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.     

Somatic embryogenesis in banana (Musa acuminata AAA cv. Grand Naine): effect of explant and culture conditions

An improved, rapid, reproducible, and simple protocol has been developed for somatic embryogenesis in banana cv. ‘Grand Naine’ using explants derived from actively growing multiple shoot cultures. Many restrictive factors remain in banana embryogenesis such as long duration, unpredictability, and a high degree of genotype dependence. In the present study, we used split shoot tips from 4-wk-old cultures as explants. Somatic embryos were induced in 15 d directly in Murashige and Skoog (MS) medium supplemented with different combinations of 0–8.28 μM picloram and 0.22–4.44 μM 6-benzylaminopurine (BA) without callus formation. Maximum embryo induction (100%) occurred when 4.14 μM picloram and 0.22 μM BA were used. Conversion of somatic embryos into plantlets occurred sporadically (2–3%) in MS medium containing α-naphthalene acetic acid (NAA; 0.53–2.68 μM) together with BA (2.22–44.39 μM), or thidiazuron (4.54 μM) plus glutamine (200 mg/L). This protocol is far superior to those already reported for fast and high frequency induction of somatic embryo. In liquid agitated culture, individual embryos separated easily and produced a large number of secondary embryos within 10 d which, upon transfer to filter paper overlaid on MS liquid medium supplemented with 4.44 μM BA, resulted in conversion (3%) into plantlets.     

Morphogenic capability of <Emphasis Type="Italic">Gentiana kurroo</Emphasis> Royle seedling and leaf explants

Experiments have been carried out on seedling and primary leaf explants of Gentiana kurroo Royle. Morphogenic capacities of cotyledons, hypocotyls and roots were investigated using MS (1962) medium supplemented with 4.64 μM kinetin and 2.26, 4.52 or 9.04 μM 2,4-D. Percentage of callusing explants for each combination was inversely proportional to numbers of obtained embryos. Cotyledons showed the highest morphogenic capabilities. To assess the morphogenic potential of leaf explants, 189 combinations of auxin (NAA, dicamba and 2,4-D) and cytokinin (kinetin, BAP, zeatin, CPPU and TDZ) in different concentrations were tested. The presence of NAA with BAP and dicamba with zeatin produced the greatest number of differentiated somatic embryos. Microscopic analysis of responsive explants led to identifying rhizogenic centers, non-embryogenic and embryogenic cells. The best embryo conversion into germlings was obtained on MS medium containing 4.46 μM kinetin, 1.44 μM GA₃ and 2.68 μM NAA or ½ MS. Both media were supplemented with 4.0% sucrose and 8.0% agar. Depending on explant origin and conversion medium, 55.8–71.0% of somatic embryos developed into germlings and plants.     

Multiple shoot induction and regeneration of whole plants from cotyledonary node and nodal explants of Sterculia urens Roxb., a gum yielding tree

Plant regeneration from the nodal explants of 1-month-old in vitro grown plants and cotyledonary node explants of 15-days-old seedlings of Sterculia urens is reported. Nodal explants were grown on MS medium supplemented with various growth regulators like BA, KIN and TDZ. For shoot induction 13.3 μM BA, 0.9 μM TDZ and 9.3 μM KIN were found optimum. Among the three growth regulators 0.90 μM TDZ was used for the growth of cotyledonary node explants. An average of 8.6 shoots per node and 11.2 shoots per cotyledonary node were observed in 4 to 5 weeks. These shoots were subsequently rooted in vitro on half strength MS medium containing various concentrations of auxins like IBA and NAA. The best concentrations for rooting of shoots were 19.7 μM IBA and 16.1 μM NAA. Plantlets were acclimatized to ex vitro conditions and established in the field.     

In vitro plantlet regeneration of Capsicum chinense Jacq. cv. ‘Bhut jalakia’: hottest chili of northeastern India

Chili (Capsicum chinense) cv. ‘Bhut jalakia’ is used in India for extraction of oleoresin and capsaicin as it is characterized by a very high capsaicin content. The conventional method of propagation of ‘Bhut jalakia’ is through seeds, but this is beset by short viability and low germination rates. Developing a suitable regeneration protocol for ‘Bhut jalakia’ was the focus of this study; as to date, in vitro regeneration for this cultivar has not been investigated. Cotyledon and shoot tip explants were cultured on Murashige and Skoog (MS) media supplemented with different concentrations of cytokinins and auxins. In the case of cotyledon explants, MS medium supplemented with 6-benzylaminopurine (BAP) at 35 μM and kinetin (KIN) at 15 μM were found to be optimal (4.00?±?0.57) for induction of multiple shoots per explant, whereas BAP at 14.8 μM and KIN at 60 μM were best (5.00?±?0.57) for growth of shoot tip explants. Shoots developed from cotyledon explants produced the maximum (8.67?±?0.32) number of roots on MS medium supplemented with low concentration (2.6 μM) of 2-naphthaleneacetic acid (NAA). Supplementation of indole-3-butyric acid (IBA) at 5 μM was found optimal for root formation (16.67?±?2.60) for shoots derived from of shoot tip explants. One month after transfer of in vitro regenerated plantlets to various potting mixes, the highest survival rate (40%) was observed in a mixture of sand, soil, and cow dung in a ratio of 1:1:1. Thus, both shoot tip and cotyledon explants may be cultured on MS medium modified with BAP, IBA, NAA, and KIN to regenerate ‘Bhut jalakia’ chili plants within 90 d.     

TDZ-induced plant regeneration in <Emphasis Type="Italic">Brassica oleracea</Emphasis> L. var. <Emphasis Type="Italic">botrytis</Emphasis>: effect of antioxidative enzyme activity and genetic stability in regenerated plantlets

The effects of various combinations of plant growth regulators on regeneration potential from seedling-derived leaf tissues of Brassica oleracea L. var. botrytis were evaluated. Callus was induced from 2-wk-old leaf explants. The explants were incubated on Gamborg’s (MSB₅) medium. The maximum frequency of callus induction (85.56%) was recorded on MSB₅ medium supplemented with 9.1 μM thidiazuron (TDZ) and 0.5 μM α-naphthaleneacetic acid (NAA). Optimum shoot induction (54.44%) was obtained on MSB₅ medium supplemented with 4.5 μM TDZ and 0.5 μM NAA. The maximum number of shoots per explant (5.33) was recorded on MSB₅ medium with 4.5 μM TDZ and 0.5 μM NAA, whereas the maximum shoot length (4.86 cm) was recorded for shoots cultured on MSB₅ medium supplemented with 4.5 μM TDZ and 5.7 μM gibberellic acid (GA₃). However, optimum root induction (71.11%) occurred on half-strength Murashige and Skoog basal medium supplemented with 4.9 μM indole-3 butyric acid (IBA). Studies on the antioxidant activity of superoxide dismutase, ascorbate peroxidase, and peroxidase in seedlings, callus, regenerated shoots, and regenerated plantlets cultured on 4.5 μM TDZ and 0.5 μM NAA medium revealed the roles of these key antioxidative enzymes in callus induction and regeneration. The genetic stability of the regenerated plantlets was assessed using inter simple sequence repeat primers. The monomorphic amplification products confirmed true-to-type in vitro regenerated plants. This in vitro regeneration method can be useful in the large-scale production of genetically uniform plants, for genetic transformation, and conservation of elite germplasm of plant species.     

The positive effect of arabinogalactan on induction of somatic embryogenesis in Quercus bicolor followed by embryo maturation and plant regeneration

This is the first report on the successful induction of somatic embryogenesis in swamp white oak from leaf and shoot apex explants excised from in vitro shoot cultures derived from 6- to 7-year-old trees. We demonstrated that arabinogalactan from larch wood (2–4 mg/L) promoted embryogenesis in the three genotypes evaluated by increasing the frequency of somatic embryogenesis, the embryogenic sites per explant, and by speeding the onset of embryo initiation. The explants were cultured sequentially on three culture media consisting of Murashige and Skoog (MS) salts and vitamins supplemented with 500 mg/L casein hydrolysate and different concentrations of α-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BA). Somatic embryogenesis induction frequencies of up to 12.4, 4.5, and 0.7 % were obtained for the three genotypes. Clonal embryogenic lines were maintained by repetitive embryogenesis following culture on MS medium containing 0.44 μM BA with or without 0.27 μM NAA. Before germination, cotyledonary-stage embryos were cultured for 4 weeks in maturation medium (MS medium with half-strength macronutrients) containing 6 % sorbitol. Germination response was significantly improved by applying a 2-month cold storage as a post-maturation treatment. The mineral formulation and plant growth regulator content of the germination medium influenced the frequency of plantlet conversion with the best results achieved on Gresshoff and Doy medium with BA (0.25–0.44 μM). This procedure resulted in over 50–60 % of germinating embryos exhibiting continuous root growth and either epicotyl elongation or shoot development.     

In vitro propagation,genetic and phytochemical assessment of <Emphasis Type="Italic">Habenaria edgeworthii</Emphasis>: an important Astavarga plant

An efficient in vitro propagation protocol for Habenaria edgeworthii Hook. f. ex. Collett using seed-derived callus was established. The maximum seed germination was observed in Murashige and Skoog (MS) medium supplemented with 1.0 μM α-naphthalene acetic acid (NAA). Induction of callus was achieved on full and ½-strength MS medium supplemented with 1.0 μM NAA. The highest number of shoot (11.9 shoots/explant) was achieved in MS medium supplemented with 0.1 μM 6-benzyladenine (BA) and 0.01 μM NAA. Further, elongated shoots when transferred to ½-strength MS rooting medium with different auxin concentrations induced roots (41.6–83.3%) and tubers (0–20.8%); however, a maximum of 87.5% rooting was achieved in a plant growth regulator (PGR)-free MS medium. Rooted shoots (plantlets) when transferred to a mixture of soil:sand:perlite (1:1:1 ratio) resulted in 68% survival. Inter-simple sequence repeats (ISSR) markers confirmed the genetic stability among regenerated plants. The phytochemical analysis of tissue culture-raised tubers showed higher phenolic content than wild tuber. The regeneration protocol developed in this study provides a basis for germplasm conservation and harnessing the total phenol and phenolic compounds of H. edgeworthii. Further, the methods can open avenues for application in other Orchidaceous plants of the Indian Himalayan region.     

Factors affecting plantlet regeneration from in vitro cultured immature embryos and cotyledons of Prunus mume “Xue mei”

Using immature embryos and cotyledons as explants, a successful immature embryo culture and efficient plant direct regeneration via organogenesis from cotyledons, which showed different patterns, was established for the “Xuemei” cultivar of Prunus mume. For immature embryo culture, high frequency plantlet forming (89.5%) from embryo axis was obtained on half-strength Murashige and Skoog (½MS) medium supplemented with 13.2 μM 6-benzyladenine (BA) and 2.7 μM 1-naphthaleneacetic (NAA). At the same time, shoots direct differentiation from cotyledons with the embryo axis development was also observed on ½MS medium containing 2.2 μM BA together with different combinations of NAA (2.7, 5.4 μM) and indole-3-butyric acid (IBA) (0, 2.5, 5.0 μM). Better results were achieved when embryo axes were removed from cotyledons and cultured on ½MS medium supplement with 13.2 μM BA, 2.7 μM NAA (72.9%) or 2.2 μM BA, 2.2 μM thidiazuron (TDZ), and 2.7 μM NAA (84.2%), respectively. Regenerated shoots were successfully rooted on ½MS or Woody Plant medium (WPM) supplemented with 2.5–5.0 μM IBA. The effect of embryo axes, BA and TDZ, on cotyledons’ regeneration were investigated in detail. The rooted plantlets were transferred to soil successfully with normal morphology.     

Propagation of mature <Emphasis Type="Italic">Quercus ilex</Emphasis> L. (holm oak) trees by somatic embryogenesis

Somatic embryogenesis from in vitro leaf and shoot apex explants excised from axillary shoot cultures established from two mature Quercus ilex trees has been developed. Somatic embryos (SE) were obtained from both explant types and genotypes evaluated, although embryogenic frequencies were influenced by the genotype, auxin concentration, and explant type. The explants were cultured on Murashige and Skoog salts and vitamins, supplemented with 500 mg L^?1 casein hydrolysate (CH) and different concentrations of indole-3-acetic acid or α-naphthalene acetic acid (NAA) in combination with 2.22 µM 6-benzylaminopurine (BA). In both genotypes, shoot apex explants were more responsive than leaf explants. The best results were obtained with apex explants of clone Q3 (11%) cultured on medium with 21.48 µM NAA plus 2.22 µM BA. This combination was also effective for initiating SE from leaf explants, although the induction rates were lower (1–3%). Embryogenic lines were maintained by repetitive embryogenesis following culture of nodular embryogenic structures on Schenk and Hildebrand medium without plant growth regulators. Low embryo multiplication rates were obtained when torpedo or early cotyledonary SE were used as initial explant for embryo proliferation, or when glutamine or CH (500 mg L^?1) was added to proliferation medium. For germination, cotyledonary-stage SE were isolated and stored at 4 °C for 2 months. After cold storage, SE were cultured on germination medium consisting of Gresshoff and Doy medium, supplemented with 0.44 μM BA and 20 μM silver thiosulphate. Under these conditions, plantlets were regenerated from 21 to 66.7% of the SE generated for both genotypes.     

A rapid and efficient in vitro shoot regeneration protocol using cotyledons of London plane tree (<Emphasis Type="Italic">Platanus acerifolia</Emphasis> Willd.)

A rapid, prolific and reproducible protocol for in vitro shoot regeneration from mature cotyledons of Platanus acerifolia has been developed. The influences of different plant growth regulator (PGR) combinations and donor seedling ages on shoot regeneration were investigated. The results showed that the application of BA in conjunction with NAA was the most effective PGR combination for the induction of shoot regeneration. When cotyledon explants of 5-day-old seedlings were incubated on MS basal medium supplemented with 4.0 mg L^?1 BA and 0.2 mg L^?1 NAA, 67.6?±?4.9% of the cotyledon segments produced adventitious shoots. These regenerated shoots were initially formed as stunted rosette cluster forms and were encouraged to elongate to produce distinct shoots by transfer onto MS medium containing 0.5 mg L^?1 BA and 0.05 mg L^?1 NAA; the resulting mean number of adventitious shoots per explant was 5.81?±?0.36. The elongated shoots were readily induced to root (i.e. 89.3% of shoots) by incubation on ½-strength MS medium supplemented with 0.1 mg L^?1 IBA. This is the first report of an efficient in vitro shoot regeneration protocol for P. acerifolia through direct organogenesis using cotyledon explants. Hence, this provides a more efficient basis for the Agrobacterium-mediated genetic transformation of Platanus than previously available.     

High-frequency plantlet regeneration and multiple shoot induction from cultured immature seeds of Rhynchostylis retusa Blume., an exquisite orchid

Seeds of an exquisite orchid, Rhynchostylis retusa, germinated in vitro on ½ Murashige and Skoog (MS) medium supplemented with different concentrations of coconut milk (CM). Of the different concentrations of CM employed for seed germination, 15% gave optimum response. On this medium a maximum of 93% cultures produced seedlings 90 days after inoculation. Individual seedlings with a length of about 0.5 cm were subcultured on MS medium supplemented with various concentrations of 6-benzylaminopurine (BA) and α-naphthalene acetic acid (NAA), with or without activated charcoal (AC), for further growth. Seedling growth was maximum on MS medium supplemented with 6 μM BA, 0.2 μM NAA, and 1 g L^?1 AC. Here a maximum seedling length of 2.3 cm was observed after 1 month of culture. The seedlings were subcultured on MS medium supplemented with kinetin (Kn) or thidiazuron (TDZ), in the presence or absence of AC, for multiple shoot induction. A maximum multiple shoot number of 8.2 was observed on MS medium supplemented with 2 μM TDZ in the presence of AC. The shoots were rooted on ½ MS medium supplemented with 2 μM indole-3-butyric acid (IBA) and successfully transplanted to soil. Of the 45 plantlets transferred to soil 40 survived. The reproducible protocol standardized here will enable rapid propagation and conservation of this precious orchid.     

Recurrent somatic embryogenesis and plant regeneration from seedlings of <Emphasis Type="Italic">Hepatica nobilis</Emphasis> Schreb.

A method for secondary somatic embryogenesis was developed on embryos derived from embryogenic callus formed on Hepatica nobilis seedlings. Somatic embryogenesis (SE) was induced on seedlings (on the hypocotyl and epicotyl parts) grown on the Murashige and Skoog (1962) medium (MS) supplemented with 1 µM naphthaleneacetic acid (NAA), and/or 0.1 µM 6-benzyladenine (BA) and on medium without plant growth regulators (PGR). The best response of embryogenic callus formation was observed on the medium containing 1 µM NAA alone or with 0.1 µM BA. Individual somatic embryos, formed on embryogenic callus on the medium without PGR (MS0), at heart, torpedo and cotyledonary stage, were transferred to the media where secondary somatic embryo formation and development into plantlets occurred. Although the most efficient repetitive cycles of secondary SE were recorded for all stages of somatic embryos (heart, torpedo, cotyledonary) on the MS0 medium (77.8–87.4 %), secondary somatic embryos were also obtained on all media supplemented with cytokinins. The best rate of somatic embryos germination was achieved on MS media with 0.2 µM NAA and 2 µM BA, and 0.1 µM NAA and 1 µM BA (48.8–52.0 %) when more mature embryos (cotyledonary stage) were used. Plantlets grown from somatic embryos were successfully acclimatized to greenhouse conditions.     

Callus Induction and Shoot Regeneration from Stem, Rachis and Leaf Explants in Beach Pea (Lathyrus japonicus Willd)

Combinations of naphthaleneacetic acid (NAA) and benzyladenine (BA) affected the callus formation from stem, rachis and leaf explants of beach pea. Mature leaf pieces were best for inducing a callus; when cultured on Murashige and Skoog’s (MS) medium supplemented with 4.4μM BA at least 87% of the explants callused regardless of the NAA concentration. Optimal induction was achieved with 5.4 to 10.7μM NAA depending on the explants used. BA at 4.4μM was always more effective than at the lower concentration of 1.1μM. Multiple buds were induced from calli and formed shoots when transferred to MS medium supplemented with 2.7μM NAA + 4.4μM BA.     

Antioxidant activity evaluation of essential oil and RAPD analysis of in vitro regenerated Blumea mollis (D. Don) Merr.

Essential oil obtained from the leaves of Blumea mollis (D. Don) Merr. was analyzed by gas chromatography and gas chromatography coupled with mass spectroscopy (GC and GC–MS). Bicyclic sesquiterpene, β-caryophyllene, was identified as a major compound which accounted for 24.54 %. Antioxidant activity of oil was significantly higher than that of methanol extract of callus. Murashige and Skoog (MS) medium supplemented with 4.4 μM BA showed multiple shoot induction after 8 weeks of culture. 4.6 μM Kin showed in vitro flowering and direct organogenesis was observed from the leaf explants on medium containing 4.4 μM BA and 5.4 μM NAA. Rooted plantlets developed on half strength MS medium fortified with 2.1 μM IBA were hardened and percentage survival was recorded up to 70. RAPD analysis revealed a little genetic variation in micropropagated plants.     

High frequency multiple shoot induction from nodal segments and rhinacanthin production in the medicinal shrub Rhinacanthus nasutus (L.) Kurz

High frequency multiple shoots have been induced from nodal segments of Rhinacanthus nasutus (L.) Kurz., a potent anticancerous ethnomedicinal plant. For initiation of cultures, nodal segments were cultured on MS medium supplemented with various concentrations (1.0–5.0 μM) of 6-benzyladenine or thidiazuron (TDZ) alone or in combination with α-naphthalene acetic acid (NAA 0.5–1.0 μM). The optimum frequency of response (85 %) and shoot number (3.3) was observed on MS medium supplemented with 4.0 μM TDZ and 0.8 μM NAA. The shoots developed on initiation media were excised and nodal segments were subcultured on MS medium supplemented with TDZ (4.0 μM) and NAA (0.5–1.0 μM). This subculturing process was repeated thrice, each with 45 days of duration and the multiple shoot formation was recorded at the end of every subculture stage. The highest frequency of response (100 %) and number of multiple shoots (24.1) per explant were recorded at the end of the third subculture passage on MS medium supplemented with 4.0 μM TDZ and 0.8 μM NAA. The optimum rooting of shoots was observed on ½ MS medium fortified with 3.0 μM indole-3-butyric acid. The rooted plants were successfully transplanted to soil. The estimation of rhinacanthin (RC) content in shoots and roots was carried out in 6-month-old ex vitro plants (i.e., plants regenerated via in vitro culture) and field grown natural plants by high performance liquid chromatography. Both shoots and roots of naturally grown plants showed slightly higher RC content than ex vitro grown plants. The highest RC content (4.6 mg/g DW RC-C, 0.14 mg/g DW RC-D and 0.10 mg/g DW RC-N) was recorded in roots of naturally grown plants.     

In vitro propagation of two Iranian commercial pomegranates (Punica granatum L.) cvs. ‘Malas Saveh’ and ‘Yusef Khani’

An efficient in vitro propagation is described for Punica granatum L. using shoot tip and nodal explants. The influence of two basal medium, WPM and MS, and different plant growth regulators was investigated on micropropagation of the Iranian pomegranate cultivars, ‘Malas Saveh’ and ‘Yousef Khani’. For proliferation stage, media supplemented with different concentrations (2.3, 4.7, 9.2 and 18.4 μM) of kinetin along with 0.54 μM NAA was used. WPM proved to be more efficient medium compared to MS. The best concentrations of kinetin were 4.7 μM for ‘Malas Saveh’ and 9.2 μM for ‘Yousef Khani’, resulting in the highest number of shoots per explants, shoot length and leaf number. For both cultivars, half-strength WPM medium supplemented with 5.4 μM NAA was most effective for rooting of shoots. Rooted plantlets were successfully acclimatized and transferred into soil. The micropropagated plants were morphologically uniform and exhibited similar growth characteristics and vegetative morphology to the mother plants.     

Plant regeneration of non-toxic Jatropha curcas—impacts of plant growth regulators, source and type of explants

Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential as a biofuel plant, however, oil and deoiled cake are toxic. A non-toxic variety of J. curcas is reported from Mexico. The present investigation explores the effects of different plant growth regulators (PGRs) viz. 6-benzyl aminopurine (BAP) or thidiazuron (TDZ) individually and in combination with indole-3-butyric acid (IBA), on regeneration from in vitro and field-grown mature leaf explants, in vitro and glasshouse-grown seedlings cotyledonary leaf explants of non-toxic J. curcas. In all the tested parameters maximum regeneration efficiency (81.07%) and the number of shoot buds per explants (20.17) was observed on 9.08 μM TDZ containing Murashige and Skoog’s (MS) medium from in vitro cotyledonary leaf explants. The regenerated shoot buds were transferred to MS medium containing 10 μM kinetin (Kn), 4.5 μM BAP and 5.5 μM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with 2.25 μM BAP and 8.5 μM IAA. Rooting was achieved when the basal cut end of elongated shoots were dipped in half strength MS liquid medium containing different concentrations and combinations of IBA, IAA and NAA for four days followed by transfer to growth regulators free half strength MS medium supplemented 0.25 mg/l activated charcoal. The rooted plants could be established in soil with more than 90% survival rate.     

Effect of plant growth regulator combination and culture period on in vitro regeneration of spinach (Spinacia oleracea L.)

The objective of this study was to develop an efficient system for the regeneration of spinach plants (Spinacia oleracea L.) by investigating the factors influencing callus and shoot induction. All plant growth regulator (PGR) combinations tested induced callus with high frequency (73–100 %), and the combination of 5 μM α-naphthaleneacetic acid (NAA), 10 μM 6-benzyladenine (BA) and 0.1 μM gibberellic acid (GA₃) had the most significant effect on callus growth in term of weight (120.98 ± 22.56 mg). A high auxin-containing medium induced competent callus for shoot formation, while high cytokinin-containing media enhanced callus growth and made callus incompetent for shoot regeneration. Longer periods of callus induction in a high auxin-containing medium were required to form competent callus and led to a high regeneration capacity. The PGR combination shift from a high auxin to cytokinin ratio (ACR) to a low ACR resulted in highly efficient regeneration. Among the regeneration systems tested, the combination of 10 μM NAA and 0.3 μM GA₃ for callus induction for 6 weeks followed by 2 μM NAA and 5 μM BA resulted in the highest plant regeneration frequency (83.33 ± 6.43 %) and the highest number of plantlets per explant (7.93 ± 1.24). Somatic embryos at cotyledonary stage and plantlets were transferred to PGR-free medium to establish whole plants. Regenerated female plants grew well to maturity in the greenhouse (77.17 ± 9.80 %) and produced seeds (175.21 ± 28.01 firm seeds per plant).     

Efficient plant regeneration from petiole explants of West Indian gherkin (Cucumis anguria L.) via indirect organogenesis

An efficient protocol for in vitro organogenesis was achieved from callus-derived immature and mature petiole explants of West Indian gherkin (Cucumis anguria L.). Calluses were induced from immature petiole explants excised on 7-day-old in vitro seedlings and mature petiole explants of 40-day-old in vivo plants. The maximum frequency of immature petiole explants (98.0 %) and mature petiole (91.5 %) produced green, compact organogenic callus in Murashige and Skoog (MS) medium with Gamborg (B5) vitamins containing 30 g l^?1 sucrose, 8.0 g l^?1 agar and 4.0 μM naphthalene acetic acid (NAA) with 2.0 μM benzyl amino purine (BAP) after two successive subculture at 11 days interval. Adventitious shoots were produced from the organogenic callus when it was transferred to MSB₅ medium supplemented with 3.0 μM TDZ, 1.0 μM NAA and 0.05 mM L-glutamine with shoot induction frequency of immature petiole 45 shoots and mature petiole 40 shoots per explant. The shoots were excised from callus and elongated in MSB₅ medium fortified with 3.0 μM gibberellic acid (GA₃). Then elongated shoots were rooted in half strength MSB₅ medium supplemented with 3.0 μM indole 3-butyric acid (IBA). Histological analyses of the regeneration process confirmed the indirect organogenesis pattern. Plantlets with well-developed shoot and root systems were successfully acclimatized (95 %) in winter season and exhibited normal morphology and growth characteristics. The survival percentage differed with seasonal variations.