4. RESULTS AND DISCUSSIONshodhganga.inflibnet.ac.in/bitstream/10603/10176/10/10... · 2015. 12....

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4. RESULTS AND DISCUSSION

Doubled haploidy (DH) breeding is the fastest route to get instant

homozygosity. When conventional breeding procedures take atleast six

generations to achieve homozygous condition, it takes only one generation. The

relevance of DHs to plant breeding has increased markedly in recent years owing

to the development of protocols for 25 species (Maluszynski et al. 2003) and

doubled haploid methodologies have now been applied to over 250 species.

Doubled haploids can be produced in vivo or in vitro. Haploid embryos are

produced in vivo by parthenogenesis, pseudogamy or chromosome elimination

after wide crossing. The haploid embryo is rescued, cultured and chromosome

doubling produces doubled haploid. The in vitro methods include gynogenesis

(ovary and flower culture) and androgenesis (anther and microspore culture).

Chromosome elimination after wide crossing is reported to be used mostly

in cereals. In barley, haploids can be produced by wide crossing with the related

species Hordeum bulbosum. Fertilization is effected, but during the early stages

of seed development the H. bulbosum chromosomes are eliminated leaving a

haploid embryo (Kasha and Kao 1970).

In oat, haploids have been obtained by crossing oat with maize (Riera-

Lizarazu et al. 1996 and Sidhu et al. 2006).

In wheat, Laurie and Bennett (1987) had given wheat x maize system of

haploid production that was genotype non-specific because of the insensitivity of

maize (2n= 20) pollen to the action of Kr1 and Kr2 genes thereby rendering the

chromosome elimination technique more efficient and practical. More recently,

Chaudhary and his associates (2005 and 2007) and Chaudhary (2008 a and b

and 2010) have reported wheat x I. cylindrica approach for obtaining high

frequency of haploid and doubled haploids in wheat, triticale and derivatives of

triticale x wheat. I. cylindrica (2n= 20) also known has cogon grass or Kunai

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grass, is a species of grass placed in the subfamily panicoideae and tribe

andropogoneae. It is a perennial rhizomatous grass native to East and Southeast

Asia, India, Australia and eastern and southern Africa. It coincides well with all

rabi cereals for flowering. As it is naturally available during winters in the bunds

and surroundings of wheat fields, separate raising of polyhouse is not needed

unlike maize. Likewise wheat, there can be a scope of induction of haploids in

other cereals viz., oat, barley, maize and rice following the I. cylindrica- mediated

system of chromosome elimination approach.

Despite immense importance and advantages of doubled haploidy

breeding, its extensive adoption in crop improvement programmes is lacking.

This may be ascribed to the reason that there is high mortality rate of

regenerated haploid plants during colchicine application for chromosome

doubling. This limitation necessitates and demands to look into other alternative

of colchicine application which can avoid the mortality of the haploids.

So, keeping in view the efficient and effective nature of I. cylindrica as a

pollen source for haploid induction (DHs) in wheat, the feasible application in

other cereals and to find out possible alternative ways of colchicine application

that might minimize the mortality rate of plants during colchicine application, the

present research programme was undertaken. The findings are discussed under

the following heads:

4.1 Induction of haploids through wide hybridization

4.1.1 Wheat x Imperata cylindrica

4.1.2 Rice x Imperata cylindrica

4.1.3 Maize x Imperata cylindrica

4.1.4 Barley x Imperata cylindrica

4.1.5 Oat x Imperata cylindrica

4.2 Enhancement of doubled haploid production

4.2.1 In vivo colchicine application

4.2.2 In vitro colchicine application

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4.1 Induction of haploids through wide hybridization

In cereals, interspecific and intergeneric hybridization (wide crosses) is

exercised so as to yield karyotypically stable hybrid plants that have been used

as starting point to widen the genetic base of a crop and uniparental genome

elimination in karyotypically instable hybrids have been utilized for cereal haploid

production.

4.1.1 Wheat x Imperata cylindrica

The performance of different wheat varieties in respect to various haploid

induction parameters are presented in Tables 4.1 and 4.2. Two spring wheat

genotypes KWS-29 and C-306 and one durum wheat genotype HW-896 were

used in the study. Durum wheat was also crossed with maize so as to compare

the performance of two pollen sources.

4.1.1.1 Percent seed formation

The performance of KWS-29 and C-306 were at par (80% and 82%

respectively) for percent seed formation (Figure 4.1a). In case of durum wheat,

the percent seed formation was comparatively lower (67.83%) but when it was

crossed with maize, the performance of percent seed formation was even lesser

(40.80%) than the I. cylindrica – mediated system. So, I. cylindrica was proved to

be better pollen source than maize in case of durum wheat (Figure 4.1b). The

results were similar to the findings of Chaudhary et al. (2010).

4.1.1.2 Percent embryo formation

In case of percent embryo formation also, I. cylindrica (25%) outperformed

the maize (19.61 %) in case of durum wheat (Table 4.2). The performance of

other two spring wheat varieties was normal (Table 4.1).

4.1.1.3 Percent regenerated plants

Similarly, for percent plant regeneration also, I. cylindrica (23.07%) gave

better results than the maize (10%) in durum wheat (Table 4.2).

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Figure 4.1a Performance of different wheat genotypes in respect of various haploid induction parameters after hybridization with Imperata cylindrica

Figure 4.1b Performance of durum wheat in respect of various haploid induction parameters after hybridization with Imperata cylindrica / maize

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Table 4.1 Performance of different wheat genotypes in respect of various haploid induction parameters after hybridization with Imperata cylindrica

Parameters Crosses

KWS 29 x I. cylindrica C-306 x I. cylindrica

Total florets pollinated 300 350

Total seed formation 240 287

Percent Seed Formation 80.00 82.00

Total embryo formation 123 150

Percent embryo formation 51.25 52.26

Regenerated plants 60 77

Percent regenerated plants 48.78 51.33

Table 4.2 Performance of a durum wheat genotype in respect of various haploid induction parameters after hybridization with Imperata cylindrica and maize

Parameters Crosses

Durum wheat (HW-896) x I. cylindrica

Durum wheat (HW-896) x Maize





Percent embryo formation 25 19.61


Percent regenerated plants 23.07 10.00

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As per the earlier studies executed by Chaudhary et al. (2005) and

Chaudhary (2008 a and b), the I. cylindrica has been identified as the superior

pollen source for generating significantly higher frequency of pseudoseeds,

haploid embryos and their regeneration in comparison to maize. Likewise, I.

cylindrica has also shown superiority over the maize in respect of induction of

higher frequency of haploids in durum wheat (AABB) in the present investigation

but the performance is significantly lesser than the hexaploid wheat (AABBDD). It

indicated that the D genome plays a pivotal role in deciding the recovery of

haploids in wheat.

4.1.2 Rice x Imperata cylindrica

Data in respect of various haploid induction parameters in rice when

hybridized with I. cylindrica are presented in Tables 4.3, 4.4, 4.5 and 4.6.


Almost all the genotypes of rice produced some seeds upon hybridization

with I. cylindrica and application of all the dosages of 2, 4-D except in HPR-1068

and Sarat (Tables 4.3, 4.4, 4.5 and 4.6). The cross Bhrigu x I. cylindrica

generated highest seed set viz., 17.21%, 18.36%, 21.23% and 20.0% with all the

dosages of 2, 4-D viz., 30, 50, 70 and 100ppm, respectively. Of all the four

dosages of 2, 4-D applied, 70 ppm was found to be the most stable dose for

seed set (Figure 4.2 a and b).


In the genotypes Bhrigu, Kunjan, Deku, Amdeng and Kabder when

hybridized with I. cylindrica, embryo like structure were obtained (Tables 4.3, 4.4,

4.5 and 4.6) but no green plant could be generated from these embryo like

structures. Some genotypes did not set seed which indicated that seed setting in

rice might be genotype-specific.

No effort in rice has ever been made anywhere in India and abroad for

doubled haploid production through chromosome elimination mediated approach

especially following maize and I.e cylindrica pollen sources. Hence, the present

investigation is the first innovative effort in this direction.

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Table 4.3 Performance of different rice genotypes in respect of various haploid induction parameters when hybridized with Imperata cylindrica (30 ppm)

Genotypes

Parameters

Total florets pollinated

Total seed formation

Seed

formation (%)

Total embryo formation

Embryo

formation (%)

Regenerated

plants

Regeneration (%)

Bhrigu 215 37 17.21 10 27.03 0 0

Varun 203 4 1.97 0 0 0 0

Kunjan 223 19 8.52 5 26.32 0 0

HPR-1068 206 0 0.00 0 0 0 0

HPR-2143. 204 10 4.90 0 0 0 0

HPR-1156 194 7 3.61 0 0 0 0

Deku 207 15 7.25 3 20.00 0 0

Amdeng 212 12 5.66 2 16.67 0 0

Sarat 197 0 0.00 0 0 0 0

Kabder 214 13 6.07 3 23.08 0 0

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Table 4.4 Performance of different rice genotypes in respect of various haploid induction parameters when hybridized with Imperata cylindrica (50 ppm)

Genotypes

Parameters



Seed formation (%)


Embryo formation (%)

Regenerated

plants

Regeneration

(%)

Bhrigu 207 38 18.36 9 23.68 0 0

Varun 200 15 7.50 0 0 0 0

Kunjan 209 17 8.13 2 11.76 0 0

HPR-1068 202 0 0 0 0 0 0

HPR-2143 204 9 4.41 0 0 0 0

HPR-1156 194 7 3.61 0 0 0 0

Deku 202 12 5.94 2 16.67 0 0

Amdeng 208 15 7.21 3 20.00 0 0

Sarat 195 0 0 0 0 0 0

Kabder 206 12 5.83 2 16.67 0 0

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Table 4.5 Performance of different rice genotypes in respect of various haploid induction parameters after hybridization with Imperata cylindrica (70 ppm)

Genotypes

Parameters



Seed formation (%)



Regenerated plants

Regeneration (%)

Bhrigu 212 45 21.23 13 28.89 0 0

Varun 194 12 6.19 0 0 0 0

Kunjan 208 32 15.38 9 28.13 0 0

HPR-1068 196 0 0 0 0 0 0

HPR-2143 204 7 3.43 0 0 0 0

HPR-1156 198 8 4.04 0 0 0 0

Deku 205 17 8.29 4 23.53 0 0

Amdeng 206 12 5.83 3 25.00 0 0

Sarat 187 0 0 0 0 0 0

Kabder 212 16 7.55 4 25.00 0 0

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Table 4.6 Performance of different rice genotypes in respect of various haploid induction parameters after hybridization with Imperata cylindrica (100 ppm)

Genotypes

Parameters



Seed formation (%)



Regenerated plants

Regeneration (%)

Bhrigu 175 35 20.00 8 22.86 0 0

Varun 200 13 6.50 0 0 0 0

Kunjan 198 27 13.64 7 25.93 0 0

HPR-1068 209 0 0 0 0 0 0

HPR-2143 196 9 4.59 0 0 0 0

HPR-1156 198 8 4.04 0 0 0 0

Deku 203 24 11.82 3 12.50 0 0

Amdeng 204 15 7.35 3 20.00 0 0

Sarat 182 0 0 0 0 0 0

Kabder 209 13 6.22 2 15.38 0 0

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a b c

d f

g h i

h. Cross-section of ovule (rice x I. cylindrica)

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Figure 4.2a Performance of different rice genotypes in respect of percent seed formation after hybridization with Imperata cylindrica

Figure 4.2b Performance of different rice genotypes in respect of percent embryo formation after hybridization with Imperata cylindrica

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4.1.2.3 Cross-sectional study of fertilized ovules

Cross sectional study of fertilized ovules preserved at 24, 48, 72 and 96

hours after pollination was also performed, so as to confirm the fertilization. The

result (Plate 4) indicated that fertilization had occurred in the cross. Torpedo

shaped embryos could be observed in the cross-sectional slide photographs. The

reasons for failure of development of embryo into green plants may be due to

lack of proper media, improper harvesting time and some other unknown

reasons.

Frequency of embryo formation was also worked out and although

crossing rice with maize was not part of the research work, it was carried

additionally so as to get more information for comparison with the performance of

I. cylindrica. The perusal of data in the Table 4.7 showed that in the beginning, at

three days after pollination, the frequency of embryo formation in maize was

higher (27.27%) but later, at five days after pollination the performance of I.

cylindrica (25%) was much better than that of maize (20%). The reason for this

fact is also not known. So, more work in this direction is also needed.

Table 4.7 Performance of rice in respect of embryo formation frequency after hybridization with Imperata cylindrica and maize at various intervals after pollination

Days after pollination

Parameters 1 3 5

Maize I. cylindrica Maize I. cylindrica Maize I. cylindrica

No. of ovules examined

18 15 11 9 5 8

Embryo no. 0 0 3 2 1 2

Frequency of embryo formation

0 0 27.27 22.22 20.00 25.00

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Plate 4. Wide hybridization in rice with I. cylindrica

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Although seed set and embryo formation in different rice varieties/

genotypes was feasible with I. cylindrica yet no green plantlet could be seen

regenerating out of the small embryo like structures cultured in the medium.

Hence there is still need to explore the possibilities of generating healthy and

large size embryos by manipulating the auxin (2, 4- D and others) application

dose and the harvesting period of the fertilized ovules. Besides, there is a need

to manipulate the culture medium so as to recover green plantlets from the

embryos.

4.1.3 Maize x Imperata cylindrica

Performance of different varieties of maize in respect of various haploid

induction parameters when hybridized with I. cylindrica are tabulated in tables

4.8, 4.9 and Plate 5. Both the varieties viz., Early Composite and Bajaura Makka

could not generate any seed or embryo upon hybridization with I. cylindrica. The

manipulation of the 2, 4-D application has also not worked. Chromosome

elimination mediated approach of DH breeding has never been tried anywhere in

the world in maize. So, there is no any report available on this aspect.

Table 4.8 Performance of maize varieties in respect of various haploid induction parameters after hybridization with Imperata cylindrica (100 ppm)

Parameters Varieties

Early Composite Bajaura Makka

Total cobs pollinated 25 25


Percent Seed Formation 0 0


Percent embryo formation 0 0


Percent regenerated plants 0 0

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Plate 5. Wide hybridization in maize with I. cylindrica

c

d e

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Table 4.9 Performance of maize varieties in respect of various haploid induction parameters after hybridization with Imperata cylindrica (150 ppm)

Parameters Varieties

Early Composite Bajaura Makka

Total cobs pollinated 25 25


Percent Seed Formation 0 0





Although many manipulations in the process of hybridization of maize with

I. cylindrica were exercised yet it seems quite cumbersome to identify the exact

stage and place/ location of pollination. Keeping in view the short style length of

the pollen tube of I. cylindrica in comparison to the style of the maize, excellent

expertise is required to get the ovule of maize fertilized. Attempt has also been

made to pollinate the female cob by removing the cob leaves but still more

concerted efforts are needed to make it fruitful.

4.1.4 Barley x Imperata cylindrica

The results obtained and data in respect of various haploid induction

parameters in barley when hybridized with I. cylindrica are presented in Plate 6

and tables 4.10, 4.11, 4.12 and 4.13.

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All the five genotypes of barley viz., Purthy, Tindi, Karpat, Dolma and

Barot exhibited proper seed set upon hybridization with I. cylindrica (Tables 4.10,

4.11, 4.12 and 4.13) in all the 2,4-D doses applied (50 ppm, 100 ppm, 150 ppm

and 200 ppm). The performance of all the genotypes of barley were at par in

respect of percent seed formation trait. However, Purthy showed highest seed

set of 73.33 percent at 50 ppm, 2,4-D dose applied (Table 4.10). Amongst all the

2,4-D doses used, lowest dose (50 ppm) was found to be the most effective for

percent seed set trait (Figure 4.3 a).

Kasha and Kao (1970) reported average seed set of 51.5 percent in nine

different lines of Hordeum vulgare when hybridized with Hordeum bulbosum.

They also reported 100 percent seed development in some spikes that were

pollinated two days after emasculation. There is no report currently available on

these aspects. Hence, the present investigation is a unique and innovative

attempt to explore the possibilities of development of haploids through I.

cylindrica- mediated system of chromosome elimination approach.


All the five genotypes of barley produced some embryo like structures

(ELS) upon hybridization with I. cylindrica in all the doses of 2, 4-D applied (50

ppm, 100 ppm, 150 ppm and 200 ppm). But, on culturing such ELS, green plants

could not be obtained. The performance of all the five genotypes of barley in

respect of percent embryo formation were at par in all the doses of 2, 4-D applied

(Figure 4.3 b). However, lower dose (50 ppm) appeared to be stable and better

for percent embryo formation. The failure to obtain green plant from ELS might

be due to improper media, harvesting time and faulty handling of the embryos.

Hence further detailed study on these aspects is needed in future. Kasha and

Kao (1970) reported 8.4% to 18.4% of embryo regenerating into plants in barley

following bulbosum technique.

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Table 4.10 Performance of different barley genotypes in respect of various haploid induction parameters after hybridization with Imperata cylindrica (50 ppm)

Genotypes

Parameters



Seed formation (%)


Embryo formation(%)

Regenerated plants

Regeneration (%)

Purthy 300 220 73.33 123 55.91 0 0

Tindi 270 186 68.89 97 52.15 0 0

Karpat 350 252 72.00 138 54.76 0 0

Dolma 312 225 72.12 130 57.78 0 0

Barot 325 235 72.31 127 54.04 0 0


Genotypes

Parameters



Seed formation (%)


Embryo formation(%)

Regenerated plants

Regeneration (%)

Purthy 306 210 68.63 116 55.24 0 0

Tindi 286 182 63.64 98 53.85 0 0

Karpa 350 240 68.57 124 51.67 0 0

Dolma 300 200 66.67 99 49.50 0 0

Barot 305 202 66.23 96 47.52 0 0

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Genotypes

Parameters



Seed formation (%)



Regenerated plants

Regeneration (%)

Purthy 310 150 48.39 80 53.33 0 0

Tindi 270 157 58.15 87 55.41 0 0

Karpat 300 187 62.33 100 53.48 0 0

Dolma 304 128 42.11 77 60.16 0 0

Barot 300 137 45.67 79 57.66 0 0


Genotypes

Parameters



Seed formation (%)


Embryo formation(%)

Regenerated plants

Regeneration (%)

Purthy 340 210 61.76 110 52.38 0 0

Tindi 300 157 52.33 80 50.96 0 0

Karpat 320 170 53.13 90 52.94 0 0

Dolma 300 150 50.00 86 57.33 0 0

Barot 310 160 51.61 87 54.38 0 0

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Figure 4.3a Performance of different barley genotypes in respect of percent seed formation after hybridization with Imperata cylindrica

Figure 4.3b Performance of different barley genotypes in respect of percent embryo formation after hybridization with Imperata cylindrica

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4.1.4.3 Cross-sectional study of fertilized ovules

The results of cross-sectional study of ovule of barley x I. cylindrica are

shown in Plate 6, which clearly indicated that fertilization had occurred and

embryo was formed. The reasons for failure to obtain regenerated green plants

by embryo culture might be due to faulty media, harvesting time and other

unknown reasons.

In barley also, crosses were attempted using maize as a pollen source for

comparison of its performance with I. cylindrica. The data of table 4.14 indicated

that in case of barley, maize (22%) performed better than the I. cylindrica

(18.75%), in respect of embryo formation frequency at five days after pollination.

Table 4.14 Performance of barley in respect of embryo formation frequency when hybridized with maize and I. cylindrica at various intervals after pollination

Parameters

Days after pollination

1 3 5

Maize I. cylindrica Maize I. cylindrica Maize I. cylindrica

No. of ovules examined

24 21 15 11 9 16

Embryo no. 0 0 0 0 2 3

No. of cells in embryo

- - - - 70 60

Frequency of embryo formation

0 0 0 0 22 18.75

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Plate 6. Wide hybridization in barley with I. cylindrica

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In all, it can be concluded that there is possibility of obtaining haploids in

barley through chromosome elimination mediated approach utilizing I. cylindrica

and maize as pollen sources but certain manipulations in media, harvesting time

of the fertilized ovules and use of auxin during hybridization are needed to obtain

fruitful results.

4.1.5 Oat x Imperata cylindrica

The data pertaining to various haploid induction parameters in oat when

hybridized with I. cylindrica are presented in tables 4.15, 4.16, 4.17 and Plate 7.


Both the genotypes viz., Palampur-1 and Naked oat produced seed upon

hybridization with I. cylindrica (Tables 4.15 to 4.17). Both the genotypes

performed at par in all the doses of 2, 4-D applied (50 ppm, 100 ppm and 150

ppm). However, 100 ppm doses of 2, 4-D was comparatively better (Figure 4.4).

Several workers have reported development of haploids by crossing oat with

maize (Riera Lizaraju et al. 1996 and Sidhu et al. 2006). However, in oat the

plant recovery frequency reported was very less, that is 1-2 percent of the florets

pollinated with maize.


No embryo was obtained in both the genotypes with all the 2, 4-D dosages

applied (Tables 4.15 to 4.17). Although maize has been utilized by a few

researchers in India and abroad for DH production in oat, yet the results obtained

are not satisfactory. I. cylindrica has never been tried as a pollen source to

generate haploids in oats. So, the present investigation is first report in this

regard. More concerted efforts are needed to make the hybridization of oat with I.

cylindrica successful. There is a need to explore the use of auxin other than 2, 4-

D during hybridization so as to retain more longevity of the embryos if formed

after hybridization.

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Plate 7. Wide Hybridization in oats with I. cylindrica

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Figure 4.4 Performance of different oat varieties in respect of percent seed formation when hybridized with Imperata cylindrica

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Table 4. 15 Performance of different oat genotypes in respect of various haploid induction parameters after hybridization with Imperata cylindrica (50 ppm)

Parameters

Genotypes

Palampur-1 Naked oat








Table 4.16 Performance of different oat genotypes in respect of various haploid induction parameters after hybridization with Imperata cylindrica (100 ppm)

Parameters

Genotypes




Percent Seed Formation 52 50.82





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Table 4.17 Performance of different oat genotypes in respect of various haploid induction parameters after hybridization with Imperata cylindrica (150 ppm)

Parameters

Genotypes




Percent Seed Formation 51.88 50





In all, it can be concluded that doubled haploidy breeding is becoming

important in crop breeding programme. It is the only fastest breeding technique

for the development of superior homozygous cultivars within shortest span of

time. Mostly, two methods viz., androgenesis and chromosome elimination

approach like bulbosum technique, wheat x maize system and wheat x I.

cylindrica system have been employed to obtain doubled haploids wheat. Anther

culture (androgenesis) has been attempted in many species but the frequency of

recovery of doubled haploids is very low compared to the large number of pollen

per floret. Besides, it is genotype specific which hampers its application in many

important crops like wheat, maize, barley, and oat. Therefore, chromosome

elimination approach has widened the horizon and enhanced the scope of

acceleration of crop improvement endevours.

The chromosome elimination approach like bulbosum technique, wheat x

maize and wheat x I. cylindrica are successfully used in cereal crops like barley

and wheat but in leading cereals like rice and maize, it is yet to be explored in a

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systematic manner so as to obtain fruitful results. As per the recent report of

Chaudhary et al. (2005) and Chaudhary (2008a and b), I. cylindrica- mediated

system of haploid production in wheat was more efficient and superior than

maize- mediated system. Kishore et al. (2010) also reported striking success

results in respect of induction of haploids in spring and winter wheat x Himalayan

rye derivatives following I. cylindrica- mediated system where maize- mediated

system failed.

The overall wide hybridization results obtained in the present investigation

in respect of different cereals like wheat, rice, maize, barley and oat with I.

cylindrica indicated that I. cylindrica perform well as a pollen source for obtaining

high frequency of haploids in bread wheat and durum wheat as reported by

earlier workers viz., Chaudhary et al. 2005 and Chaudhary (2008 a and b). There

is possibility of producing haploids in other cereals like maize, rice, barley and

oat using this newly emerged system but in order to obtain more fruitful results,

more exhaustive research efforts associated with modification of culture media,

standardization of harvesting time and manipulation of auxin application during

hybridization are needed to be exercised.

4.2 Enhancement of doubled haploid production

Doubled haploids are produced by employing various methods like anther

culture and chromosome elimination techniques viz., bulbosum technique, wheat

x maize system and wheat x I. cylindrica system. But, in all these methods higher

mortality rate of haploid plants due to lethal action of colchicine hampers the

appreciable recovery of doubled haploids (DHs). Keeping in view the impact of

this hurdle, various modifications/manipulations in colchicine doses and

application methods has been executed, which provided an opportunity to

eliminate the constraint of lethality to some extent and enhance the efficiency of

DHs production. The manipulation of colchicine was executed at both the in vivo

(Plate 8) and in vitro level.

4.2.1 In vivo colchicine application

Data pertaining to effect of different concentration of colchicine applied in

vivo and without 2, 4-D at various interval of pollination with I. cylindrica in wheat

on various haploid induction parameters were recorded and arranged in two way

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table and student’s t-test was performed to check the significance of

concentration of colchicine over treatments and vice-versa on various haploid

induction parameters.

4.2.1.1 Percent pseudo seed formation

The results analysed following student’s t-test with control (without

colchicine) indicated that almost all the concentration of colchicine (100 ppm to

10,000 ppm) applied exhibited negative significant effect over all the treatments

on pseudo seed formation (Table 4.18) with exception in 200 ppm in T3, T4 and

T8, 300 ppm and 500 to 700 ppm in T7 where it was at par with the control.

Similarly, all the treatments (T1 to T12) showed negative significant effect over all

the concentration on pseudo seed formation (Table 4.19) with exception in 1500

ppm and 2000 ppm in T2, 100 ppm and 200 ppm in T3, 200 ppm in T4, 300 ppm

and 500 to 700 ppm in T7, 200 ppm and 500 ppm in T8, 300 ppm and 2000 ppm

in T9 and 500 ppm in T12 where it was at par with the control.

Perusal of Figure 4.5 illustrated that the percent pseudo seed formation

was negatively affected by the application of colchicine (different doses and

application method) but the effects were random. Although the seeds were

formed with all the concentration of colchicine in all the 12 treatmetns yet the

seed setting was lower in all concentration and treatments as compared to the

control. Such attempt has also been made by Sood et al. (2003) in wheat by

utilizing maize as a pollen source.


The student’s t-test with control showed that almost all the concentration

(100 ppm to 10,000 ppm) of colchicine over all the treatments exhibited negative

significant effect on percent embryo formation trait (Table 4.20) with few

exception in 100 ppm in T1 to T4, 300 ppm inT3, 400 ppm in T2 and T3, and 600

ppm in T3, T5 and T8 where it was at par with control. Similarly, all the treatments

(T1 to T12) exhibited negative significant effect over all the concentrations on

percent embryo formation (Table 4.21) with exceptions in 100 to 400 ppm

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Table 4.18 Effect of different concentration of colchicine applied in vivo with and without 2, 4-D at various intervals of pollination with I. cylindrica in wheat on pseudo seed formation

Treat- ments

Colchicine Concentration (ppm)

100 200 300 400 500 600 700 800 900 1000 1200 1500 2000 3000 5000 7000 10000

T1 75.00 67.24 70.59 73.53 74.19 70.59 78.16 78.77 37.21 69.57 75.30 76.54 76.62 38.85 61.49 47.96 65.31

T2 82.69 61.76 74.19 82.86 38.98 71.88 80.12 80.14 78.57 68.99 67.86 87.82 86.11 80.92 51.79 81.82 81.91

T3 86.67 92.31# 83.87 78.33 72.22 72.22 83.77 70.83 62.50 58.24 53.68 78.26 72.60 68.42 71.65 60.20 79.31

T4 80.21 90.00# 74.63 77.17 73.53 75.00 80.90 41.77 50.67 79.65 82.50 76.35 68.00 74.32 53.52 83.33 51.16

T5 81.25 71.00 80.00 77.08 79.49 67.65 82.46 64.61 74.43 68.45 66.87 77.34 79.27 66.00 65.58 57.78 40.82

T6 72.60 81.82 75.00 78.13 85.71 67.14 76.79 74.36 50.00 63.69 75.00 79.21 78.67 75.44 56.55 46.77 58.95

T7 82.26 72.73 90.82# 73.53 90.63# 90.63# 91.07# 66.84 59.29 68.90 82.47 70.69 78.68 78.41 60.63 80.43 66.67

T8 78.13 89.39# 86.32 70.59 88.89 75.53 83.74 50.59 71.43 75.00 59.20 74.34 86.18 76.51 76.32 67.39 64.66

T9 73.53 71.74 86.67 82.86 70.59 80.56 65.91 54.76 65.43 68.18 82.89 82.10 86.61 41.18 68.90 80.00 74.19

T10 68.75 80.56 80.00 68.75 67.65 78.57 58.72 79.63 57.06 60.34 70.22 70.83 75.95 64.29 36.31 71.28 72.34

T11 72.39 83.33 85.29 80.00 71.43 70.59 73.17 74.56 63.07 81.25 70.12 84.97 73.91 70.51 58.86 48.91 63.33

T12 81.25 83.82 73.53 80.88 90.00 70.31 72.29 85.96 74.12 75.58 81.48 78.05 77.95 67.09 67.61 53.23 50.00

Control 91.95 91.95 91.95 91.95 91.95 91.95 91.95 91.95 91.95 91.95 91.95 91.95 91.95 91.95 91.95 91.95 91.95

Mean 78.98 79.82 80.99 78.13 76.56 75.59 78.39 70.37 64.29 71.52 73.81 79.11 79.42 68.76 63.17 67.00 66.20

SE ± 1.79 2.78 1.95 1.67 3.96 2.21 2.58 4.00 3.94 2.54 2.95 1.73 1.84 4.08 3.71 4.31 3.91

P≤0.05; # = Non significant; No symbol= Significantly negative

72

Table 4.19 Effect of different concentration of colchicine applied in vivo with and without 2, 4-D at various intervals of pollination with Imperata cylindrica in wheat on pseudo seed formation

Colchicine oncentration (ppm)

Treatments

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12

100 75.00 82.69 86.67# 80.21 81.25 72.60 82.26 78.13 73.53 68.75 72.39 81.25

200 67.24 61.76 92.31# 90.00# 71.00 81.82 72.73 89.39# 71.74 80.56 83.33 83.82

300 70.59 74.19 83.87 74.63 80.00 75.00 90.82# 86.32 86.67# 80.00 85.29 73.53

400 73.53 82.86 78.33 77.17 77.08 78.13 73.53 70.59 82.86 68.75 80.00 80.88

500 74.19 38.98 72.22 73.53 79.49 85.71 90.63# 88.89# 70.59 67.65 71.43 90.00#

600 70.59 71.88 72.22 75.00 67.65 67.14 90.63# 73.53 80.56 78.57 70.59 70.31

700 78.16 80.12 83.77 80.9 82.46 76.79 91.07# 83.75 65.91 58.72 73.17 72.29

800 78.77 80.14 75.83 41.77 64.61 74.36 66.84 50.59 54.76 79.63 74.56 85.96

900 37.21 78.57 62.50 50.67 74.43 50.00 59.29 71.43 65.43 57.06 63.07 74.12

1000 69.57 68.99 58.24 79.65 68.45 63.69 68.90 75.00 68.18 60.34 81.25 75.58

1200 75.30 67.86 53.68 82.50 66.87 75.00 82.47 59.20 82.89 70.22 70.12 81.48

1500 76.54 87.82# 78.26 76.35 77.34 79.21 70.69 74.34 82.10 70.83 84.97 78.05

2000 76.62 86.11# 72.60 68.00 79.27 78.67 78.68 86.18 89.61# 75.95 73.91 77.95

3000 38.85 80.92 68.42 74.32 66.00 75.44 78.41 76.51 41.18 64.29 70.51 67.09

5000 61.49 51.79 71.65 53.52 65.58 56.55 60.63 76.32 68.90 36.31 58.86 67.61

7000 47.96 81.82 60.20 83.33 57.78 46.77 80.43 67.39 80.00 71.28 48.91 53.23

10000 65.31 81.91 79.31 51.16 40.82 58.95 66.67 64.66 74.19 72.34 63.33 50.00

control 91.95 91.95 91.95 91.95 91.95 91.95 91.95 91.95 91.95 91.95 91.95 91.95

Mean 68.27 75.02 74.56 72.48 71.78 71.54 77.59 75.79 73.95 69.62 73.20 75.28

SE ± 3.32 3.13 2.62 3.33 2.68 2.85 2.54 2.60 2.97 2.84 2.48 2.61


73

Figure 4.5 Effect of colchicine on percent seed formation in the in vivo colchicine manipulation experiment

74

Plate 8. In vivo colchicine manipulation experiment

75

Table 4.20 Effect of different concentration of colchicine applied in vivo with and without 2, 4-D at various intervals of pollination with Imperata cylindrica in wheat on percent embryo formation

Treat- ments


100 200 300 400 500 600 700 800 900 1000 1200 1500 2000 3000 5000 7000 10000

T1 44.44# 41.03 41.67 42.00 39.13 41.67 32.35 35.65 34.38 25.78 18.40 15.32 19.67 16.95 17.76 17.02 9.38

T2 44.19# 40.48 41.30 48.28# 43.48 39.13 32.56 34.19 35.61 26.61 19.30 25.55 20.33 16.13 16.83 15.28 14.29

T3 44.23# 39.58 44.23# 44.64# 43.59 43.59# 34.11 28.57 36.92 27.27 17.81 24.07 6.73 22.64 4.32 5.08 0

T4 42.86# 33.33 30.00 42.25 40.00 37.50 32.64 34.85 31.58 21.17 12.88 28.32 10.00 29.41 15.79 12.00 13.64

T5 40.38 42.25 35.42 37.84 35.48 45.65# 33.33 42.61 28.24 28.70 10.81 34.34 15.15 33.08 14.85 15.38 12.50

T6 31.13 37.78 35.42 30.00 31.67 34.04 31.01 31.03 22.35 25.23 9.01 21.28 10.47 21.19 11.58 6.90 8.93

T7 39.22 35.20 35.96 38.00 43.10 32.76 31.37 33.86 31.33 35.40 11.81 21.95 8.70 35.51 7.22 5.41 6.25

T8 34.00 31.36 39.02 39.58 39.06 44.00# 35.07 30.23 40.00 30.16 29.13 27.43 10.24 34.35 11.21 9.68 9.33

T9 46.00 37.88 38.46 43.10 39.58 39.66 35.34 28.26 37.40 30.48 27.78 16.54 17.86 16.67 10.62 5.56 8.70

T10 32.73 32.76 38.33 39.39 36.96 38.18 32.67 26.36 23.71 28.57 19.20 15.13 11.11 26.67 9.84 4.48 0

T11 34.02 32.00 37.93 33.93 37.50 35.42 32.50 34.13 23.42 27.69 29.57 32.31 7.27 25.21 3.23 4.44 2.63

T12 46.15 42.11 40.00 41.82 38.89 33.33 41.67 26.14 24.60 36.15 31.82 21.88 11.32 13.13 9.20 6.06 0

Control 46.25 46.25 46.25 46.25 46.25 46.25 46.25 46.25 46.25 46.25 46.25 46.25 46.25 46.25 46.25 46.25 46.25

Mean 40.43 37.85 38.77 40.54 39.59 39.32 34.68 33.24 31.98 29.96 21.83 25.41 15.01 25.94 13.75 11.81 10.15

SE ± 1.56 1.29 1.16 1.37 1.07 1.29 1.22 1.65 2.03 1.75 2.94 2.41 2.89 2.66 2.98 3.14 3.32


76

Table 4.21 Effect of different concentration of colchicine applied in vivo with and without 2, 4-D at various intervals of pollination with Imperata cylindrica in wheat on percent embryo formation


Treatments

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12

100 44.44 # 44.19 # 44.23 # 42.86 # 40.38 31.13 39.22 34.00 46.00 # 32.73 34.02 46.15 #

200 41.03 # 40.48 # 39.58 # 33.33 42.25 # 37.78 35.20 31.36 37.88 32.76 32.00 42.11 #

300 41.67 # 41.30 # 44.23 # 30.00 35.42 35.42 35.96 39.02 38.46 38.33 37.93 40.00 #

400 42.00 # 48.28 # 44.64 # 42.25 # 37.84 30.00 38.00 39.58 43.10 # 39.39 33.93 41.82 #

500 39.13 43.48 # 43.59 # 40.00 35.48 31.67 43.10 # 39.06 39.58 36.96 37.50 38.89 #

600 41.67 # 39.13 43.59 # 37.50 45.65 # 34.04 32.76 44.00 # 39.66 38.18 35.42 33.33

700 32.35 32.56 34.11 32.64 33.33 31.01 31.37 35.07 35.34 32.67 32.50 41.67 #

800 35.65 34.19 28.57 34.85 42.61 # 31.03 33.86 30.23 28.26 26.36 34.13 26.14

900 34.38 35.61 36.92 31.58 28.24 22.35 31.33 40.00 37.40 23.71 23.42 24.60

1000 25.78 26.61 27.27 21.17 28.70 25.23 35.40 30.16 30.48 28.57 27.69 36.15

1200 18.40 19.30 17.81 12.88 10.81 9.01 11.81 29.13 27.78 19.20 29.57 31.82

1500 15.32 25.55 24.07 28.32 34.34 21.28 21.95 27.43 16.54 15.13 32.31 21.88

2000 19.67 20.33 6.73 10.00 15.15 10.47 8.70 10.24 17.86 11.11 7.27 11.32

3000 16.95 16.13 22.64 29.41 33.08 21.19 35.51 34.35 16.67 26.67 25.21 13.13

5000 17.76 16.83 4.32 15.79 14.85 11.58 7.22 11.21 10.62 9.84 3.23 9.20

7000 17.02 15.28 5.08 12.00 15.38 6.90 5.41 9.68 5.56 4.48 4.44 6.06

10000 9.38 14.29 0 13.64 12.50 8.93 6.25 9.33 8.70 0 2.63 0

Control 46.25 46.25 46.25 46.25 46.25 46.25 46.25 46.25 46.25 46.25 46.25 46.25

Mean 29.94 31.10 28.54 28.58 30.68 24.74 27.74 30.01 29.23 25.69 26.64 28.36

SE ± 2.88 2.80 3.77 2.75 2.81 2.71 3.22 2.84 3.14 3.10 3.12 3.51


77

Figure 4.6 Effect of colchicine on percent embryo formation in the in vivo colchicine manipulation experiment

78

78

and 600 ppm in T1, 100 to 500 ppm in T2, 100 to 600 ppm in T3, 100 ppm and

400 ppm in T4, 200 ppm, 600 ppm and 800 ppm in T3, 500 ppm in T7, 600 ppm in

T7, 600 ppm in T8, 100 ppm and 400 ppm in T9 and 100 ppm to 500 ppm and 700

ppm in T12 where it was at par with the control.

Figure 4.6 indicated that with the increase in dose of colchicine there was

decline in embryo formation. These results showed that higher dose of colchicine

adversely affect the frequency of embryo formation.

No any report has been made in this regard until now. So, the present

endeavour is an innovative and unique attempt in respect of doubled haploid

production efficiency enhancement in bread wheat.

4.2.1.3 Percent regenerated plants

The analysis of data following student’s t-test in respect of all colchicine

concentration (100 ppm to 10,000 ppm) over all the treatments (T1 to T12) and

vice-versa revealed both positive and negative significant effects on percent

regenerated plants with exceptions in some cases viz., 300 ppm in T2 and T3,

400 ppm in T2, T4, T5 and T8, 3000 ppm in T2, T3 and T8, 5000 ppm in T2, 7000

ppm in T1, T3, T4 and T10 and 10,000 ppm in T2, and T6 in concentration over

treatments (Table 4.22) and 3000 ppm and 7000 ppm in T1, 300 ppm, 500 ppm,

1000 ppm, 1500 ppm, 3000 ppm and 5000 ppm in T2, 500 ppm in T3 and T4, 400

ppm, 600 ppm, 800 ppm and 3000 ppm in T5, 500 ppm, 600 ppm, 800 ppm, 900

ppm and 10,000 ppm in T6, 800 ppm, 1200 ppm, 1500 ppm and 10,000 ppm in

T7, 400 ppm,1500 ppm and 3000 ppm in T8, 300 ppm, 900 ppm and 7000 ppm

inT10, 900 ppm, 3000 ppm and 5000 ppm inT11 and 700 ppm and 3000 ppm in

T12 in treatments over concentration (Table 4.23) where it was at par with the

control.

These results also indicated that some of the concentration of colchicine

exhibited positive significance over some treatments for percent regeneration

of plants viz., 400 ppm in T6 and T7 and 10000 ppm in T7 and T8 (Table 4.22).

79

Table 4.22 Effect of different concentration of colchicine applied in vivo with and without 2, 4-D at various intervals of pollination with Imperata cylindrica in wheat on percent regenerated plants

Treat- ments


100 200 300 400 500 600 700 800 900 1000 1200 1500 2000 3000 5000 7000 10000

T1 10.00 18.75 15.00 9.52 27.78 25.00 4.55 24.39 72.73 6.06 21.74 5.26 25.00 33.33 21.05 37.5 # 16.67

T2 21.05 5.88 36.84# 28.57# 40.00 11.11 11.90 12.50 68.09 37.93 22.73 45.71 28.00 44.00# 35.29# 27.27 27.27#

T3 13.04 5.26 43.48# 19.05 17.65 23.53 11.36 29.92 16.67 33.33 46.15 11.54 16.67 42.86# 16.67 33.33# 0

T4 9.09 23.81 33.33 50.00# 35.00 16.67 4.26 26.09 4.17 13.79 11.76 25.00 20.00 18.18 8.33 33.33# 16.67

T5 9.52 3.33 17.65 35.71# 31.82 33.33 6.38 34.69 13.51 6.06 8.33 52.94 6.95 33.33 26.67 25.00 0

T6 3.03 5.88 29.41 73.33* 42.11 37.50 7.50 38.89 47.37 25.93 10.00 20.00 12.00 22.22 18.18 0 40#

T7 5.00 5.88 25.00 68.42* 20.00 15.79 10.42 44.19 69.23 2.05 33.33 37.04 7.89 25.00 14.29 0 50*

T8 17.65 21.62 21.88 42.11# 20.00 18.18 31.91 61.54 25.00 2.63 26.67 45.16 4.44 38.46# 7.69 16.67 57.14*

T9 8.70 8.00 13.33 20.00 26.32 34.78 9.76 30.77 19.57 18.75 31.43 13.64 21.74 20.00 8.33 25.00 0

T10 8.33 5.26 47.83 26.92 17.65 23.81 12.12 88.24 34.78 13.33 4.17 0 12.5 18.18 16.67 33.33# 0

T11 9.09 12.5 27.27 15.79 6.67 17.65 17.95 23.26 34.62 16.67 29.41 4.76 10.00 37.5 33.33 0 100.00

T12 4.17 8.33 25.00 13.04 14.29 6.67 36.00 87.50 16.13 8.51 69.05 7.14 15.38 33.33 18.18 0 0

Control 40.54 40.54 40.54 40.54 40.54 40.54 40.54 40.54 40.54 40.54 40.54 40.54 40.54 40.54 40.54 40.54 40.54

Mean 12.25 12.70 28.97 34.08 26.14 23.43 15.74 41.73 35.57 17.39 27.33 23.75 17.01 31.30 20.40 20.92 26.79

SE± 2.74 2.99 3.03 5.64 3.11 2.91 3.41 6.56 6.36 3.66 4.95 5.10 2.78 2.63 2.95 4.36 8.39

P≤0.05; # = Non significant; No symbol = Significantly negative; *= significantly positive

80

Table 4.23 Effect of different concentration of colchicine applied in vivo with and without 2, 4-D at various intervals of pollination with Imperata cylindrica in wheat on percent regenerated plants


Treatments

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12

100 10.00 21.05 13.04 9.09 9.52 3.03 5.00 17.65 8.70 8.33 9.09 4.17

200 18.75 5.88 5.26 23.81 3.33 5.88 5.88 21.62 8.00 5.26 12.5 8.33

300 15.00 36.84 # 43.48 # 33.33 17.65 29.41 25.00 21.88 13.33 47.83 # 27.27 25.00

400 9.52 28.57 19.05 50.00* 35.71 # 73.33* 68.42* 42.11 # 20.00 26.92 15.79 13.04

500 27.78 40.00 # 17.65 35.00 # 31.82 42.11 # 20.00 20.00 26.32 17.65 6.67 14.29

600 25.00 11.11 23.53 16.67 33.33 # 37.50 # 15.79 18.18 34.78 23.81 17.65 6.67

700 4.55 11.90 11.36 4.26 6.38 7.50 10.42 31.91 9.76 12.12 17.95 36.00 #

800 24.39 12.50 26.92 26.09 34.69 # 38.89 # 44.19 # 61.54* 30.77 88.24* 23.26 87.50*

900 72.73 68.09* 16.67 4.17 13.51 47.37 # 69.23 * 25.00 19.57 34.78 # 34.62 # 16.13

1000 6.06 37.93 # 33.33 13.79 6.06 25.93 2.50 2.60 18.75 13.33 16.67 8.51

1200 21.74 22.73 46.15 # 11.76 8.33 10.00 33.33 # 26.67 31.43 4.17 29.41 69.05*

1500 5.26 45.71 # 11.54 25.00 52.94* 20.00 37.04 # 45.16 # 13.64 0 4.76 7.14

2000 25.00 28.00 16.67 20.00 6.98 12.00 7.89 4.44 21.74 12.50 10.00 15.38

3000 33.33 # 44.00 # 42.86 # 18.18 33.33 # 22.22 25.00 38.46 # 20.00 18.18 37.50 # 33.33 #

5000 21.05 35.29 # 16.67 8.33 26.67 18.18 14.29 7.69 8.33 16.67 33.33 # 18.18

7000 37.50 # 27.27 33.33 33.33 25.00 0 0 16.67 25.00 33.33 # 0 0

10000 16.67 27.27 0 16.67 0 40.00 # 50.00 # 57.14* 0 0 100.00* 0

Control 40.54 40.54 40.54 40.54 40.54 40.54 40.54 40.54 40.54 40.54 40.54 40.54

Mean 23.05 30.26 23.23 21.67 21.43 26.33 26.36 27.74 19.48 22.43 24.28 22.40

SE ± 3.86 3.60 3.26 3.03 3.62 4.50 5.08 3.98 2.52 5.05 5.26 5.58

P≤0.05; # = Non significant; No symbol = Significantly negative; *= significantly positive

81

Figure 4.7 Effect of colchicine on percent plant regenerated in the in vivo colchicine manipulation experiment

82

82

Similarly, some treatments showed positive significance over some concentration

for percent plant regeneration viz., T2 in 900 ppm, T4 in 500 ppm, T5 in 1500

ppm , T6 in 400 ppm, T7 in 400 and 900 ppm, T8 in 800 and 10000 ppm, T10 in

800 ppm, T11 in 10000 ppm and T12 in 800 and 1200 ppm (Table 4.23).

Perusal of Figure 4.7 indicated that application of colchicine had also

adverse effects on percent regenerated plants but in few cases the percent

regeneration was more than the control indicating that colchicine effects was

meager or negligible. The reasons for such incidents may be due to lesser or non

absorption of colchicine by the plants, improper application (leakage) and some

other unknown reasons.

4.2.1.4 Identification of ideal dose and time of in vivo application of

colchicine

In order to work out the concentration where doubling of chromosomes

initiated, cytological investigation was carried out from the fixed and preserved

roots of regenerated plants following the standard protocol (3.2.4).The results of

cytological studies (Plate 8) indicated that chromosome doubling started at

concentration 2000 ppm applied after various interval of pollination. The doubling

concentration ranged from 2000 ppm to 10,000 ppm (Table 4.24). Mostly the

chromosome doubling was observed in treatments like T2, T3 and T7. In T2,

colchicine was applied at 48 hours after pollination while in T3 at 72 hours and in

T7 two injections at 48 and 72 hours consecutively (Table 3.5). The in vivo

colchicine experiment exhibited that the dose from 2000 to 10,000 ppm resulted

in doubling of chromosomes, by and large in treatments like T2, T3 and T7.

These findings indicated that the chromosome doubling was effective only when

colchicine was administered at 48 and 72 hours after pollination. The

performance in respect of all the haploid induction parameters in T2 at 2000ppm

was appreciable compared to the T3 and T7.Besides, single application of 2000

ppm colchicine at 48 hours after pollination (T2) was found to be most stable for

chromosome doubling as confirmed through cytological investigation (Plate 10)

suggesting that it is the most efficient and effective dose of colchicine for

chromosome doubling at in vivo application.

83

Plate 9. Preparation of slide for cytological investigation

84

84

Table 4.24 Response of various colchicine concentration (injected in vivo to the I. cylindrica fertilized wheat ovules) on chromosome doubling of the haploid embryos

Colchicine Concentration (ppm) Status of chromosome doubling as

confirmed through cytology

100 No

200 No

300 No

400 No

500 No

600 No

700 No

800 No

900 No

1000 No

1200 No

1500 No

2000 Yes

3000 Yes

5000 Yes

7000 Yes

10000 Yes

Control No

85

Plate 10. Cytological investigation of regenerated plantlets from the wheat x I. cylindrica derived embryos with in vivo/ in vitro colchicine application

86

86

4.2.2 In vitro colchicine application

The results of in vitro colchicine application are presented in tables 4.25,

4.26 and Figure 4.8 in respect of percent plant regeneration. Highest green plant

regeneration was obtained in the doses 100 ppm to 500 ppm. In higher dosage

like 3000 ppm, most of the embryos were unable to transform into green plants

and remained in the callus form. The chromosome doubling started at 300 ppm

(Table 4.25) at 48, 72 and 96 hours of colchicine treatment duration. Matzk and

Mahn (1994) also reported chromosome doubling in vitro on medium containing

0.02 percent colchicine for 30 hours treatment in wheat while Hensen and

Andersen (1998) obtained high frequency of green plants and fertile plants from

48 hours colchicine treatment in wheat. Chen et al. (2002) also reported to

obtained 98.2 percent doubling frequency in case of colchicine treatment by

immersing leaves and roots of plants in colchicine solutions (500 mg/L).

These colchicine manipulation experiments will not only enhance the

production efficiency of DHs but also save time and energy required for the

chromosome doubling in the haploid regenerated plantlets. The outcome of

present endeavour bear far reaching implications in the acceleration of genetic

upgradation and gene mapping population development efforts in wheat and

other cereals.

87

87

Table 4.25 Percent regenerated plants in the in vitro colchicine

manipulations experiment


Treatments

T1 T2 T3 T4 T5

100 40 40 20 40 20

200 20 40 20 20 20

300 20 40 20 20 40

400 20 40 20 20 0

500 20 40 20 20 20

600 20 20 20 20 20

700 20 40 20 20 0

800 20 20 20 0 0

900 20 20 20 0 0

1000 20 20 20 0 0

1500 20 20 0 0 0

2000 20 20 0 0 0

2500 0 20 0 0 0

3000 20 20 0 20 0

Control 40 40 40 40 40

88

88

Table 4.26 Response of various colchicine concentration (in vitro colchicine application) on chromosome doubling of the haploid embryos

Colchicine Concentration (ppm) Status of chromosome doubling as

confirmed through cytology

100 No

200 No

300 Yes

400 Yes

500 Yes

600 Yes

700 Yes

800 Yes

900 Yes

1000 Yes

1500 Yes

2000 Yes

2500 Yes

3000 Yes

Control No

89

Figure 4.8 Effect of colchicine on percent plant regenerated in the in vitro colchicine manipulation experiment

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