tip tips tip BLSC purple purple green purple purple 8 9 Culm number >25 >40 24 >30 >40 8 9 Panicle exertion just exerted well exerted well exerted just exerted well exerted 8 I

Panicle length 15.3cm 19.04cm 22.73cm 19.05cm 15.7cm I I Panicle shattering high high - high high 8 9 Panicle type open compact-open compact open compact-open 8 9 Seed coat color dark brown brown black dark brown brown 8 9 Stigma color dark purple dark purple white dark purple dark purple 8 9

BLSC = basal leaf sheath coloration

153

Appendix. 4.3e. Phenotypic characters relationship between parents and their hybrids

Characters Male parent Female parent Female parent Dominance of

O. nivara (8) Pokhreli (5) Jethobudo (4) F1 (5/8) F1(4/8) character of

F1(5/8) F1(4/8)

Apiculus color dark brown dark purple dark purple dark brown dark brown 8 8

200 grain wt. 4.9gm 3.45gm 3.1gm 4.0gm 3.98gm 8 8

Awn density long and - very short long and long and 8 8

fully and partly fully fully

Awn length 6.01cm - 0.4cm 5.09cm 5.38cm 8 8

Branching of culm present(2-3) - - present (2-3) present(2-3) 8 8

Heading days 80 112 109 104 96 I I

Height 57cm 146.42cm 119cm 101cm 98.57cm I I

Internode color purple light green pale green purple lines purple lines 8 8

lines

Leaf color dark green green green dark green dark green 8 8

with light with purple

purple tip tips

Panicle exertion just exerted well exerted well exerted just exerted just exerted 8 8

Panicle length 15.3cm 22.8cm 23.6 20.05cm 19.46 I I

Panicle shattering high - - high high 8 8

Panicle type open compact intermediate open open 8 8

Seed coat color dark brown golden reddish light golden dark brown dark brown 8 8

Stigma color dark purple white white dark purple dark purple 8 8

BLSC purple green green purple purple 8 8

Culm number >25 16 22 >25 >25 8 8

BSLC = basal leaf sheath coloration

154

Appendix. 4.3f. Phenotypic characters relationship between parents and their hybrids

Characters Male parent Female parent Dominance of

O. rufipogon (9) IR64 (7) F1(7 / 9) character of

F1(7/9)

Apiculus color black light golden black 9

Awn density long and very short long and 9

fully and partly fully

Anther large small large

Anther color deep yellow light yellow deep yellow 9

Awn length 4.02. cm - 0.5mm 3.65cm

Branching of culm present(2-3) - present (2-3) 9

Heading days 125 104 115 9

Culm angle procumbent erect procumbent 9

Height 92cm 112.28cm 123cm H

Internode color purple lines light green purple lines 9

Leaf color dark green, green dark green,

purple tip purple tip 9

Panicle exertion well exerted well exerted well exerted -

Panicle length 19.04cm 23.84cm 22.61cm 6

Panicle shattering high - high 9

Panicle type compact - open intermediate open 9

Seed coat color dark brown straw yellow dark brown 9

Stigma color dark purple white dark purple 9

BLSC purple green purple 9

Culm number >40 22 >35 9

BLSC = basal leaf sheath coloration

155

Appendix. 4.3g. Phenotypic characters relationship between parents and their hybrids

Characters Male parent Female parent Female parent Dominance of

O. officinalis (10) Kalanamak(3) Manshara (1) F1 (3 / 10) F1 (1 / 10) character of

F1(1/8) F1(2/8)

Apiculus color black black brown weak red black black 10 10

Awn density short and - - short and short and 10 10

partly partly partly

Anther color reddish grey light yellow light yellow light yellow light yellow 10 10

Awn length 0.82. cm - - 0.38cm 0.37cm 10 10

Branching of culm present(3-4) - - present (2-3) present (2-3) 10 10

Heading days 110 109 96 107 107 I I

Height 107.14cm 132.72cm 112.14cm 130.2 cm 123cm H 10

Internode color light green with pale green pale green dark dark 10 10

dense dark green with green with green with I I

dots green dots purple lines purple lines

Leaf color dark green green green dark green dark green 10 10

Panicle length 27.16cm 22.73cm 19.00cm 19.67cm 22.61cm 10 10

Panicle shattering high - - high high 10 10

Panicle type open compact intermediate open open 10 10

See d coat color brownish black dark reddish - - 10 10

grey yellow

Stigma color dark purple white white dark purple dark purple 10 10

with white tip

Under ground stem present - - present present 10 10

BLSC brownish green green green BG BG 10 10

Culm number >35 24 25 >35 >35 10 10

BG = brownish green

156

Appendix. 4.3h. Phenotypic characters relationship between parents and their hybrids

Characters Male parent Female parent Female parent Dominance of

O. officinalis (10) Pokhreli (5) Jhinuwa (2) F1 (5 / 10) F1 (2 / 10) character of

F1(1/8) F1(2/8)

Apiculus color black dark purple black light red black 10 10

Awn density short and - - short and short and 10 10

partly partly partly

Anther color light yellow yellow yellow light yellow light yellow 10 10

Awn length 0.82. cm - - 0.32cm 0.38cm 10 10

Branching of culm present(3-4) - - present (2-3) present (2-4) 10 10

Heading days 110 112 102 108 105 I -

Height 107.14cm 146.42cm 127.43cm 130.2 cm 123cm I I

Internode color light green with light green pale green light light green 10 10

dense dark green with dark green with with

dots green dots green dots purple lines

Leaf color dark green green green dark green dark green 10 10

Culm number >35 16 15 >40 >35 10 10

Panicle length 27.16cm 22.8cm 23.6cm 22.98cm 24.76cm I I

Panicle shattering high - - high high 10 10

Panicle type open compact intermediate open open 10 10

Stigma color dark purple white white dark purple dark purple 10 10

with white tip

Under ground stem present - - present present 10 10

BLSC BG green green BG BG 10 10

Culm number >35 16 15 >40 >35 10 10

BLSC = basal leaf sheath color, BG = brownish green

157

BIOGRAPHICAL SKETCH

The author was born in 1974, August 1 at Wolane V.D.C, Panchthar, East Nepal.

He got his primary education partly from Shree Wolane Primary School and partly from

Shree Janajyoti Primary School, Damak- 7, nearby his village. He joined his secondary

level of education at Shree Manohar Janta M.V., Madhumalla, Morang, but he completed

S.L.C. with first division in 1990 from Shree Radhika M.V., Uralabari, Morang, Nepal.

He joined IAAS at Rampur, Chitwan and graduated in first division with plant

breeding as major subject in 1999. Immediately the author enrolled in 1999 at the IAAS,

Rampur to pursue his Master of Science in Agriculture (Plant Breeding) as general student.

During MSc he was awarded research assistantship for two years under the CDR/USAID

Wheat breeding and Rockefeller Foundation funded Rice Breeding Projects, and he gained

valuable knowledge in many aspects of genetics and plant breeding. In addition, he was

also actively engaged in volunteer teaching for undergraduate and graduate student on his

subject matter. He also presented some experimental findings in a scientific journal. Now

he is very interested in molecular biotechnology and cytology, and his future research

dream is on apomictic breeding in rice.

Raj Kumar Niroula