Bulblet Regeneration Research Articles

Establishment of Artificial Rapid Propagation System of Fritillaria crassicaulis

Fritillaria crassicaulis S. C. Chen is a precious traditional Chinese medicine, but the number of populations has declined rapidly due to overexploitation. An artificial rapid propagation system was established to screen the suitable plant regeneration method and to explore the efficient propagation method, useful for propagation technology or for further research and development of F. crassicaulis. This study selected scale as the experimental material, set Murashige and Skoog (MS) medium as the basic medium, and optimized the types and proportions of plant growth regulator (PGR) suitable for callus induction, bulblet differentiation and proliferation, and plant regeneration by means of single-factor, full-factorial, and L9 (3)4 orthogonal experiments. Results demonstrate that in the experiment with single exogenous PGR, the high concentration of 6-benzylaminopurine (6-BA) was significantly better than kinetin (KT) to induce bulblets, 2, 4-dichloroacetic acid (2, 4-D) had a significant effect on callus induction, and a higher concentration of naphthaleneacetic acid (NAA) was beneficial to the occurrence and growth of bulbs, but the rooting effect promoted by indole butyric acid (IBA) was preferable to that by NAA. In MS medium with 0.5 mg/L 2, 4-D and 1.5 mg/L 6-BA, a large number of yellowish-green compact calli could be induced from the scales with the calli induction frequency at 93.3%, and about 11.4% materials directly differentiated bulblets. In the subsequent orthogonal experiment, after the scales were cultured in MS medium with 2.0 mg/L 6-BA, 0.5 mg/L 2, 4-D, and 0.1 mg/L NAA for 20 days, the small yellow and white globular protuberances formed near the incision, but no callus appeared, and many protuberances appeared on the surface of the scales. After 60 days, the protuberances at the incision developed into bulblets directly, while protuberances on the surface of the scales developed into few bulblets but crowded “leaf spines,” which gradually died and disappeared in the later culture; the proliferation coefficient was ∼6.30 then. Experimental results indicate that the optimal rooting medium for bulblets was 1/2MS medium with 2.0 mg/L IBA and 1.0 mg/L activated carbon (AC), with the rooting rate at 95.6%. This study identifies bulblet regeneration of F. crassicaulis, and an efficient direct organogenesis method was established: regenerated bulblets could be induced from scales in one step, so a large number of regenerated plants with the same genotype could be obtained in a short time.

Full-length transcriptome analysis revealed that 2,4-dichlorophenoxyacetic acid promoted in vitro bulblet initiation in lily by affecting carbohydrate metabolism and auxin signaling.

Bulblet initiation, including adventitious bud initiation and bulblet formation, is a crucial process for lily and other bulbous flowers that are commercially propagated by vegetative means. Here, by a hybrid strategy combining Pacific Biosciences (PacBio) full-length sequencing and Illumina RNA sequencing (RNA-seq), high-quality transcripts of L. brownii (Lb) and its variety, L. brownii var. giganteum (Lbg), during in vitro bulblet initiation were obtained. A total of 53,576 and 65,050 high-quality non-redundant full-length transcripts of Lbg and Lb were generated, respectively. Morphological observation showed that Lbg possessed a stronger capacity to generate bulblets in vitro than Lb, and 1 mg L-1 2,4-dichlorophenoxyacetic acid (2,4-D) significantly increased bulblet regeneration rate in two lilies. Screening of differentially expressed transcripts (DETs) between different stages and Mfuzz analysis showed 0 DAT to 1 DAT was the crucial stage with the most complex transcriptional change, with carbohydrate metabolism pathway was significantly enriched. In addition, 6,218 and 8,965 DETs were screened between the 2,4-D-treated group and the control group in Lbg and Lb, respectively. 2,4-D application had evident effects on the expression of genes involved in auxin signaling pathway, such as TIRs, ARFs, Aux/IAAs, GH3s and SAURs. Then, we compared the expression profiles of crucial genes of carbohydrate metabolism between different stages and different treatments. SUSs, SUTs, TPSs, AGPLs, GBSSs and SSs showed significant responses during bulblet initiation. The expression of CWINs, SUTs and SWEETs were significantly upregulated by 2,4-D in two lilies. In addition, 2,4-D increased the expression of starch degradation genes (AMYs and BAMs) and inhibited starch synthesis genes (AGPLs, GBSSs and SSs). SBEs were significantly upregulated in Lbg but not in Lb. Significant co-expression was showed between genes involved in carbohydrate metabolism and auxin signaling, together with transcription factors such as bHLHs, MYBs, ERFs and C3Hs. This study indicates the coordinate regulation of bulblet initiation by carbohydrate metabolism and auxin signaling, serving as a basis for further studies on the molecular mechanism of bulblet initiation in lily and other bulbous flowers.

Effects of Morphological Characteristics, Nutritional Status and Light on the Scale Propagation of Lilium

Scaling is the most commonly used technique to artificially propagate lilies. Scales from different positions of the donor bulb vary in regeneration efficiency; however, the mechanism underlying bulblet formation remains unclear. To investigate the relationship between scale morphological characteristics, initial nutrient status and bulblet regeneration capacities during scale propagation of Lilium, we performed comprehensive morphological and correlation analyses using scales from three lily cultivars. Principal component analysis clearly distinguished middle scale (MS) from outer scale (OS) by morphological characteristics alone. Morphological results indicated that MS and OS differ significantly in terms of scale width, facial area, basal area, volume, length-to-width ratio and width-to-thickness ratio. Correlation analysis showed that scale width was significantly positively correlated with both the quantity and quality of regenerated bulblets. Among the cultivars, starch and soluble sugars accounted for 50–80% of scale DW. And a higher initial ratio of sucrose to starch in scales was more conducive to the bulblets formation. Although light had no effect on the incidence of bulblets, the formation of bulblets was positively enhanced, and better morphological consistency was obtained. This present study achieved a comprehensive morphological and nutritional analysis focused on bulblet formation capacities of scales from different positions of lily bulbs via scaling propagation, laying a foundation for future molecular studies on bulblet formation.

Bulblet Regeneration of Endemic Muscari adilii Güner & Duman on MS Media Having 6-Benzylaminopurine-α-Naphthaleneacetic

Bulblet Regeneration of Endemic Muscari adilii Güner & Duman on MS Media Having 6-Benzylaminopurine-α-Naphthaleneacetic

Early Sucrose Degradation and the Dominant Sucrose Cleavage Pattern Influence Lycoris sprengeri Bulblet Regeneration In Vitro.

Bulblet formation and development determine the quantitative and qualitative traits, respectively, of bulb yield for most flowering bulbs. For Lycoris species, however, the underlying molecular mechanism remains elusive. Here, clonal bulblets of Lycoris sprengeri (Ls) derived from the same probulb were used as explants to establish efficient and inefficient in vitro regeneration systems by adjusting the 6-benzyladenine (BA) concentrations in media. BA application did not change the biological processes among groups but led to earlier decreases in sucrose and total soluble sugar (TSS) contents. Correlation analyses showed that the BA treatments changed the interaction between carbohydrate and endogenous hormone contents during bulblet regeneration. We found that two sucrose degradation enzyme-related genes, cell wall invertase (CWIN) and sucrose synthase, exhibited exactly opposite expression patterns during the competence stage. In addition, the regeneration system that obtained more bulblets showed significantly higher expression of LsCWIN2 than those that obtained fewer bulblets. Our data demonstrate the essential role of BA in accelerating sucrose degradation and the selection of a dominant sucrose cleavage pattern at the competence stage of in vitro bulblet regeneration. We propose that a relatively active CWIN-catalyzed pathway at the competence stage might promote bulblet regeneration, thus influencing bulb yield.

In vitro regeneration of bulblet using two and four bulb-scales explants of summer snowflake (Leucojum aestivum L.)

Abstract Leucojum aestivum is a valuable and endangered plant species with bulb scales best suited as explants in micropropagation. In the current study, its micropropagation was investigated by using two different explants and various concentrations and combinations of plant growth regulators (PGRs). Bulbs were first disinfected with benomyl® for 5 hours. After meeting the chilling requirements, two-scale and four-scale explants were provided for direct and indirect organogenesis. Explants were exposed to hot water, 70% ethanol and 2.5% sodium hypochlorite for further disinfestation. Four-scale explants were treated with different concentrations and combinations of naphthaleneacetic acid (NAA), 6-benzyladenine (BA), and kinetin (Kin) for bulblet regeneration. For callogenesis, 0.5 mg L-1 of BA combined with 1, 2, 3, 4, 5 or 6 mg L-1 of 2,4-dichlorophenoxyacetic acid (2,4-D) were applied. Regarding two-scale explants, different combinations and concentrations of BA, Indole-3-butyric acid (IBA) and NAA were used for bulblet induction, and various combinations of Indoleacetic acid (IAA), NAA, 2, 4-D and BA were used for callus induction. None of the two-scale explants responded to the bulblet regeneration and callus induction media. Unlike, four-scale explants regenerated bulblets and roots in the control medium and MS media enriched with different PGRs. Callus was generated on MS medium supplemented with 2,4-D and BA, and indirect regeneration was observed in some cases. On the control medium, the regenerated roots had a natural form, but in PGRs-rich media, they were deformed. Regarding the regeneration percentage, bulblet number and length and root length, no significant differences were found between the control and the best PGR-treatment in each case. Therefore, it seems logical suggesting not to use PGRs, which will considerably reduce the costs at large-scale production.

Clonal bulblet regeneration and endophytic communities profiling of Lycoris sprengeri, an economically valuable bulbous plant of pharmaceutical and ornamental value

Lycoris Herb. is valuable as a natural source of starch, broad-spectrum fungicides, and natural compounds with antimicrobial, anticancer and is an antiviral abilities, and for ornamental and urban landscaping plant. The unlimited massive exploitation of Lycoris bulbs has led to a considerable threat to its wild resources and natural habitats. In this study, an efficient protocol for clonal bulblet regeneration of Lycoris sprengeri via seed explants was developed for resource protection and to satisfy the needs of the pharmaceutical and ornamental industries. Contamination-free probulbs were obtained at a rate of approximately 88.7% by using a combination of treatment with HgCl2 for 10 min and NaClO for 5 min to achieve surface sterilization. The endophytic community profiling of seed explants provided a comprehensive overview of changes in the Lycoris endophytes before and after effective surface sterilization. Additionally, the survival of the majority of endophytes without causing secondary contamination suggests the potential utilization of endophytes for the production of novel natural products in vitro. Well-rooted probulbs obtained from Murashige and Skoog’s (MS) medium containing 3% sucrose and 0.2 mg l-1 α-naphthaleneacetic acid (NAA) were selected as secondary explants for multiplication. Cross-cutting without complete separation was superior to complete separation for bulblet multiplication. Maximum bulblet multiplication was achieved in 4.0 mg l−1 6-benzyladenine (BA) and 1.0 mg l-1 NAA with 3% sucrose via direct shoot organogenesis. We were able to transplant approximately 95% of the rooted regenerated bulblets in a natural field after acclimatization. Genetic stability assessment of the in vitro regenerated bulblets was performed using simple sequence repeat (SSR) markers and flow cytometry analysis, with no observed difference compared to seed-germinated probulbs.

Effect of photoperiod and plant growth regulators on in vitro mass bulblet proliferation of Narcissus tazzeta L. (Amaryllidaceae), a potential source of galantamine.

Narcissus tazetta L., a bulbous plant belongs to the Amaryllidaceae family, contains alkaloid galantamine (GAL) with acetylcholinesterase inhibitory activity which has been recently considered to treat Alzheimer’s disease (AD). In the current work, the effect of photoperiod (16/8 h light/dark and 24 h dark) and various concentrations of NAA, BAP, and GA3 (0, 0.5, 1 and 2 mg l‒1) on the in vitro mass bulblet regeneration of N. tazetta was studied. The GAL production ability of the regenerated bulblets was assessed by HPLC-UV-MS. Light treatments significantly affected the number of bulblet and leaf, the ratio of bulblet/leaf, and leaf length. The maximum number of bulblet (31.0 ± 1.58) and leaf (13.3 ± 1.33) was recorded from the cultures fortified with NAA and BAP (2 mg l‒1) kept in 16/8 h light/dark, while the maximum leaf length (2.1 ± 0.92 cm) was measured on the MS medium containing 0.5 mg l‒1 NAA and 2 mg l‒1 BAP incubated in the same photoperiod. The average ratio of bulblet proliferation per explant was significantly different between studied photoperiod (1.1 ± 0.86) and 24 h dark (0.62 ± 0.31). The regenerated bulblets contained 40 and 20 µg g‒1 DW GAL underexposed photoperiod and 24 h dark, respectively. This information could be useful in the commercial production of GAL as a valuable anti-AD compound through in vitro mass bulblet proliferation of N. tazetta.

In vitro propagation of a cut flower variety Muscari armeniacum Leichtlin ex Baker through direct bulblet proliferation pathways

Muscari armeniacum is one of the important ornamental cut flowers in floriculture industry, which is native to Southern Europe, North Africa, Western Asia and Asia Minor. In this study, bulb explants (basal plate of bulb having meristems), bulb scales, and leaf segments from in vitro derived bulblets were cultured in Murashige and Skoog (MS) medium with different plant growth regulator concentrations and combinations to assess their effects on different bulblet organogenesis pathways. The results demonstrated that cytokinin in combination with auxin is required for both axillary and adventitious bulblet regeneration. Benzyladenine (BA) combined with α-naphthalene acetic acid (NAA) showed significant effects compared to other growth regulator combinations tested. The 4.0 µM BA + 2.0 µM NAA was found to be the best for axillary bulblet formation from bulb explants. Likewise, bulb and leaf segments showed the best response in adventitious bulblet organogenesis when they were cultured in BA-NAA combinations. Out of several concentrations of BA with NAA, 4.0 µM BA + 1.0 µM NAA was optimum for adventitious bulblet regeneration. Bulblets, properly isolated from both axillary and adventitious proliferation systems, showed maximum percentage of rooting on half strength MS medium containing 2.0 µM indole-3-butyric acid (IBA). However, all the auxins at more than 2.0 µM concentration showed reduced root formation through either callus formation at the base of shoots or malformation of roots. All the in vitro regenerated plantlets were successfully acclimatized under ex vitro environment with 60% survival rate using garden soil.

Efficient somatic embryogenesis and bulblet regeneration of the endangered bulbous flower Griffinia liboniana

Using flower organs as primary explants and via somatic embryogenesis, we developed an efficient protocol for bulblet regeneration from in vitro-derived seedlings (bulblets) of Griffinia liboniana. Callus induction was tested on five types of floral organ (perianth, filament, pedicel, ovary and anther) in the presence of three combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (6-BA). Filament constituted the most responsive primary explant for regenerative callus induction, and the highest frequencies of callus induction (63.0 ± 1.9%) and numbers of differentiated buds (3.7 ± 0.3 buds/callus) were found on Murashige and Skoog (1962) medium (MS) supplemented with 1.0 mg L−1 2,4-D and 1.0 mg L−1 6-BA. Starting with in vitro-derived bulblets (0.8–1.5 cm in diameter), somatic embryo (SE) formation occurred within 6 weeks, followed by 8 weeks for SE germination and development on PGR-free media. The highest percentage (78.9 ± 2.2%) of embryogenesis was obtained on MS media supplemented with 0.5 mg L−1 6-BA and 1.5 mg L−1 2,4-D, with an average of 28.0 ± 2.1 bulblets/explant. Well-rooted bulblets were successfully acclimated to ex vitro conditions. A stable ploidy level of the regenerated bulblets was confirmed by flow cytometry (FCM) analysis. This is the first report about micropropagation methods of G. liboniana and constitutes an efficient and reusable method for bulblet regeneration of this endangered species. Additionally, this protocol enables large-scale vegetative production, germplasm preservation and genetic engineering of endangered Griffinia species.

In Vitro Bulb Regeneration from Stem Explants of Endemic Geophyt Muscari aucheri (Boiss.) Baker

Muscari aucheri (Boiss.) Baker bears beautiful and attractive purplish blue flowers bloom between May and June; it grows stony slopes, mountain pastures an altitude of 1000-3000 meters. The natural populations of Muscari aucheri , is seriously affected by increased environmental pollution, urbanization and grazing. Therefore, this study aimed to accelerate multiplication by devising a strategy for an efficient in vitro bulblet regeneration system of Muscari aucheri using stem explants on MS medium containing 1, 3, 5 mg/l TDZ plus 0, 0.1, 0.2, 0.4 mg/l NAA (12 combinations). The stem explants induced direct bulblet regeneration on explants. Maximum mean number of bulblets per explant was noted on MS medium containing 3 mg/l TDZ + 0.4 mg/l NAA and maximum mean number of bulb diameter was noted on MS medium containing 5 mg/l TDZ + 0.2 mg/l NAA. The regenerated bulblets were isolated from stem explants and cultured on MS medium containing 40 g/l sucrose; where they gained diameter and rooted

Advancement in protocol for in vitro seed germination, regeneration, bulblet maturation, and acclimatization of Fritillaria persica

The present study addressed the successful in vitro seed germination, bulblet regeneration, increased bulblet size, hardening, and acclimatization of Persian lily (Fritillaria persica). Seed germination rate was recorded as 86.7% and 96.7% after 2 and 3 months of cold treatment at 4 °C, respectively. Bulblet explants taken from a germination experiment were cultured on Murashige and Skoog (MS) medium supplemented with 0.5-2.0 mg/L thidiazuron (TDZ). Maximum bulblet regeneration frequency and bulblets per explants were achieved on a medium containing 2.0 mg/L TDZ. Leaf disc and leaf scale explants isolated from germinated seedlings were also cultured on MS medium containing 2-6 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) with a constant concentration of 0.2 mg/L kinetin. Both explants yielded the highest bulblet regeneration on MS medium containing 2 mg/L 2,4-D and 0.2 mg/L kinetin. A combination of 1.5 g/L agar and 1.6 g/L GELRITE resulted in a maximum bulblet diameter with a higher number of roots. The addition of 10 g/L NaCl to the culture medium resulted in the best root and leaf formation and secondary bulblet regeneration. Exposure of regenerated bulblets to 10 g/L NaCl at 4 °C for 2 months on a medium with 20 g/L sucrose was also found to be beneficial for acclimatization.

Effect of Different Plant Growth Regulators on Direct Bulblet Regeneration from Scale Explants of Fritillaria imperialis

Effect of Different Plant Growth Regulators on Direct Bulblet Regeneration from Scale Explants of Fritillaria imperialis

Retraction: Uranbey S. In vitro bulblet regeneration from immature embryos of endangered and endemic Muscari azureum. Arch biol sci. 2011; 63(1):209-15 doi: 10.2298/ABS1101209U

This is a notice of retraction of the article: In vitro bulblet regeneration from immature embryos of endangered and endemic Muscari azureum, published in the Archives of Biological Sciences in 2011, Vol. 63, Issue 1. The Editor-in-Chief has been informed that this paper is a duplicate of an earlier paper: Uranbey S. In vitro bulblet regeneration from immature embryos of Muscari azureum. Afr J Biotechnol. 2010;9(32):5121-5. This claim is correct and the entire paper is a verbatim copy of the earlier one. After confirmation of this fact, the Editor-in-Chief of the Archives of Biological Sciences has decided to retract the paper immediately. We apologize to the readers of the journal that it took so many years to notice this error and to retract the paper. We request readers of the journal to directly get in touch with the editorial office and the editors of the journal for similar cases in the future, so that they can be handled promptly. <br><br><font color="red"><b> Link to the retracted article <u><a href="http://dx.doi.org/10.2298/ABS1101209U">10.2298/ABS1101209U</a></b></u>

Factors affecting efficient in vitro micropropagation of Muscari muscarimi Medikus using twin bulb scale

Endemic Muscari muscarimi Medikus is the most fragrant plant among Muscari species and has a high ornamental potential. The natural populations of M. muscarimi, are severely affected by increased environmental pollution and urbanization. There is a need to develop a micropropagation method that should serve effectively for commercial propagation and conservation. Therefore, the study targeted to set up a strategy for efficient in vitro bulblet regeneration system of M. muscarimi using twin scale bulb explants on 1.0×MS medium containing 4.44, 8.88, 17.76μM BAP (6-Benzylaminopurine) plus 2.685, 5.37, 10.74μM NAA (α-Naphthalene acetic acid). Maximum number of 19 daughter axillary bulblets and 16 daughter adventitious bulblets per twin bulb scale explant was regenerated on 1.0×MS medium containing 17.76μM BAP plus 10.74μM NAA and 17.76μM BAP plus 2.685μM NAA respectively. The daughter bulblets regenerated on twin bulb scales on 8 out of 9 regeneration treatment could be easily rooted on 1.0×MS medium containing 4.9μM IBA (Indole-3-butyric acid). The daughter bulblets regenerated on 9th treatment (1.0×MS medium containing 17.76μM BAP plus 10.74μM NAA) were transferred to 1.0×MS medium containing 30g/l sucrose to break negative carry over effect of this dose of BAP–NAA, where they grew 2–3 roots of variable length. Daughter bulblet diameter was increased by culturing them on 1.0×MS medium containing 4.44μM BAP plus 5.37μM NAA. The results verified that both age and the source of explants had significant effect on regeneration. In another set of experiments, twin scales were obtained from in vitro regenerated daughter bulblets, although they induced bulblets, yet their bulblet regeneration percentage, mean number of bulblets per explant and their diameter were significantly reduced. In vitro regenerated bulblets were acclimatized in growth chamber under ambient conditions of temperature and humidity on peat moss, where they flowered. The study provides important information about selection of suitable micropropagation medium, strategies to improve bulblet diameter and rooting of M. muscarimi which offers a scope for commercial propagation.

In vitro micropropagation from immature embryos of the endemic and endangered Muscari muscarimi Medik.

An efficient in vitro bulblet production procedure from immature zygotic embryos of endemic and endangered Muscari muscarimi Medik. was described in the current study. Zygotic embryos were first isolated from immature seeds and cultured on different nutrient media compositions supplemented with various combinations of \alpha-naphthalene acetic acid (NAA), picloram, dicamba, 6-benzylaminopurine (BAP), and thidiazuron (TDZ). The best bulblet regeneration (59 bulblets per explant) was achieved in Murashige and Skoog (MS) medium containing 4 mg/L BAP and 0.5 mg/L NAA after 1 year of culture initiation. Regenerated bulblets were then transferred into MS medium without plant growth regulators for rooting. Bulblets produced well-developed root systems and increased their size on this medium after 2 months. All rooted bulblets were successfully transplanted into a potting mixture and acclimatized to ambient conditions.

Improved in vitro regeneration and propagation of Tunceli garlic (Allium tuncelianum L.)

SummaryOver-exploitation of endemic and threatened Tunceli garlic [Allium tuncelianum (Kollman) N. Ozhatay, B. Mathew & Siraneci] for household purposes has threatened the species and requires that a reliable, improved tissue culture protocol be developed for its conservation. Leaf tips, the middle portions of leaves, leaf bases, vertically-sectioned halved or quartered bulbs, horizontally-sectioned upper and lower bulb halves, and root tip explants were cultured on 1.0× Murashige and Skoog (MS) medium containing 1.0, 2.0, 3.0, 4.0, or 5.0 mg l–1 2,4-dichlorophenoxyacetic acid (2,4-D) or 1.0, 2.0, 3.0, 4.0, or 5.0 mg l–1 6-benzylaminopurine (BAP) plus 0.5 mg l–1 α-naphthaleneacetic acid (NAA). The results indicated that root tip explants were most suitable for bulblet regeneration on 1.0× MS medium containing 5.0 mg l–1 BAP plus 0.5 mg l–1 NAA. All other explants failed to regenerate on different concentrations of BAP plus NAA, or on 2,4-D. The regenerated bulblets were acclimatised at 24º ± 1ºC and 80.0% relative humidity under growth chamber conditions, then transferred to pots containing peat-moss in a greenhouse. The results will be important for garlic breeders and researchers.

Direct and indirect shoot and bulblet regeneration from cultured leaf explants of Lilium pumilum, an endangered species

Alternative methods of in vitro cloning that involve both adventitious (direct) and callus intermediate (indirect) pathways were investigated for the endangered species Lilium pumilum. Plantlet regeneration was obtained from leaf explants, cultured on Murashige and Skoog (MS) basal medium supplemented with various combinations of auxins and cytokinins at different concentrations. About 30% of the explants directly formed adventitious shoots on MS medium containing 8.88 μM 6-benzyladenine (BA) and 2.69 μM α-naphthaleneacetic acid (NAA). For production of regenerable callus, callus formation followed by shoot induction was best when explants were initially cultured on MS medium supplemented with 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Regenerable calli were yellow or purple and readily regenerated shoots when subcultured onto MS medium containing 2.22 μM BA and 1.61 μM NAA. About 78% of the calli were able to produce adventitious shoots. Shoots were rooted on half-strength MS medium supplemented with 1.34 μM NAA and were successfully acclimatized to greenhouse conditions. This report describes an efficient method for the in vitro multiplication of whole plants from leaf explants of the endangered species L. pumilum.

Effects of Hormonal and Basal Nutrient Medium on In vitro Regeneration of an Ornamental Plant - Muscari armeniacum Leichtlin. ex Baker

In vitro propagation system has been developed for an important ornamental and medicinal plant, Muscari armeniacum Leichtil. ex Bak. A range of a cytokinin and auxin concentration has been investigated for axillary bulblet proliferation, and direct and indirect adventitious bulblet regeneration from the explants whole bulb, one fourth part of bulb, bulb-scale of ex vitro (field grown mature bulb), and only leaf-sheath explants of in vitro grown bulblet. Axillary bulblet regeneration occurred on MS containing 2.0 - 8.0 ?M BAP or Kn. Direct adventitious bulblets were induced successfully on MS basal medium supplemented with various concentrations of BAP or Kn (1.0 - 4.0 ?M) in combi-nation of either NAA, IBA, or 2,4-D (0.5 - 4.0 ?M). The maximum frequency of adventitious bulblets regeneration occurred from both bulb-scale and leaf-sheath explants on MS with 4.0 ?M BAP and 2.0 ?M NAA, IBA, or 2,4-D. The highest frequency (95.5%) of indirect adventitious bulblets was obtained from in vitro grown leaf-sheathderived callus on MS containing 4.0 ?M BAP with 1.0 ?M 2,4-D whereas, highest number (80.2) and average length (55.5 cm) of bulblets were obtained on MS supplemented with 4.0 ?M BAP and 1.0 ?M NAA. In vitro grown bullets was rooted successfully on MS with 0.5 - 4.0 ?M of IBA, NAA, or IAA. The rooted bulblets were transferred to garden soil and successfully established under ex vitro environment. DOI: http://dx.doi.org/10.3329/ptcb.v22i2.14200 Plant Tissue Cult. & Biotech. 22(2): 113-126, 2012 (December)

In vitro propagation of Lilium (Lilium longiflorum)

Lilium longiflorum has a wide applicability in the floral industry as cut flower and potted plants. Micropropagation in bulb plants is an alternative to the conventional methods for vegetative propagation which attracts much attention because of its advantages such as many fold multiplication rate. In present study an efficient micropropagation protocol in L. longiflorum has been developed. The highest bulblet regeneration (94%), maximum number of shoots (22), maximum shoot length (12 cm) and heaviest bulblets with average fresh weight (365 mg) were obtained in MS medium supplemented with BA 2.0 mg/l + NAA 0.5 mg/l. The treatment of MS medium with NAA 2.0 mg/l was most effective for rooting and gave maximum rooting (93%), maximum number of roots (19) and highest root length of 5.2 cm as compared to other treatments. The micropropagation from inoculation of scales to hardening was completed only in six months.