Indirect Organogenesis from the Leaf Explants of Medicinally Important Plant Curculigo orchioides Gaertn

Curculigo orchioides Gaertn is an important medicinal plant of commercial value. A protocol for indirect regeneration from the leaf explants was developed. The leaf explants on MS (Murashige & Skoog) basal medium fortified with 6-benzylaminopurine (9μM) gave rise to proliferating green callus. The calli regenerated shoots on subculturing in the MS medium with 1.1μM 6-benzylaminopurine which was the optimum concentration for multiplication. Rooting was observed in MS medium supplemented with α-napthaleneacetic acid (5.3μM), indole-3-butyric acid (1.2μM), and on MS basal medium free of plant growth regulators. The rooted plants were hardened and transferred to field with 80% survival.     

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.     

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.     

High-Frequency Regeneration by Abscisic Acid (ABA) from Petiole Callus of Jatropha curcas

Jatropha curcas L. is attaining worldwide interest as an important biofuel crop. Experiments were conducted to improve the prevailing micropropagation technique as well as to develop a new ex vitro rooting method for J. curcas plant regeneration. Regeneration and ex vitro rooting efficiency was enhanced by augmenting the culture medium with abscisic acid (ABA). Different concentrations of 6-benzylaminopurine (BAP) and indole-3-butyric acid (IBA) were tested for callus generation from both in vitro and in vivo explants (leaf and petiole) on Murashige and Skoog (MS) medium. The best regenerative callus was achieved on MS medium supplemented with BAP (4.44 μM) and IBA (2.45 μM) from in vitro-cultured petioles. Highest regeneration (91%) was achieved by culturing petiole callus on MS medium supplemented with BAP (8.88 μM), IBA (0.49 μM), and ABA (1.9 μM), whereas 61% regeneration was obtained from in vitro leaf callus. Shoot proliferation and elongation was achieved on BAP (2.22 μM) and IAA (8.56 μM) with 10–13 shoots per explants. Highest rooting (65%) was achieved from M1 shoots (BAP, IAA, and ABA) on MS medium supplemented with IBA (2.45 μM), naphthaleneacetic acid NAA (0.54 μM), and 0.02% activated charcoal. Ex vitro rooting of 1-mo-old M1 shoots obtained from the charcoal-containing medium resulted optimum rooting (>72%) when transferred to polybags containing sterile sand. The plantlets were successfully acclimatized in soil with more than 98% survival rate in the greenhouse.     

Micropropagation and in vitro flowering of endemic and endangered plant Ceropegia attenuata Hook

Factors affecting in vitro propagation were evaluated for Ceropegia attenuata Hook., an endemic and endangered plant having ornamental potential but a limited reproductive capacity. Rapid shoot multiplication from nodal explants was established using varying concentrations of cytokinins and auxins either alone or in combinations. The highest frequency of shoot induction was achieved when nodal explants were inoculated on Murashige and Skoog (MS) medium supplemented with 13.31 μM 6-benzylaminopurine with a mean of 12.9?±?0.5 shoots per explant. High concentrations of TDZ (6.81–11.35 μM) and KN (6.78–11.61 μM) resulted in stunted and vitrified shoots. Factors implicated in the promotion of floral transition of the C. attenuata have been identified which are 4-amino-3, 5, 6-trichloropicolinic acid (picloram), 6-benzylaminopurine, sucrose and photoperiod. The highest frequency of flowering (100%) was obtained when axillary shoot explants were transferred to MS medium supplemented with picloram (4.14 μM) within 4 weeks of culture. Transfer of in vitro regenerated shoots to half strength MS medium with 2.46 μM indole-3-butyric acid (IBA) showed maximum root induction. The in vitro grown plantlets were successfully acclimatized in the glasshouse with 85% of survival and showed normal development. The developed protocol provided a simple, cost-effective approach for the conservation of endangered plant C. attenuata for replenishing its declining populations.     

In vitro propagation of <Emphasis Type="Italic">Cymbidium goeringii</Emphasis> Reichenbach fil. through direct adventitious shoot regeneration

The influence of 2,4-dichlorophenoxyacetic acid (2,4-D), benzyladenine (BA), and thidiazuron (TDZ) on direct rhizome induction and shoot formation from rhizome explants of Cymbidium goeringii was explored. Rhizome segments obtained from in vitro seed cultures of C. goeringii were placed on Murashige and Skoog (MS) medium incorporated with 5, 10, 20, or 40 µM 2,4-D and 1, 2, 4, or 8 µM BA or TDZ alone or in combination with 20 µM 2,4-D. The explants developed only rhizomes on MS medium with or without 2,4-D. The highest percent of rhizome formation (100%) was obtained on MS medium incorporated with 20 μM of 2,4-D. The morphology and number of rhizomes varied with the level of 2,4-D in the medium. Direct adventitious shoot formation was achieved on medium incorporated with BA or TDZ. The adventitious shoots produced per explant significantly increased with the supplementation of 2,4-D to cytokinin-containing medium. The highest mean of 21.8 ± 1.8 shoot buds per rhizome segment was obtained in medium fortified with 20 μM 2,4-D and 2 μM TDZ. The greatest percent of root induction (100%) and the mean of 5.3 ± 1.1 roots per shoot were achieved on ½ MS medium incorporated with 2 μM of α-naphthaleneacetic acid. About 97% of the in vitro-produced plantlets acclimatized in the greenhouse. An efficient in vitro propagation protocol was thus developed for C. goeringii using rhizome explants.     

Enhancement of in vitro shoot regeneration from leaf explants of apple rootstock G.41

Apple (Malus domestica) rootstock G.41 is an excellent member of the Geneva series because it has traits for resistance to abiotic and biotic stresses. A simple and efficient protocol for obtaining shoots from leaf explants was established by optimizing the combinations of plant growth regulators, mode of wounding, and explant orientation on the culture medium. The best shoot multiplication index (2.58) was obtained from successful subculture medium that was the standard Murashige and Skoog (MS) medium supplemented with 7.5 g L^?1 agar, 3.55 μM N ⁶-benzyladenine, 0.16 μM indole-3-butyric acid, and 30 g L^?1 sucrose. Regeneration rates were highest (99%) when MS medium was supplemented with 2.7 μM thidiazuron and 0.9 μM 1-naphthaleneacetic acid, and cut-wounding explants before placing the abaxial surface in contact with the medium. The best rooting percentage (80%) was obtained on MS medium supplemented with 4.92 μM indole-3-butyric acid. Plantlets were rooted in vitro and survived acclimatization in the laboratory and greenhouse.     

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.     

Efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Vigna mungo</Emphasis> using immature cotyledonary-node explants and phosphinothricin as the selection agent

Herbicide (Basta®)-tolerant Vigna mungo L. Hepper plants were produced using cotyledonary-node and shoot-tip explants from seedlings germinated in vitro from immature seeds. In vitro selection was performed with phosphinothricin as the selection agent. Explants were inoculated with Agrobacterium tumefaciens strain LBA4404 (harboring the binary vector pME 524 carrying the nptII, bar, and uidA genes) in the presence of acetosyringone. Shoot regeneration occurred for 6 wk on regeneration medium (MS medium with 4.44 μM benzyl adenine, 0.91 μM thidiazuron, and 81.43 μM adenine sulfate) with 2.4 mg/l PPT, explants being transferred to fresh medium every 14 d. After a period on elongation medium (MS medium with 2.89 μM gibberellic acid and 2.4 mg/l PPT), β-glucuronidase-expressing putative transformants were rooted in MS medium with 7.36 μM indolyl butyric acid and 2.4 mg/l PPT. β-Glucuronidase expression was observed in the primary transformants (T₀) and in the seedlings of the T₁ generation. Screening 128 GUS-expressing, cotyledonary-node-derived, acclimatized plants by spraying the herbicide Basta® at 0.1 mg/l eliminated nonherbicide-resistant plants. Southern hybridization analysis confirmed the transgenic nature of the herbicide-resistant plants. All the transformed plants were fertile, and the transgene was inherited by Mendelian genetics. Immature cotyledonary-node explants produced a higher frequency of transformed plants (7.6%) than shoot-tip explants (2.6%).     

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.     

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.     

Optimization of regeneration and Agrobacterium-mediated transformation of immature cotyledons of chickpea (Cicer arietinum L.)

Immature cotyledons collected at different time intervals from four genotypes of chickpea (C 235, BG 256, P 362 and P 372) were cultured adaxially on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine, thidiazuron, kinetin, zeatin and dimethylallylaminopurine (2-iP), either alone or in combination with indole-3-acetic acid (IAA) or α-napthoxyacetic acid (α-NOA) for dedifferentiation and regeneration of adventitious shoots. Morphogenesis was achieved with explants cultured adaxially on MS medium with 13.68 μM zeatin, 24.6 μM 2-iP, 0.29 μM IAA and 0.27 μM α-NOA. Explants prepared from pods of 21 days after pollination, responded favourably to plant growth regulator treatment in shoot differentiation. Histological studies of the regenerating explants, revealed the initiation of meristematic activity in the sub-epidermal region during the onset of morphogenesis, which can be correlated with elevated activity of cytokinin oxidase-dehydrogenase, for cytokinin metabolism. The regenerated shoots were efficiently rooted in MS medium supplemented with 2.46 μM indole-3-butyric acid and acclimatized under culture room and glasshouse conditions for normal plant development leading to 76–80 % survival of the rooted plantlets. The immature cotyledon explants were used for Agrobacterium-mediated transformation with critical manipulation of cultural conditions like age of explant, O.D. of Agrobacterium suspension, concentration of acetosyringone, duration of sonication and co-cultivation for successful genetic transformation and expression of the reporter gene uidA (GUS). Integration of transgene was confirmed by molecular analysis. Transformation frequency up to 2.08 % was achieved in chickpea, suggesting the feasibility of using immature cotyledon explants for Agrobacterium-mediated transformation.     

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.     

Plant regeneration via somatic embryogenesis in Drimia robusta

A simple efficient in vitro plant regeneration system was developed by direct and indirect somatic embryogenesis of Drimia robusta, a medicinal plant extensively used in South African traditional medicine. Different developmental stages of somatic embryos (SEs: globular embryos, partial pear-shaped embryos and club-shaped embryos), club-shaped cotyledon initiation, plumule initiation and plantlets were directly obtained from leaf explants on Murashige and Skoog (MS) medium containing 3.5 % (w/v) sucrose and different plant growth regulators (PGRs). In MS medium containing 3.5 % (w/v) sucrose and supplemented with 10 μM picloram, 1 μM thidiazuron (TDZ) and 20 μM glutamine, a higher number of SEs and plantlets were achieved. These were established onto half-strength MS medium followed by successful acclimatization (100 %) in the greenhouse. Liquid somatic embryo medium (SEM_L) containing 500 mg of friable embryogenic callus on MS medium supplemented with different concentrations and combinations of PGRs and organic elicitors produced different stages of SEs. Somatic embryo production was enhanced by 0.5 μM picloram, 1 μM TDZ and mebendazole treatment. The highest number of plantlets (9.0 ± 0.70) was obtained in SEM_L containing 0.5 μM picloram, 1 μM TDZ and 25 mg l^?1 haemoglobin. All the cotyledon and plumule embryos germinated on half-strength MS medium, however 90 % of SEs germinated on half-strength MS medium containing 0.5 μM naphthaleneacetic acid. All plantlets were successfully acclimatized in the greenhouse. This first report of D. robusta somatic embryogenesis provides an opportunity to control extinction threats, ensure germplasm conservation and provides a system for analysis of bioactive compounds and bioactivity.     

In vitro organogenesis from leaf and transverse thin cell layer derived callus cultures of Talinum triangulare (Jacq.) Willd.

Talinum triangulare is an important medicinal herb used traditionally in the treatment of various diseases. The present study was intended to develop a rapid and efficient protocol for indirect organogenesis from leaf discs and transverse thin cell layer (tTCL) of internodal explants of T. triangulare. Best callusing response (100 %) was observed with tTCL explants on Murashige and Skoog (MS) medium supplemented with 4.44 μM 6-benzyl amino purine and 5.37 μM α-naphthalene acetic acid (NAA). High frequency shoot regeneration (96.67 %) was obtained from tTCL derived calli on MS medium supplemented with a combination of 0.45 μM thidiazuron and 0.27 μM NAA, by producing 9.20 ± 0.35 shoots with a shoot length of 2.74 ± 0.03 cm. In vitro rooting of the microshoots was recorded on half-strength MS medium containing 2.46 μM indole-3-butyric acid by eliciting 15.20 ± 0.27 roots with a length of 4.25 ± 0.11 cm. The rooted shoots were acclimatized on garden soil, sand and coco pith (1:1:3 v/v) planting substrate. The plantlets were successfully established under field conditions with 100 % survival rate. The hardened plants exhibited homogeneity and no observable morphological variations were detected among the regenerants and the mother plants of T. triangulare.     

Rapid in vitro multiplication and ex vitro establishment of Caribbean copper plant (Euphorbia cotinifolia L.): an important medicinal shrub

An efficient micropropagation protocol was developed for E. cotinifolia by utilizing mature nodal segments for axillary shoot proliferation. The nodal explants from a 2-year-old plant were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations (0.5, 2.5, 5.0, 7.5 and 10.0 μM) of 6-benzyladenine (BA), Kinetin and 2-isopentenyl adenine singly as well as in combination with α-naphthalene acetic acid (NAA) or Indole-3-butyric acid (IBA) (0.1, 0.5 and 1.0 μM). The highest regeneration frequency (92 %) with multiple shoots (13.0 ± 1.15) and shoot length (4.23 ± 0.14 cm) was achieved on MS medium supplemented with 5.0 μM BA and 0.1 μM NAA after 8 weeks of culture. Further experiments were performed to test the effects of medium type, medium strength, pH and subculture passages on shoot induction and proliferation. An enhancement in average number of shoots (16.6 ± 0.45) per explant was obtained after four subculture passages. Micro shoots exhibited in vitro rooting on half strength MS medium containing 2.5 μM Indole-3-butyric acid (IBA) after 4 weeks of culture. The in vitro raised healthy plantlets with well-developed roots and shoots were successfully acclimatized in plastic cups containing sterile soilrite for 8 weeks under culture room conditions (150 PPFD) prior to field transfer. Through the acclimatization period (0–56 days), photosynthetic pigments (Chlorophyll a, b and Carotenoid content) decreased during the initial 2 weeks followed by significant increase during the successive period (21–56 days) of acclimatization. At the same time, all the tested antioxidant enzymes (SOD, CAT, APX and GR) exhibited an increasing trend throughout the acclimatization period. The culture room acclimatized plantlets were successfully established in earthen pots containing garden soil in greenhouse with 70 % survival rate.     

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.     

Somatic embryogenesis and plantlet regeneration from mature zygotic embryos of Manchurian ash (Fraxinus mandshurica Rupr.)

Using mature cotyledonary explants of Fraxinus mandshurica, an efficient plant regeneration system was developed via somatic embryogenesis. More than 67 % of mature cotyledons of zygotic embryos yielded 23–159 somatic embryos (SEs) per explant when incubated on medium consisting of half-strength Murashige and Skoog (MS) salts and vitamins (MS1/2) supplemented with 8.88 μM 6-benzyladenine (BA), 26.84 μM naphthaleneacetic acid (NAA), 75 g L^?1 sucrose, and 400 mg L^?1 casein hydrolysate (CH). Approximately, 82 % of induced SEs were observed on browning cotyledonary explants. Histological studies of cotyledon explants at various stages of somatic embryogenesis revealed that the SEs originated from single epidermal cells and developed to the globular, heart, torpedo, and cotyledonary stage embryos. Secondary somatic embryos (SSEs) formed on the surface of radicle tips of the SEs. Addition of low concentrations of NAA and 200–400 mg L^?1 CH to MS1/2 medium increased SSE induction. Cotyledonary SSEs were cultured on MS1/2 medium with 10 mM abscisic acid in the presence of light to promote maturation, and >92 % of mature SSEs were able to germinate with normal shoots. After 8 weeks in culture in the presence of light on medium with one-third of the MS macroelements as well as 0.06 μM NAA, >94 % of the germinated SSEs converted into plantlets. Plantlets acclimatized successfully to ex vitro conditions and developed normal phenotypes under field conditions.     

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.     

Somatic embryogenesis in Alnus glutinosa (L.) Gaertn

Induction of somatic embryos and plant regeneration was demonstrated for the first time in Alnus glutinosa. Somatic embryos were initiated from zygotic embryos collected 1–3 weeks post-anthesis (WPA), i.e., when they were at globular or early cotyledonary stage and were 0.5–1 mm in length. Induction frequency (16.6 %) and the mean number of somatic embryos (4.5 embryos/explant) were highest after culture of zygotic embryos, collected at 3 WPA, on Murashige and Skoog medium (MS) supplemented with 0.9-μM 2,4-dichlorophenoxyacetic acid and 2.22-μM benzyladenine (BA). No embryogenic induction was observed on medium with BA alone. Initial somatic embryos differentiated indirectly from callus tissue formed at the surface of the zygotic embryos. Embryogenic competence was maintained by secondary embryogenesis, which was affected by explant type, plant growth regulators and genotype. Secondary embryogenesis was induced by culture of small groups of whole somatic embryos or isolated cotyledon explants on medium consisting of MS medium (half-strength macronutrients) supplemented with 0.44-μM BA. Histological study of isolated cotyledon explants revealed that secondary embryos developed directly from differentiated embryogenic tissue on the surface of cotyledons. Somatic embryos at successive stages of development, including cotyledonary-stage embryos with shoot and root meristems, were evident. For plantlet conversion, somatic embryos were transferred to maturation medium supplemented with 3 % maltose, followed by 6 weeks of culture in Woody Plant Medium supplemented with 0.44-μM BA and 0.46-μM Zeatin (Z). This novel protocol appears promising for mass propagation, conservation and genetic transformation of black alder.