Bulblet Formation Research Articles

大理百合的组织培养研究

采用离体组织培养的方法对大理百合的快速繁殖进行了研究，结果表明：以大理百合的鳞片为外植体进行离体培养，可获得再生植株，并初步建立了其快速繁殖体系。由试验结果可知，适宜大理百合鳞片诱导不定芽和愈伤组织的培养基为MS + 2.0 mg/L6-BA + 0.5 mg/L NAA + 3%蔗糖；而MS + 0.5 mg/L 6-BA + 0.1 mg/L NAA + 3%蔗糖更有利于不定芽的诱导；不定芽增殖的培养基以MS + 1.0 mg/L 6-BA + 0.1 mg/L NAA + 3%蔗糖效果较好；不定芽形成小鳞茎的培养基以MS + 1.0 mg/LNAA + 3%蔗糖效果较好；小鳞茎膨大的培养基以1/2MS + 0.3 mg/L NAA + 0.1 mg/L IBA + 12%蔗糖效果较好。 Rapid propagation protocol of Lilium taliense Franch. by in vitro tissue culture was investigated. The results indicated that plant regeneration can be obtained from the bulb scales of L. taliense and a rapid propagation system was established efficiently. Among the media investigated, MS + 2.0 mg/L6-BA + 0.5 mg/LNAA + 3% sucrose is suitable for induction of adventitious bud and callus while MS + 0.5 mg/L6-BA + 0.1 mg/LNAA + 3% sucrose is better for induction of adventitious buds. The medium MS + 1.0 mg/L 6-BA + 0.1 mg/L NAA + 3% sucrose is proper for adventitious bud multiplication; MS + 1.0 mg/L NAA + 3% sucrose is optimum for bulblet formation from the adven-titious buds, while 1/2MS + NAA 0.3 mg/L + IBA0.1 mg/L + 12% sucrose is suitable for the growth of bulblets.

Transcriptome analysis of carbohydrate metabolism during bulblet formation and development in Lilium davidii var. unicolor.

BackgroundThe formation and development of bulblets are crucial to the Lilium genus since these processes are closely related to carbohydrate metabolism, especially to starch and sucrose metabolism. However, little is known about the transcriptional regulation of both processes. To gain insight into carbohydrate-related genes involved in bulblet formation and development, we conducted comparative transcriptome profiling of Lilium davidii var. unicolor bulblets at 0 d, 15 d (bulblets emerged) and 35 d (bulblets formed a basic shape with three or four scales) after scale propagation.ResultsAnalysis of the transcriptome revealed that a total of 52,901 unigenes with an average sequence size of 630 bp were generated. Based on Clusters of Orthologous Groups (COG) analysis, 8% of the sequences were attributed to carbohydrate transport and metabolism. The results of KEGG pathway enrichment analysis showed that starch and sucrose metabolism constituted the predominant pathway among the three library pairs. The starch content in mother scales and bulblets decreased and increased, respectively, with almost the same trend as sucrose content. Gene expression analysis of the key enzymes in starch and sucrose metabolism suggested that sucrose synthase (SuSy) and invertase (INV), mainly hydrolyzing sucrose, presented higher gene expression in mother scales and bulblets at stages of bulblet appearance and enlargement, while sucrose phosphate synthase (SPS) showed higher expression in bulblets at morphogenesis. The enzymes involved in the starch synthetic direction such as ADPG pyrophosphorylase (AGPase), soluble starch synthase (SSS), starch branching enzyme (SBE) and granule-bound starch synthase (GBSS) showed a decreasing trend in mother scales and higher gene expression in bulblets at bulblet appearance and enlargement stages while the enzyme in the cleavage direction, starch de-branching enzyme (SDBE), showed higher gene expression in mother scales than in bulblets.ConclusionsAn extensive transcriptome analysis of three bulblet development stages contributes considerable novel information to our understanding of carbohydrate metabolism-related genes in Lilium at the transcriptional level, and demonstrates the fundamentality of carbohydrate metabolism in bulblet emergence and development at the molecular level. This could facilitate further investigation into the molecular mechanisms underlying these processes in lily and other related species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0358-4) contains supplementary material, which is available to authorized users.

Propagation and conservation of Lilium polyphyllum D. Don ex Royle

In the present study conventional and in vitro propagation protocols have been developed for Lilium polyphyllum D. Don ex Royle. Sodium hypochlorite (4%) treatment improves seed germination (82±7.64%). For vegetative propagation, IBA 100ppm treatment produced 3±1.07 bulblets/scale. We achieved multi-bulblet formation through in vitro seed germination and callus culture using bulb scales on MS medium. The study reports 91% in vitro germination using chipped seed, treated with IBA 100ppm within 14–18 days of inoculation. This is the first report of germination >90% within <20 days in the genus Lilium having hypogeal germination. After 18 days of germination, the in vitro seedlings showed multi-bulblet structures. Bulb scales maximum callusing (69±6.94%) and bulblet formation (1.7±0.18 bulblets/scale) were observed in MS medium supplemented with IBA 25μM. Bulblet formation was observed after four weeks following two sub-cultures on the same medium. Later individual bulblets were hardened with >40% survival after a year. The study can serve as a tool of mass multiplication for in situ and ex situ conservation of the species.

High-frequency Plant Regeneration from Cultured Flower Bud Receptacles of Allium hookeri L.

Allium hookeri L. (Alliaceae family) is an important ethnomedicinal plant native to the Himalayan region of Asia. The aim of this research was to establish a high-frequency plant regeneration system for in vitro propagation of A. hookeri. Among the tissue types examined, receptacle explants derived from immature flower buds showed the highest regeneration rate of shoots (<TEX>$93.33{\pm}4.63%$</TEX>), roots (<TEX>$76.67{\pm}7.85%$</TEX>), and calli (<TEX>$80.00{\pm}7.43%$</TEX>) when cultured on Gamborg B5 (B5) medium containing <TEX>$10{\mu}M$</TEX> 6-benzylaminopurine (BA) + <TEX>$1{\mu}M$</TEX> naphthalene acetic acid (NAA), <TEX>$0.5{\mu}M$</TEX> BA + <TEX>$5{\mu}M$</TEX> NAA, and <TEX>$1{\mu}M$</TEX> BA + <TEX>$10{\mu}M$</TEX> NAA, respectively. Shoot multiplication was superior when cultured in liquid rather than on solid medium and relatively high concentrations of BA, ranging from 5 to <TEX>$10{\mu}M$</TEX>. Efficient bulblet formation following root induction from shoot clumps was achieved with culture in liquid B5 medium containing 7% (w/v) sucrose. Regenerated bulblets were successfully acclimatized to ex vitro conditions with a greater than 95% survival rate. By this method, a maximum of 62 plantlets per receptacle could be propagated within 9 weeks of initial culture. The in vitro propagation system established in this study will promote A. hookeri biotechnology, including large-scale production of healthy and aseptic clones, preserving parental genotypes with desirable traits, and genetic manipulation to enhance medicinal value.

English

Water yam (Dioscorea alata L.) is an important source of food for millions of people in Africa, Asia, South America, Caribbean and the South Pacific islands. Genetic mapping populations of this species have been produced as part of efforts to raise the efficiency of breeding through development and use of molecular markers. In this study, an F1 mapping population (AM1) of D. alata, exhibited segregation for both tuber yield- and quality- related traits when evaluated in the field for 12 agronomic characters: days to shoot emergence, number of primary vines per plant, days to flowering, flower sex, flowering intensity, days to tuber initiation, number of tubers per plant, tuber yield per plant, tuber shape, bulbil formation, tuber browning and reaction to anthracnose (Colletotrichum gloeosporioides Penz) infection. The number of tubers/plant and the number of primary vines/plant were moderately positively correlated, while most of the other quantitative traits were weakly correlated with each other. There were more males (49%) than females (19.9%) identified in the population, however more than a quarter of the population (31%) did not flower and their sexes could not be determined. 20% of the progeny was resistant or tolerant to field infection by anthracnose. Population AM1 will be a valuable resource as a mapping population for genetic analysis and molecular marker development for tuber quality and several other agronomic traits in D. alata.&nbsp; &nbsp; Key words: Flowering intensity, marker assisted breeding, mapping population, phenotypic variation and trait segregation, tuber yield and quality, water yam.

Callus culture, somatic embryogenesis and direct bulblet formation of Allium rotundum L

Wild leek (Allium rotandum L.) is a perennial plant of Allium genuse and in the family of Alliaceae known to be a rare medicinal plant. The current study used Ilam and Kordestan genotypes, MS basic medium with the different auxin growth regulators (NAA, 2, 4-D) and cytokinin (BAP, 2ip, Kin) Meanwhile, the attributes of direct bulblet formation, callus induction, and somatic embryogenesis were inspected. The results of inspecting the analyzed attributes showed that Kordestan genotype started to induction callus in a shorter time in comparison with that of Ilam’s, furthermore, it has more callus in an environment containing 1 milligram per liter 2,4-D and 0.5 milligram per liter Kin. In this test, the attribute of direct bulblet formation was also scrutinized. The results of experiment indicate that the most number and percentage of direct bulblet formation were found in Kordestan genotype in the culture medium C (0.24 milligrm per 2, 4-D liter, and 0.5 milligram per 2ip liter).Embryogenesis was not observed in both culture mediums C (0.24 milligram per liter and 0.5 milligram in BAP liter) and B (6 milligrams in 2,4-d liter and 3 milligrams in BAP liter). The highest percentage of somatic embryogenesis was in the use of 2,4-D with twice more cytokinin.

Developmental response of hybrid lilies to bulb removal date

The effect of different bulb removal date on the development of underground organs particularly bulblets of four lily hybrids was investigated. Three treatments were used included the bulbs were not removed (control) or removed 6 and 8 weeks after planting .The test hybrids were Royal show (LA hybrids), White heaven (L. longiflorum hybrid), Sorbonne and Premium blond (Oriental hybrids).The results indicated that the development of underground organs of plant was greatly influenced by bulb removal. However, both 6, 8 weeks treatments significantly increased the bulblet production compared to control treatment, but 8 weeks treatment recorded the highest production. The six weeks treatment had stronger induced effect on the emerged shoots from bulblets. The hybrid lilies differed significantly for all observed traits. However, the Royal show cultivar recorded the highest values of bulblets number and weight. The White heaven cultivar showed the best capability of the shoots emergence from the bulblets and from the daughters which grown inside the mother bulbs. Both Sorbonne and Premium blond cultivars had more growth of stem roots, less bulblets, no emerged shoots, compared to other cultivars. All these observations suggest that the technique of bulb removal have a potential use to stimulate the plant to produce more bulblets and the tested hybrids of lilies showed significant differences in their capability for bulblets formation and in their response to bulb removal. Key words: Hybrid lilies, underground organs, bulb removal date, bulblets.

Minor differences with big consequences: Reproductive patterns in the genus Gagea (Liliaceae)

Reproductive patterns in ten species of Gagea Salisb. were compared by counts and measurements of bulbs, bulbils and flowers in large cohorts including all life stages. Two types of bulbils were found: taxa with “type I bulbils” start to develop a single to several bulbils as soon as the replacement bulb has reached a certain diameter and then continue to form them indefinitely throughout the life of the plant. “Type II bulbils” are only temporarily produced in immature, non-flowering plants of some species, but not in fully grown, flowering individuals, a phenomenon termed “reproductive switch”. Patterns of bulbil formation are species-specific: G. davlianidzeae, G. nigra, G. peduncularis, G. pratensis, and G. spathacea produce only type I bulbils; G. angelae, G. fedschenkoana and G. lutea develop only type II bulbils. Both bulbil types occur simultaneously in G. fragifera and G. villosa. The quantitative investigations demonstrate the existence of species-specific thresholds for the development of bulbils as well as flowers. Compared to the adult volume of the replacement bulb (where 90% of all plants flower), both types of bulbils have usually low thresholds: 0–5% (type I, all but one species) and 3–13% (type II). Inflorescences develop if plants attain between 38 and 60% of the adult bulb volume. Minor changes in patterns of bulbil formation and thresholds for their development may ensure survival of highly sterile taxa (e.g. G. spathacea, G. fragifera). This, in turn, can facilitate speciation in the genus driven by both hybridization and polyploidization.

Regulation of protein turnover by heat shock proteins

The effects of different media, temperatures and plant growth regulators on the ex vitro propagation of twin-scale cuttings as well as on the relationship between starch degradation and bulblet development of Hippeastrum vittatum cv. ‘Red lion’ were studied. Sawdust was the most optimal medium for the propagation of twin-scale cuttings resulting in the lowest percentage of rotting scales and the best quality bulblets. Furthermore, 25 °C was a more suitable temperature than 30 or 20 °C. Gibberellic acid (GA3), indole-3-butyric acid (IBA) or 1-naphthyleneacetic acid (NAA) did not result in scale propagation and bulblet development. In particular, NAA treatment significantly increased the frequency of rotten scales. During bulblet formation and development, starch content in the mother scales declined earlier and more rapidly, especially in the outer scale of twin scales. The culture of scales at 30 °C resulted in a distinct increase in the consumption of starch in the scale. The starch content in bulblets increased during development. Starch accumulation in bulblets at 25 °C was most remarkable at the last stage of development. The dominant soluble sugars in the bulbs of H. vittatum were fructose, sucrose and glucose, fructose content being highest in the mother scales. However, the key soluble sugar in the bulblets was sucrose and its content increased linearly over the first 50 days.

Micropropagation of Lilium candidum L. : a rare and native bulbous flower of Turkey

Lilium candidum bulb scales were cultured on MS containing different doses and combinations of NAA, BA, Kn and 2iP. Maximum bulblet formation was 88.2% in MS supplemented with 0.1 mg/l NAA + 0.01 mg/l BA and the average number of bulblets per explant was 2.9. DOI: http://dx.doi.org/10.3329/bjb.v42i1.15913 Bangladesh J. Bot. 42(1): 185-187, 2013 (June)

Effects of temperature, plant growth regulators and substrates and changes in carbohydrate content during bulblet formation by twin scale propagation in Hippeastrum vittatum ‘Red lion’

The effects of different media, temperatures and plant growth regulators on the ex vitro propagation of twin-scale cuttings as well as on the relationship between starch degradation and bulblet development of Hippeastrum vittatum cv. ‘Red lion’ were studied. Sawdust was the most optimal medium for the propagation of twin-scale cuttings resulting in the lowest percentage of rotting scales and the best quality bulblets. Furthermore, 25°C was a more suitable temperature than 30 or 20°C. Gibberellic acid (GA3), indole-3-butyric acid (IBA) or 1-naphthyleneacetic acid (NAA) did not result in scale propagation and bulblet development. In particular, NAA treatment significantly increased the frequency of rotten scales. During bulblet formation and development, starch content in the mother scales declined earlier and more rapidly, especially in the outer scale of twin scales. The culture of scales at 30°C resulted in a distinct increase in the consumption of starch in the scale. The starch content in bulblets increased during development. Starch accumulation in bulblets at 25°C was most remarkable at the last stage of development. The dominant soluble sugars in the bulbs of H. vittatum were fructose, sucrose and glucose, fructose content being highest in the mother scales. However, the key soluble sugar in the bulblets was sucrose and its content increased linearly over the first 50 days.

Optimizing in vivo bulblet formation on leaf cuttings in Eucomis spp.

Optimizing in vivo bulblet formation on leaf cuttings in Eucomis spp.

Effects of Different Light Intensities on the Photosynthesis and the Tubers Yield of Pinellia Ternata (Thunb.) Breit

Pinellia ternata (thunb.) Breit. from three different areas (Jiangsu, Shandong, and Hebei) under field conditions were used as study materials. The influences of different levels of shade (0%, 30%, 50%, and 70%) on the chlorophyll content, rate of photosynthesis, and yield of tubers were examined. The results showed that the daily change of photosynthetic rate exhibited typical bimodal curves at shade level 0-50%, and unimodal curves at shade level 70%. As the level of shade increased, the maximum values of the daily rate of photosynthesis of Pinellia from all three habitats appeared later in the day and at significantly decreased levels. The rates of photosynthesis during different stages of growth and the total yield of Pinellia and the tuber yield by dry weight also decreased significantly. The yields of first- and second-grade Pinellia tubers grown under full light were both significantly higher than those grown under shaded conditions. Shade favored the formation of Pinellia bulbils. The yield of Pinellia from Shandong was higher than those from the other two sources. Pinellia-producing areas have rather high yields under full light. The adaptability of Pinellia to light merits further study. Differences in the results of experiments on Pinellia yield in the literature may be related to differences in cultivation techniques or to differences in the responses of Pinellia from different areas to light. In the present study, long-term shade in the field conditions were adopted, and Pinellia from three production areas were planted as study materials in the same place. The effects of shade on the rate of photosynthesis and yield were examined, and the responses of Pinellia from different area were evaluated. The goal was to provide a basis for planting and introducing Pinellia to new areas.

The Comparison of Plant Regeneration between Jerusalem Artichoke and Purple Potato Cultured on MS Media with Different Concentrations and Combinations of Plant Growth Regulators

A deeper understanding of the influence of culture media and different germplasm is crucial to propagate the plants in vitro conditions. To focus this, two tuberous plant species; Jerusalem artichoke (Helianthus tuberosus) and Purple potato (Solanum tuberosum) were selected. In the present study, stem segments from two species selected as explant source were cultured on Murashige and Skoog’s media containing various concentrations (0.2 and 0.5 mg L-1 NAA) (0.2 and 0.5 mg L-1 IAA) of auxin and (0.2 and 0.5 mg L-1 BA) (0.2 and 0.5 mg L-1 KIN) of cytokinins. Callus induction from stem segments of Jerusalem artichoke occured on most of the media tested, but the most callus formation (100%) took place on MS media containing only NAA for explants of purple potato. When the level of NAA increased in the medium, bulblet formation and shoot proliferation decreased for both species. On the other hand, lower concentrations of KIN induced shoot formation for the explants of both species. The present study reports on the effective regeneration protocol in tuberous plants tested and the outcome provides for further genetic research on Jerusalem artichoke and purple potato.

POLYPLOIDY INDUCTION OF THREE LILIUM SPECIES ENDEMIC TO CHINA (LILIUM PUMILUM, L. SARGENTIAE, L. TSINGTAUENSE)

This study was conducted to obtain tetraploids of lily plants in three Lilium species (L. pumilum, L. sargentiae, L. tsingtauense) by treating with colchicine (0, 0.02, and 0.04%) and oryzalin (0, 0.006, and 0.01%) for 24 and 48 hours. In colchicine treatments, the highest percentage of tetraploids was obtained for the 24 hour treatment with 0.04% colchicine for L. pumilum. Tetraploids were obtained by treating with colchicine at 0.02 and 0.04% for 48 hours for L. sargentiae and L. tsingtauense. The optimal oryzalin concentration for the induction of tetraploids was 0.01% for L. tsingtauense and L. sargentiae with 24 or 48 hours treatment, while 0.01% with 24 hours and 0.006% with 48 hours were the optimal oryzalin treatment for L. pumilum. Oryzalin was more efficient than colchicine in inducing tetraploids in the lily species investigated and may be considered as an alternative reagent of colchicine. Duration of treatment and concentration of reagent both had significant effects on the survival rate, bulblet formation, and chromosome doubling. Morphological and anatomical characteristics of leaves and stomatal characteristics correlated with ploidy of lily plants. Chromosome counts and flow cytometry confirmed these findings.

Regeneration and Agrobacterium-mediated transformation of multiple lily cultivars

To pursue genetic improvement of lily, efficiency of both regeneration and transformation from callus cultures induced from different explants were evaluated in multiple cultivars. Thirty-five callus lines induced from filaments or styles and one control callus line derived from bulb scales of in total twenty lily cultivars representing Lilium longiflorum, Oriental × Trumpet and Longiflorum × Asiatic hybrids were maintained on a medium with 8.3 μM picloram (PIC). In this study, they were tested for their regeneration potential by transferring them onto a regeneration medium supplemented with 0.4 μM PIC and 0.044 μM 6-benzyladenine. Regeneration was obtained in all cultivars examined and the percentage varied from zero to 89 % in the 36 callus lines. Regeneration frequency was significantly influenced by the genotype (cultivar). Subculturing the calli every 4 weeks by refreshing the regeneration medium contributed positively to bulblet formation, when compared to an eight week subculture frequency. It was found that the regeneration ability generally decreased with an increasing age of the callus cultures for all cultivars. The origin of the callus (style or filament) did not lead to significant differences in regeneration frequency, but there was an interaction between callus origin and genotype. Calli of eight randomly chosen cultivars were co-cultivated with Agrobacterium tumefaciens strain AGL0 carrying binary vectors with the gus gene as reporter and putative transgenic plants were produced. GUS histochemical assays demonstrated transient and stable expression of the gus gene in both calli and regenerated lily plants. Transient expression frequencies ranged from 0.3 to 20.6 % while stable transformation was much lower, only 1.4 % as the maximum.

Somatic embryogenesis and bulblet regeneration in snakehead fritillary (Fritillaria meleagris L.)

Induction of in vitro morphogenesis of mature zygotic embryos of Fritillaria meleagrisL. was investigated. Somatic embryogenesis and whole plant regeneration were achieved. Isolated zygotic embryos were cultured on MS medium that contained 3% sucrose, 0.7% agar, 250.0 mg/l casein hydrolysate, 250.0 mg/l L-proline, 80 mg/l adenine sulfate and 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) or thidiazuron (TDZ). Embryogenic callus was derived from mature zygotic embryos after 4 weeks on TDZ containing medium. Somatic embryos at the early stages of development arose from the surface of the embryogenic callus. Multiplication of somatic embryos, formation of bulblets and shoot development were observed on the same medium. Scale sections prepared from the formed bulblets were cultured on MS media supplemented with 2,4-D (0.1 to 10.0 mg/l) and TDZ (0.05 to 2.0 mg/l). Somatic embryos and bulblets were frequently produced from the scale sections. This is a successful report of plant regeneration through somatic embryogenesis for this very important medicinal and horticultural plant. Histological observation revealed that plants of F. meleagris L. were regenerated via somatic embryogenesis. Key words: Bulbous plant, medicinal plant, snakehead fritillary, somatic embryos.

Effects of Sucrose Concentration on the Bulblet Formation and Growth of Three Lilies &lt;i&gt;In Vitro&lt;/i&gt;

To study the effect of different sucrose concentration on bulblet formation, three kinds of Lilium plantlets, Lilium davidii var. unicolor, L. longiflorum ‘White Heaven’ and L. OT ‘Yelloween’ were inoculated on media containing different sugar concentration. It was found that L. davidii var. unicolo had 100% percent in bulblet formation at the sucrose concentration 100g/L, while the diameter and weight increment of bulblets came to the maximum. Both ‘White Heaven’ and ‘Yelloween’ had 100% percent of bulblets formation on the sucrose concentration at 80g/L and 100 g/L. But the diameter and weight increment of bulblets in different varieties had different results. The maximum of ‘White Heaven’ bulblets could be obtained at sucrose concentration 60g/L, while ‘Yelloween’ at 80g/L. It was concluded that sucrose concentrations at 40~80g/L had a positive impact on these 3 lilies bulblets formation in vitro cultures.

Mutualisms matter: pollination rate limits the distribution of oil‐secreting orchids

There are at least two immediate reasons why it is important to determine the role of biotic interactions, such as pollination, in limiting species distribution ranges. Firstly, if range limits are imposed by biotic factors, current and future distribution ranges might not be constrained by climate. Secondly, if biotic interactions limit the distribution ranges of species, anthropogenic impacts on these interactions are likely to have a major effect on biodiversity. Here we test the role of pollination in limiting plant distributions by studying plant community assembly in a guild of 15 oil‐secreting orchids (Coryciinae) along a pollination gradient. In all members of the guild, seed production depends on pollination by the oil‐collecting bee Rediviva peringueyi (Melittidae). While the mode of aboveground reproduction is uniform across the guild, the orchid species differ widely in their capacity for belowground clonal reproduction through the formation of bulbils, and hence span a range of predicted dependence on pollination (and subsequent seed set) for population persistence. Pollination rate by R. peringueyi varied across the landscape from 0 to 98% of flowers pollinated. With decreasing pollination, species richness of the orchid guild declined, and species were lost by the successive deletion of the least clonal species. Thus, pollination is shown to act as a biotic filter, excluding non‐clonal species from pollinator‐poor communities. The findings are consistent with the idea that pollination mutualisms matter ecologically by limiting the distribution of non‐clonal plants. Conversely, the results suggest that clonality allows some plant species to escape from the range of their pollinators.

Effect of alar and CCC on induction and germination of bulbils in Curculigo orchioides grown in shake flask cultures

Bulbil formation in Curculigo orchioides is followed by asynchronous germination. The effect of alar and CCC incorporated in Murashige and Skoog (MS) medium has been studied on bulbil induction from leaf explants and subsequent germination of bulbils. MS medium contained 1 mg/l BA and 0.1 mg/l morphactin for bulbil induction while germination medium contained 1 mg/l gibberrelic acid and both the media contained alar or CCC (0.5–5.0 mg/l). Growth retardants markedly reduces the bulbil formation, yield and fresh weight of bulbils. Incorporation of retardants resulted in 60% germination inhibition, thereby prolonging the storage conditions.