ORIGINAL ARTICLE

Multiple shoot proliferation, bulblet induction and evaluationof genetic stability in Asiatic hybrid lily (Lilium sp.)

Rakesh Yadav • Neha Yadav • Minakshi Pal •

Umesh Goutam

Received: 15 June 2013 / Accepted: 14 December 2013 / Published online: 29 January 2014

� Indian Society for Plant Physiology 2014

Abstract An efficient multiple shoot cluster regeneration

and micro-bulb induction protocol was developed for

Asiatic hybrid lily (Lilium sp.). Highest multiple shoot

cluster formation of 78.4 % was observed on MS medium

supplemented with 0.25 mg l-1 BAP and 1.0 mg l-1 GA3.

The growth regulators, i.e., abscisic acid, daminozide

and chlorocholine chloride, significantly affected bulblet

quality parameters such as average size, number, fresh

mass, and scale count. A dose of 0.1 mg l-1 abscisic acid,

50 mg l-1 daminozide, and 10 mg l-1 chlorocholine chloride

exhibited highest micro-bulb regeneration rate per explant of

6.0 ± 0.28, 5.6 ± 0.17, and 6.0 ± 0.34, respectively. A high

sucrose concentration of 40 g l-1 also produced highest

number of bulblet per explant (6.1 ± 0.05). Amongst the 10

arbitrary decamer primers used to analyze the samples,

only 6 primers amplified clear reproducible bands. The 6

primers produced a total of 36 (an average of 6 bands per

primer) scorable bands. Phenotypic observations and ran-

domly amplified polymorphic DNA (RAPD) analysis

established that the regenerated micro-bulbs did not

develop any detectable genetic aberration. Thus, the results

indicate that in vitro produced bulblets of Lilium spp.

(Asiatic hybrid) were clonally identical to their donor plant

and were genetically stable.

Keywords Bulblet � Lilium � Genetic stability � RAPD

Introduction

Lilies are well known perennial bulbous plants belonging

to the monocotyledonous family Liliaceae. Worldwide,

over 100 species reside in Northern Hemisphere and

thousands of genotypes are cultivated at commercial scale

as floricultural crops, mainly because of their large,

attractive flowers (Wang et al. 2009). Bulbs are cultivated

commercially for use in the cut flower and potted-plant

industries. Additionally, Lilies have also been described for

their medicinal values as a stomachic, stimulant and aro-

matic drug (Malla 1994). The premier commercial lilies

principally belong to three hybrid groups: the Asiatic

hybrids (A), the Oriental hybrids (O), and the Longiflorum

group (L). The establishment of a reproducible plant

regeneration system is necessary to supply true-to-type lily

plants for commercial purposes and also for unconven-

tional improvement approaches such as genetic transfor-

mation and the fusion procedures.

Some reports are available for tissue culture for mem-

bers of the genus Lilium for various species and hybrids

such as L. longiflorum (Nhut 1998), L. rubellum (Niimii

et al. 1997), L. lancifolium (Marinengeli and Curvetto

1997), L. auratum (Takayama and Misawa 1979), L. tes-

taceum (Wozniewski et al. 1991), L. formosanum (Nakano

et al. 2000), L. davidii (LingFei et al. 2009), L. oxypetalum

(Joshi and Dhar 2009) L. ledebourii (Bakhshaie et al. 2010)

and Lilium hybrids (Novak and Petru 1981; Marinengeli

and Curvetto 1997). However, there is no reliable

R. Yadav (&) � N. Yadav

Department of Bio and Nano Technology, Guru Jambheshwar

University of Science and Technology, Hisar 125001, Haryana,

India

e-mail: ry.yadav01@gmail.com

M. Pal

Centre for Plant Biotechnology, Haryana State Council for

Science and Technology, CCSHAU New Campus,

Hisar 125001, Haryana, India

U. Goutam

Department of Biotechnology, Lovely Professional University,

Phagwara 144402, Punjab, India

123

Ind J Plant Physiol. (October–December 2013) 18(4):354–359

DOI 10.1007/s40502-014-0060-4

regeneration protocol that can be applied to all major lily

cultivars.

Since, variations appear in tissue culture-raised plants

because of occurrence of mutational events in the somatic

cells; production of true-to-type plants through micro-

propagation techniques is important in order to retain

genotypic and phenotypic similarities of donor plant. The

incongruities are usually inherited and are therefore,

redundant in clonal propagation. Therefore, a molecular

marker based system for determination of genetic fidelity is

rapid and reliable method. Keeping in view, a protocol for

rapid clonal multiplication of Lilium spp. (Asiatic hybrid

cultivar) along with establishment of genetic stability

through RAPD has been described herein.

Materials and methods

Explant preparation and surface sterilization

Fresh field-grown bulbs (circumference 18–20 cm) of Li-

lium spp. (Asiatic hybrids) were collected and stored at

4 �C in refrigerator until use. The roots and brown scales

were excised from the bulbs and these were washed thor-

oughly under running tap water for 10 min. A hot water

treatment was given in water bath for 40 min followed by

overnight air drying. Dried bulbs were incised to obtain

individual scales. Scales were surface sterilized by soaking

in 15 % laboline solution containing two drops of Tween-

20 for 5 min, followed by a treatment with 0.5 % Bavistin

(50 % w/v Cabandazim) solution for 15 min. Explants

were then rinsed 2–3 times with autoclaved distilled water

and transferred to laminar flow cabinet. For further disin-

fection, bulbs were again soaked in 0.1 % HgCl2 solution

for 5 min followed by a quick dip in rectified spirit for

about 30 s. Eventually, the bulbs were rinsed in sterile

distilled water 4–5 times to remove any traces of chemicals

affixed during various surface disinfection treatments as

reported by Yadav et al. (2009). Before inoculation, the

bulbs were dried onto sterile filter paper discs.

Culture medium and conditions

Basal MS medium (Murashige and Skoog 1962) solidified

with 0.8 % (w/v) agar, containing sucrose at a concentra-

tion of 30 mg l-1 was used for initial in vitro regeneration

of bulb-scales. Effect of six BAP concentrations viz. 0.1,

0.25, 0.5, 0.75, 1.0, 2.0 mg l-1 was studied at a constant

concentration of 0.5 or 1.0 or 2.0 mg l-1 GA3 on the initial

multiple shoot induction response of in vitro raised

adventitious buds. The pH of all the medium was adjusted

to 5.8 with 1 N NaOH or 1 N HCl before autoclaving at

121 �C and 108 kPa for 15 min. Cultures were maintained

in Jam-bottles in plant tissue culture room at 25 ± 2 �C

under a 16 h photoperiod. White light was provided by

fluorescent tubes at a photon flux density of 45 lmol m-2

s-1. Cultures were transferred to a fresh medium after

every 3–4 weeks to supplement nutrients.

In vitro microbulb regeneration and growth of bulblets

To study the effect of sucrose and growth retardants on

differentiation, microbulb formation and growth of bul-

blets, MS medium was supplemented with various con-

centrations of sucrose (10, 20, 30 and 40 g l-1). In other

experiments, MS media were supplemented with growth

retardants such as chlorocholine chloride (CCC 2-chlor-

oethyltrimethylammonium chloride; 1, 10, and 50 mg l-1),

daminozide (B-NINE 1, 10, and 50 mg l-1) and abscisic

acid (ABA 0.1, 0.5 and 1 mg l-1) to study their effects on

bulblet regeneration.

Random amplification polymorphic DNA (RAPD)

analysis of the regenerated bulblets

After 18–20 weeks of culture, nine microbulbs were ran-

domly selected among various events of in vitro regener-

ated bulblets to carry out RAPD analysis along with the

mother control plant (M). Young scales (*120 mg) were

ground in liquid nitrogen followed by total genomic DNA

extraction by CTAB method with minor modification. The

DNA quality and yield were analyzed by gel electropho-

resis and Nanodrop spectrophotometer (Nanodrop�, ND-

1000, Nanodrop Technologies, Wilmington, Delaware,

USA), respectively, followed by dilution to 20 ng ll-1

with Tris EDTA buffer. RAPD analysis with ten decamer

arbitrary primers (OPT-1, OPT-2, OPT-3, OPT-4, OPT-5,

OPT-6, OPT-7, OPT-8, OPT-9 and OPT-11) was per-

formed according to Punia et al. (2009) with minor mod-

ification. A PCR reaction mixture (15 ll) containing 30 ng

genomic DNA, 1.5 mM MgCl2, 0.4 mM each of the

deoxynucleotide triphosphates (dATP, dGTP, dCTP,

dTTP), 1.0 U Taq DNA polymerase, and 0.1 lM of primer

was subjected to PCR amplification in a i-cyclerTM ther-

mocycler (BioRad, USA), with the initial denaturation at

94 �C for 3 min, followed by 45 cycles at 94 �C for 60 s, at

37 �C for 90 s and at 70 �C for 2 min and 1 final extension

cycle at 72 �C for 10 min. Two independent amplification

reactions were performed with all RAPD primers.

The PCR amplified products were separated on 1 %

agarose gel augmented with 0.5 mg ml-1 ethidium bro-

mide in 19 TAE buffer (40 mM Tris acetate, 1 mM

EDTA, pH 8.2) using Minipack-250, a submerged hori-

zontal gel electrophoretic system (GeNeiTM) at 70 V for

2 h. A 1 kb DNA ladder was used as molecular weight

standard. Separated DNA fragments were visualized and

Ind J Plant Physiol. (October–December 2013) 18(4):354–359 355

123

photographed with UV light on gel documentation system/

bioimaging system (GeneGenius, Syngene, UK).

Observations, data recording and statistical analysis

The in vitro experiments were carried out in a complete

randomized design with three replications, each containing

eight explants. Data were recorded at 8, 10 and 12 weeks

after culture. Data scored were subjected to one way

Analysis of Variance (ANOVA) with a 0.05 % significance

level by Duncan’s equal variance. These tests were con-

ducted using the statistical software package SPSS for

windows (v. 13.0 SPSS Inc USA).

Only discernible and reproducible bands obtained on

gels were scored by binary matrix based on presence and

absence. Data were subjected to the NTSys PC version

2.02j (www.exetersoftware.com) statistical analysis pack-

age, and cluster analysis was conducted. Similarity esti-

mates were calculated (Nei and Li 1979), and cluster

analysis was carried out using unweighted pair grouping

method with arithmetic mean (UPGMA).

Results and discussion

The percentage of shoot regenerating explants ranged from

4.3 to 78.4 % as shown in Fig. 1. The data indicates sig-

nificant differences among the culture media. Out of three

concentrations of GA3 tested in combination with various

concentrations of BAP, 1.0 mg l-1 GA3 concentration was

highly responsive with a minimum of 14.2 % shoot

regeneration at 1.0 mg l-1 BAP and maximum of 78.4 %

shoot regeneration at 0.25 mg l-1 BAP. Both lower and

higher doses of GA3 than 1.0 mg l-1 resulted into stumpy

regeneration rates and also affected shoot proliferation

potential. Similar results were reported by Bacchetta et al.

(2003) in Asiatic hybrid cultivars of lily onto BAP and IAA

supplemented MS medium.

Inclusion of GA3 in tissue culture media can suppress

callusing of cultures but favor healthier growth and dif-

ferentiation even at very low concentration of 0.1 mg l-1

(Morel et al. 1968). In combination with BAP, GA3 has

been shown to be essential to raise whole plants from

cultured meristem tips (Kartha et al. 1974).

After 3 weeks of culture, bulblet induction begun and

thickets of bulbs with burgeoning leaves were visible by

5 weeks of culture (Fig. 2c). Increased concentration of

ABA from 0.1 to 1.0 mg l-1 caused an adverse effect on

the microbulb regeneration potential of the culture as

illustrated in the Table 1. However, increase in the con-

centration of B-NINE from 1 to 50 mg l-1 enhanced an

average number of bulblet regeneration per explant from

2.8 ± 0.03 to 5.6 ± 0.17 as well as fresh mass of bulblets.

In case of CCC, highest microbulb regeneration rate of

6.0 ± 0.34 was observed at 10 mg l-1 concentration.

Further increase or decrease in CCC concentration

decreased microbulb regeneration potential of shoot clus-

ters (Table 1).

ABA-mediated signaling mechanisms play an important

part in plants to inhibit the synthesis of kinetin nucleotide

(Miernyk 1979) and also down-regulates enzymes needed

for photosynthesis (Chandler and Robertson 1994). Being

growth retardant, even very low concentration of ABA

(*0.1 mg l-1) has triggered reduction in shoot growth rate

and induced microbulb differentiation in lily as observed in

our results (Table 1). B-NINE is a synthetic plant growth

regulator that was first approved for use in the USA in

1963, primarily, for applying on apples. Low concentra-

tions of B-NINE (1 mg l-1) used in this study was insuf-

ficient to induce significant growth retardation and that too

bulblet regeneration. CCC is another chemical agent that

serves as antigibberellin growth retardant, with its gibber-

ellins biosynthesis restraining-based mechanism in plant

tissues. In present study, 10 mg l-1 dose of CCC was

found potent for highest microbulb regeneration rate

(Table 1).

Amongst various concentrations tested, 40 g l-1 sucrose

(w/v) showed highest microbulb regeneration rate of

6.1 ± 0.05 per explant through axillary shoot bud culture

(Table 1). The medium with inclusion of the same con-

centration of sucrose also regenerated bulblets with highest

fresh weight. Bulblets further grew in size during a

3–4 week subculture phase. Average fresh mass and

diameter of in vitro bulblets increased at higher concen-

tration of sucrose (Yadav and Beniwal 2008). Basically,

high sucrose dose is known to stimulate differentiation and

Fig. 1 Effect of various concentrations of BAP in the presence of 0.5

(filled diamond) or 1.0 (filled square) or 2.0 mg l-1 (filled triangle)

GA3 on percentage plant regeneration of Lilium spp. (Asiatic hybrids)

by adventitious buds; filled diamonds, at constant amount of

0.5 mg l-1 GA3; filled squares, at constant amount of 1.0 mg l-1

GA3; filled triangles, at constant amount of 2.0 mg l-1 GA3

356 Ind J Plant Physiol. (October–December 2013) 18(4):354–359

123

formation of storage structures such as bulbs as revealed in

earlier studies in lilies (Marinengeli and Curvetto 1997;

Kumar et al. 2005), gladiolus (Dantu and Bhojwani 1987)

and potatoes (Garner and Blake 1989). An increase in

bulblet size at higher concentration of sucrose was

achieved because of the increase in starch and total car-

bohydrates content (Langens-Gerrits et al. 1997).

Somaclonal variations are very recurrent in tissue cul-

ture based micropropagation systems. The extent of vari-

ation in tissue culture relies upon the type of explant, its

source and the mode of regeneration (Larkin et al. 1989).

Out of the 10 primers tested, 6 generated clear, scorable

and reproducible amplifications (Table 2). Amongst these

6 amplified primers, three produced monomorphic patterns;

whereas, the others generated polymorphic bands. The

number of bands for each primer varied from 4 (OPT-06)

to 8 (OPT-08) and ranged in size from 150 bp to 2.0 kb.

Figure 3 showed RAPD amplification pattern with primers

OPT-1, OPT-8, and OPT-11 which reveals monomorphism

in gel profiles. All amplified primers produced a total of 36

bands with an average of 6 bands per primer. Out of the 36

scorable bands, 30 were monomorphic (83.4 %); while,

only 6 were polymorphic (16.6 %; Table 2).

Cluster analysis using the UPGMA clustering method

was performed on the basis of similarity matrix calculated

from the RAPD results. UPGMA phenogram generated

through RAPD similarity profile were confined in two

major groups, which were clustered at similarity coeffi-

cients of 0.925–0.948 with a mean value of 0.936. These

analyses indicated that regenerated plants resembled the

donor control genetic profiles, based on RAPD report. As

depicted in Fig. 3, the regenerated progeny shared identical

banding patterns as that of the explant-source mother plant,

signifying their genetically similarity. The present findings

substantiate earlier reports of the genetic stability in lilies

(Varshney et al. 2001).

Fig. 2 Mass proliferation and bulblet induction in Lilium spp. a In

vitro regeneration of Lilium bulb-scale on MS basal media; b Multiple

shoot cluster developing from one adventitious bud of Lilium on MS

medium supplemented with 0.25 mg l-1 BAP and 1.0 mg l-1 GA3;

arrows indicate multi-shoots with axillary buds; c Adventitious

bulblet induction in Lilium cultures on MS medium supplemented

with 0.1 mg l-1 ABA after 3 weeks of culture; arrows indicate

bulblet formation

Table 1 Effect of three growth retardants and sucrose on bulblet

regeneration of in vitro cultures of Asiatic hybrid of lily after

10 weeks of incubation period

Treatments

(mg l-1)

Number of

bulblet per

explant

Fresh

weight

(mg)

Diameter

(mm)

Number of

scales

ABA

0.1 6.0 ± 0.28 182 ± 1.15 5.2 ± 0.17 11.2 ± 0.11

0.5 4.3 ± 0.23 165 ± 1.73 5.0 ± 0.28 10.4 ± 0.05

1.0 3.7 ± 0.28 138 ± 2.30 4.4 ± 0.11 9.2 ± 0.11

B-NINE

1 2.8 ± 0.03 126 ± 0.57 3.8 ± 0.11 7.8 ± 0.17

10 4.6 ± 0.11 173 ± 1.73 4.2 ± 0.05 9.4 ± 0.05

50 5.6 ± 0.17 191 ± 1.15 5.0 ± 0.34 10.8 ± 0.11

CCC

1 3.8 ± 0.23 136 ± 1.15 4.7 ± 0.11 8.6 ± 0.17

10 6.0 ± 0.34 187 ± 0.57 5.4 ± 0.11 11.9 ± 0.05

50 4.2 ± 0.17 168 ± 2.30 4.0 ± 0.05 9.1 ± 0.17

Sucrose

10,000 2.7 ± 0.11 140 ± 4.61 3.9 ± 0.11 8.7 ± 0.11

20,000 4.1 ± 0.49 156 ± 1.73 4.8 ± 0.12 10.2 ± 0.11

30,000 4.9 ± 0.17 191 ± 1.73 5.6 ± 0.17 11.8 ± 0.17

40,000 6.1 ± 0.05 198 ± 1.15 5.3 ± 0.11 11.7 ± 0.17

Data represent means ± standard errors for three replicates

Ind J Plant Physiol. (October–December 2013) 18(4):354–359 357

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It is established that a combination of 0.25 mg l-1 BAP

and 1.0 mg l-1 GA3 is highly efficient for multiple shoot

formation. The bulblets developed from microshoots serve

as candidate-propagules for both successful ex vitro accli-

matization, as well as, prolonged storage adaptation. The

results also underline efficient doses of growth retardants,

ABA, B-NINE, and CCC, which are stimulatory for larger

number of microbulb regeneration per explant.

Acknowledgments Authors thank Dr. Ashok Dhawan for critical

reading, valuable comments and suggestions on an earlier version of

this manuscript. We are grateful to the Department of Biotechnology,

New Delhi and the Department of Science and Technology, New

Delhi, Govt. of India for the financial assistance.

References

Bacchetta, L., Remotti, P. C., Bernardini, C., & Saccardo, F. (2003).

Adventitious shoot regeneration from leaf explants and stem nodes

of Lilium. Plant Cell, Tissue and Organ Culture, 74, 37–44.

Bakhshaie, M., Babalar, M., Masoud, M., & Khalighi, A. (2010).

Somatic embryogenesis and plant regeneration of Lilium ledeb-

ourii (Baker) Boiss., an endangered species. Plant Cell, Tissue

and Organ Culture, 102, 229–235.

Chandler, P. M., & Robertson, M. (1994). Gene expression regulated by

abscisic acid and its relation to stress tolerance. Annual Review of

Plant Physiology and Plant Molecular Biology, 45, 113–141.

Dantu, P. K., & Bhojwani, S. S. (1987). In vitro propagation of

gladiolus. Gartenbauwissenschaft, 52, 90–93.

Garner, N., & Blake, J. (1989). The induction of potato microtubers

in vitro on media free growth regulatory substances. Annals of

Botany, 63, 663–674.

Joshi, S. K., & Dhar, U. (2009). In vitro propagation from axenic

explants of Lilium oxypetalum (D. Don) Baker, an endemic

bulbous plant of high altitude Himalaya. Acta Physiologiae

Plantarum, 31, 833–838.

Kartha, K. K., Gamborg, O. L., Constabel, F., & Shyluk, J. P. (1974).

Regeneration of cassava plants from apical meristems. Plant

Science Letters, 2, 107–113.

Kumar, S., Kashyap, M., & Sharma, D. R. (2005). In vitro regen-

eration and bulblet growth from lily bulbscale explants as

affected by retardants, sucrose and irradiance. Biologia Planta-

rum, 49, 629–632.

Table 2 RAPD primers employed for verifying genetic fidelity in Lilium spp.

Primers Primer sequences (50–30) Melting temperatures

Tm (�C)

No. of bands

scored

No. of polymorphic

bands

% Age

Polymorphism

OPT-01 GGTCCTCTCA 41.0 5 0 0

OPT-02 GGAGCGACTC 42.9 – – –

OPT-03 TCCACTACTA 42.2 7 2 28.5

OPT-04 CACATAGGGT 42.8 6 0 0

OPT-05 CGGTTTAGCA 45.8 – – –

OPT-06 CTAGGGCAGA 44.1 4 0 0

OPT-07 GGAAGGCTGT 40.3 – – –

OPT-08 AACGGCGATA 43.0 8 3 37.5

OPT-09 CACCAATGAG 45.1 – – –

OPT-11 TTCGACGCGA 42.6 6 1 16.6

Total 36 6 16.6

Fig. 3 Monomorphic gel profiles of RAPD amplifications with primers OPT-1, OPT-8, and OPT-11; L, molecular size marker (100 bp DNA

ladder); M, mother plant; 1–9, nine randomly selected tissue culture raised bulblets

358 Ind J Plant Physiol. (October–December 2013) 18(4):354–359

123

Langens-Gerrits, M., Lilien-Kipnis, H., Croes, T., Miller, W.,

Kolloffel, C., & De Klerk, G. J. (1997). Bulb growth in lily

regenerated in vitro. Acta Horticulturae, 430, 267–273.

Larkin, P. J., Banks, P. M., Bhati, R., Bretell, R. I. S., Davis, P. A.,

Ryan, S. A., et al. (1989). From somatic variation to variant

plants: Mechanism and applications. Genome, 31, 705–711.

LingFei, X., FengWang, M., & Dong, L. (2009). Plant regeneration

from in vitro cultured leaves of Lanzhou lily (Lilium davidii var.

unicolor). Scientia Horticulturae, 119, 46–458.

Malla, SB. (1994). Medicinal herbs in the Bagamati zone. ADPI Ser

No. 8 (pp. 8–28). Kathmandu: ICIMOD.

Marinengeli, P., & Curvetto, N. (1997). Increased sucrose and salt

concentration in culture medium improved growth of micro-

propagated Lilium bulblets. Biocell, 21, 161–164.

Miernyk, J. A. (1979). Abscisic acid inhibition of kinetin nucleotide

formation in germinating lettuce seeds. Physiologia Plantarum,

45, 63–66.

Morel, G., Martin, C., & Muller, J. F. (1968). La guerison des pomme

de terre atteintes maladies a virus. Annales de Physiologie

Vegetale, 10, 113–139.

Murashige, T., & Skoog, F. (1962). A revised medium for rapid

growth and bioassays with tobacco tissue cultures. Physiologia

Plantarum, 15, 473–497.

Nakano, M., Sakakibara, T., Suzuki, S., & Saito, H. (2000). Decrease

in the regeneration potential of long-term cell suspension

cultures of Lilium formosanum Wallace and its restoration by

the auxin transport inhibitor, 2,3,5-triiodobenzoic acid. Plant

Science, 158, 129–137.

Nei, M., & Li, W. H. (1979). Mathematical model for studying

genetic variation in terms of restriction endonucleases. Proceed-

ings of National Academy of Sciences, 76, 5269–5273.

Nhut, D. T. (1998). Micropropagation of lily (Lilium longiflorum).

Plant Cell Reports, 17, 913–916.

Niimii, Y., Nakano, M., & Saito, S. (1997). Production of commercial

Lilium rubellum Baker bulbs: effects of volume and renewal of

liquid medium on in vitro growth, bulb rot infection during cold

treatment, and post-in vitro growth of bulblets. Journal of the

Japanese Society for Horticultural Science, 66, 113–119.

Novak, F. J., & Petru, E. A. (1981). Tissue culture propagation of

Lilium hybrids. Scientia Horticulturae, 14, 191–199.

Punia, A., Yadav, R., Arora, P., & Chaudhury, A. (2009). Molecular

and morphophysiological characterization of superior cluster

bean (Cymopsis tetragonoloba) varieties. Journal of Crop

Science and Biotechnology, 12, 143–148.

Takayama, S., & Misawa, M. (1979). Differentiation in Lilium bulb

scales grown in vitro. Effects of various cultural conditions.

Physiologia Plantarum, 46, 184–190.

Varshney, A., Lakshmikumaran, M., Srivastava, P. S., & Dhawan, V.

(2001). Establishment of genetic fidelity of in vitro-raised lilium

bulblets through RAPD markers. In Vitro Cellular & Develop-

mental Biology: Plant, 37, 227–231.

Wang, J., Huang, L., Bao, M., & Liu, G. (2009). Production of

interspecific hybrids between Lilium longiflorum and L. lopho-

phorum var. linearifolium via ovule culture at early stage.

Euphytica, 167, 45–55.

Wozniewski, T., Blaschek, W., & Franz, G. (1991). In vitro

propagation of Lilium testaceum and structural investigation of

the storage b-1,4-glucomannan. Plant Cell Reports, 10, 457–460.

Yadav, R., Arora, P., Kumar, D., Katyal, D., Dilbaghi, N., &

Chaudhury, A. (2009). High frequency direct plant regeneration

from leaf, internode, and root segments of Eastern Cottonwood

(Populus deltoides). Plant Biotechnology Reports, 3, 175–182.

Yadav, R. & Beniwal, V. (2008). In vitro clonal propagation and

influence of sucrose concentration on bulblet formation in Lily

(p. 18). In: 1st International Society BioTechnology Conference,

Majitar, Sikkim.

Ind J Plant Physiol. (October–December 2013) 18(4):354–359 359

123