Plantlet regeneration through indirect shoot organogenesis and somatic embryogenesis in Justicia gendarussa Burm. f., a medicinal plant

A protocol for the regeneration of a large number of plantlets via indirect shoot organogenesis and somatic embryogenesis has been developed from the stem and leaf explants of Justicia gendarussa Burm. f. The callus was efficiently induced from the explants using Murashige and Skoog (MS) medium supplemented with α-Naphthalene acetic acid (NAA) + Benzyl amino purine (BAP) (1.0?+?0.1 mg/l). The highest number of plantlets through indirect shoot organogenesis was obtained when the callus was subcultured to MS medium with BAP + NAA (0.1?+?1.0 mg/l). The maximum number of plantlets via somatic embryos was obtained in the medium with BAP + NAA (1.0?+?0.1 mg/l) for stem derived calli and Kinetin (Kn) + NAA (2.0?+?0.1 mg/l) for leaf derived calli. The in vitro developed shoots were rooted well in half strength MS medium supplemented with 0.5 mg/l of Indole-3-acetic acid (IAA). The in vitro regenerated plantlets were hardened using a mixture of sterile sand:soil:manure (1:1:1). The present study is the first report on the regeneration of plants through somatic embryogenesis from stem and leaf derived calli of J. gendarussa.     

Direct somatic embryogenesis of potato [<Emphasis Type="Italic">Solanum tuberosum</Emphasis> (L.)] cultivar ‘Kufri Chipsona 2’

Direct somatic embryo induction was achieved from leaf and internodal explants of Solanum tuberosum (L.) cultivar ‘Kufri Chipsona 2’ on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium containing 10.0 µM silver nitrate (MS1 medium) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D; 2.5 µM) and 6-benzyladenine (BA; 1.0 µΜ). It was observed that in absence of AgNO₃, friable callus was induced from cut ends of the explants, which does not develop into any kind of organised structure; thus highlighting the requirement of AgNO₃ for somatic embryogenesis in potato. Furthermore, the effect of medium strength, sucrose concentration and heat shock treatment on somatic embryogenic potential of explants was also investigated. When the strength of basal medium was reduced to half, the frequency of internodal segments differentiating somatic embryos was almost double in comparison to full strength MS medium. Sucrose concentration and heat shock treatment were found to have interactive effect on somatic embryo induction. Explants subcultured on medium containing 174 mM sucrose and subjected to heat shock (1 h; 50 °C) showed maximum somatic embryo differentiation. Although, the percent explants differentiating somatic embryos decreased sharply with increase in sucrose concentration (>?174 mM), yet the number of somatic embryos differentiated per explant were found to increase with further increase in sucrose concentration. Histological observations revealed that somatic embryos directly developed from epidermis of leaf explant and cut ends of internodal segments progressed from globular to cotyledonary stage after passing through intermediate embryogenic stages (heart shaped and torpedo shaped). Conversion of somatic embryos into plantlets (92%) was achieved on MS1 medium supplemented with BA (10.0 µM) and gibberellic acid (15.0 µM) and all regenerated plants were found to be phenotypically alike.     

Somatic embryogenesis,acclimatization and genetic homogeneity assessment of regenerated plantlets of <Emphasis Type="Italic">Anoectochilus elatus</Emphasis> Lindl., an endangered terrestrial jewel orchid

An efficient in vitro plant regeneration system was established through somatic embryogenesis for Anoectochilus elatus Lindley, an endangered jewel orchid. Direct somatic embryogenesis was achieved from nodal explants (17.4 embryos per explant with 63.4% response) on Mitra medium supplemented with Morel vitamins, thidiazuron (4.54 µM) and ∞-naphthaleneacetic acid (2.69 µM). Simultaneously, a protocol was developed for indirect somatic embryogenesis from internodal explant, produced embryogenic calli and embryos (31.3 embryos with 76.4% response) on same medium amended with 50 mg/L peptone and 5% coconut water. Both types of embryogenic pathways, produced morphologically similar globular embryos in the form of protocorm like bodies and successfully germinated on hormone free Mitra medium supplemented with Morel vitamins. Morpho-histological investigation of the embryo revealed the initiation and developmental features of somatic embryos. In vitro regenerated plantlets were successfully established from heterotrophic to a photoautotrophic stage by reducing the nutrient content in culture media, adjusting temperature and humidity through three step method. During the process, no morphological and physiological abnormalities were observed. Hardened plantlets were successfully acclimatized at poly tunnel chamber with 95% of survival rate. Further, inter simple sequence repeats (ISSRs) molecular markers were used to analyse the genetic homogeneity of regenerated plants. Analysis with this method showed that the homogeneity is comparatively higher in direct somatic embryo regenerated plants (94.22%) as compared to plants elevated from an indirect somatic embryo (93.05%). The present study provides morpho-histological and genetically stable plants for germplasm conservation and further utility of this endangered jewel orchid.     

Establishment of embryogenic cell suspension cultures and Agrobacterium-mediated transformation in banana cv. ‘Dwarf Cavendish’ (Musa AAA): effect of spermidine on transformation efficiency

An efficient method has been developed for somatic embryogenesis, plant regeneration and transformation of the important banana cultivar ‘Dwarf Cavendish’ (Musa AAA). A high embryogenic response was obtained in 1.36 % of immature male flower explants. Once embryogenic structures were transferred to liquid medium, embryogenic cell suspensions (ECSs) with high regeneration capacity were obtained. ECSs were incubated under different conditions with Agrobacterium tumefaciens strain EHA101 harboring vector pFAJ3000 that contains pNos-nptII-tOcs and p35S-uidAintron-t35S expression cassettes. The effect of spermidine and infection time on transformation efficiency was examined. The highest efficiency was obtained when ECSs were infected for 6 h, in medium supplemented with 200 μM acetosyringone and 1.0 mM spermidine, with more than 600 independent lines/~50 mg FW of settled cells. Spermidine showed an enhancing effect, increasing significantly the transient Gus expression and the number of transformed embryo colonies and regenerated plants in comparison with the same treatments without this polyamine. This is the first report showing efficient Agrobacterium tumefaciens mediated transformation using embryogenic cell suspension cultures in the ‘Dwarf Cavendish’ banana cultivar.     

Somatic embryogenesis and synthetic seed production in Rhinacanthus nasutus (L.) Kurz.

Young healthy cotyledon and leaf explants of Rhinacanthus nasutus (L.) Kurz. were incubated on Murashige and Skoog (MS) medium supplemented with 1.0–5.0 mg/l 2, 4-dichlorophenoxyacetic acid (2,4-D) either alone or in combination with 0.3–1.5 mg/l indole-3-butyric acid (IBA). The optimum callus induction (100 %) was observed from cotyledon explants on MS medium supplemented with 4 mg/l 2, 4-D and 0.5 mg/l IBA. The friable, embryogenic callus when subcultured on half strength MS medium supplemented with IBA (3.0–5.0 mg/l) produced several somatic embryos at various stages of development (globular, heart, torpedo) after 45 days of culture. The highest frequency of callus embryogenesis was observed on ½MS medium supplemented with 4.0 mg/l IBA. Moreover, 47 % of incubated callus responded with a mean number of 16.3 somatic embryos per gram callus. For germination, somatic embryos at the torpedo stage were isolated and subcultured on ½MS medium supplemented with 0.5 mg/l each of 6-benzyladenine and indole-3-acetic acid. After 45 days of culture, plantlets developed with mean lengths of 3.8 cm. Somatic embryos at the torpedo stage were collected and suspended in a matrix of MS medium containing sodium alginate (3 % W/V), dropped into 100 mM calcium chloride (CaCl₂·2H₂O) solution for the production of synthetic seeds. Optimum growth ability of synthetic seed was obtained on MS medium supplemented with 0.2 mg/l gibberellic acid (GA₃). Well developed healthy plantlets derived from somatic embryos and synthetic seeds were hardened and successfully transplanted to soil.     

Somatic embryogenesis and in vitro flowering of 3 species of bamboo

Plant regeneration via somatic embryogenesis was achieved in callus cultures derived from nodal explants of in vitro grown seedlings and excised mature zygotic embryos of three bamboo species on Murashige and Skoog's (MS) basal medium supplemented with 0.5 mg/l kinetin (Kn), 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 10 mg/l adenine sulphate (Ads) and 3% (w/v) sucrose incubated in the light or in the dark. Somatic embryos germinated (95–98%) into normal plants and were transferred to soil with 95% success. In vitro flowering was induced on shoots developed from nodal explants taken from somatic embryo regenerated plants of Bambusa vulgaris, Dendrocalamus giganteus and Dendrocalamus strictus on half-strength MS basal medium supplemented with 0.25 mg/l indole-3-butyric acid (IBA), 0.5 mg/l Ads, 0.5 mg/l gibberellic acid (GA₃) and 3% sucrose.Abbreviations BAP 6-benzylaminopurine - Kn kinetin - Ads adenine sulphate - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) basal medium - GA₃ gibberellic acid     

Plant regeneration through direct somatic embryogenesis from immature zygotic embryos of the medicinal plant,Paris polyphylla Sm.

A protocol for induction of direct somatic embryogenesis and subsequent plant regeneration for the medicinally important and endangered plant Paris polyphylla Sm. has been developed for the first time. Immature zygotic embryos (IZEs) were cultured on different media namely Gamborg (B5), ½ B5, Murashige and Skoog (MS), ½ MS, Chu et al. (N6), ½ N6, Schenk and Hildebrandt (SH) and ½ SH. Highest frequency of somatic embryogenesis (32.6 %) and mean number of somatic embryos (SEs) per explant (28.7 ± 1.7) were obtained on ½ MS medium directly without an intermediate callus phase. The frequency of SE induction was significantly increased to 40.7 % when ½ MS medium was solidified with gelrite compared to agar (32.6 %). Secondary somatic embryos (SSEs) appeared on the primary SEs in a repetitive way on plant growth regulator-free ½ MS medium but with a gradual decrease in embryogenic potential during subsequent subcultures. Plasmolyzing pre-treatment of SSEs with 1.0 M mannitol for 12 h effectively maintains its embryogenic capacity. Primary embryos at the elongated dimpled and early cotyledonary stage displayed the highest embryo forming capacity of 26.94 and 27.87, respectively. High frequency of SE germination (94.0 %) occurred on ½ MS medium with 0.5 mg/l gibberellic acid. Highest percentage of seedling to plantlet conversion was observed in the medium supplemented with 0.05 mg/l 6-benzylaminopurine and 0.1 mg/l α-naphthalene acetic acid. Regenerated plants displayed morphological characteristics similar to that of the wild plants. Flow cytometry analysis showed ploidy stability of the regenerated plants.     

Plant regeneration from leaf explants of Aloe barbadensis Mill. and genetic fidelity assessment through DNA markers

An efficient plant regeneration protocol was developed from leaf explants of Aloe barbadensis Mill on Murashige and Skoog’s (MS) medium supplemented with 2.0 mg/l 6-benzyladenine (BA) or Kinetin (Kn), 0.25–0.5 mg/l NAA (1-napthalene acetic acid) and 3 % (w/v) sucrose within 4 weeks of culture. The maximum number of shoot buds were obtained on MS medium supplemented with 2.0 mg/l BA, 0.5 mg/l NAA, 40 mg/l Ads (adenine sulphate) within 4–6 weeks of subculture. Inclusion of 0.25–0.50 mg/l gibberellic acid into the medium, the shoot buds became elongated. Repeated subculture on regeneration medium induces higher rate of shoot regeneration. The root induction from excised microshoots was achieved on half-strength MS medium supplemented with 0.25–1.0 mg/l NAA or indole-3-butyric acid (IBA) and 2 % (w/v) sucrose. Maximum percentage of rooting was achieved on medium having 0.5 mg/l NAA with 3 % (w/v) sucrose. About 80 % of in vitro raised plantlets were hardened in the greenhouse and successfully established in the soil. Both Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers were used to detect the variability among the regenerated plants developed in vitro. The results showed that there was no polymorphism among the regenerated plantlets. This study will help for propagation of quality planting material of Aloe barbadensis for commercialization.     

Production and conversion of haploid embryos in chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) anther cultures using high 2,4-D and silver nitrate containing media

Due to recalcitrant nature of chickpea (Cicer arietinum L.) to androgenesis, the production of double haploid plants has been only reported by Grewal et al. (Plant Cell Rep 28:1289–1299, 2009) using some physical stresses such as anther centrifugation and electrical shock. In the present study, we successfully obtained haploid plants from cultured anthers of two chickpea cultivars, Bivanij and Arman, using high 2,4-D and silver nitrate containing media without applying of these time and labor consuming stresses. For induction of androgenesis, different concentrations of 2, 4-D (0, 2, 5 and 10 mg/l) and silver nitrate (0, 5, 10, 15, 25 and 50 mg/l) were used in embryo development medium. In Bivanij cultivar, anther induction medium containing 10 mg/l 2,4-D and 15 mg/l silver nitrate produced the highest number of embryos (0.188) and regenerated plants (0.1) per each cultured anther, while the highest frequencies of embryos (0.1) and regenerated plants (0.075 and 0.063) were obtained from Arman cultivar when 10 mg/l 2,4-D was combined with 15 and 50 mg/l silver nitrate in anther culture medium, respectively. In second part of this study, different cold (4 °C for 4 and 7 days) and heat (30 °C for 10 days, 32 °C for 2 days and 35 °C for 8 h) pretreatments were applied on cultured anthers of Bivanij cultivar. Incubation of cultured anthers at 32 °C for 2 days significantly enhanced the rate of embryo formation up to 0.222 embryos per each anther, while the highest number of regenerated plants/anther (0.0332) was obtained when cold treated anthers at 4 °C for 7 days incubated at 30 °C for 10 days. Taken together, these results provide a good basis for large-scale generation of DH plants in this important legume species.     

Somatic embryogenesis from callus cultures of <Emphasis Type="Italic">Terminalia chebula</Emphasis> Retz.: an important medicinal tree

Somatic embryogenesis was obtained from cotyledon and mature zygotic embryo callus cultures of Terminalia chebula Retz. Callus cultures of cotyledon and mature zygotic embryo were initiated on induction medium containing Murashige and Skoog (MS) nutrients with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) either 0.01 or 0.1 mg/l Kinetin and 30 g/l sucrose. Induction of somatic embryogenesis, proliferation and development was obtained through different culture passages. Embryogenic cotyledon callus with globular somatic embryos was obtained on MS basal medium supplemented with 50 g/l sucrose. Globular somatic embryos were observed from mature zygotic embryo callus on induction medium. Different stages of somatic embryo development from cotyledon and mature zygotic embryo calluses were observed on MS basal medium supplemented with 50 g/l sucrose after 4 weeks of culture. Histological studies have revealed the developmental stages of somatic embryos. A maximum of 40.3±1.45 cotyledonary somatic embryos/callus was obtained from mature zygotic embryo compared to 7.70±0.37 cotyledonary somatic embryos/callus initiated from cotyledons. Germination of somatic embryos and conversion to plants were achieved. Highest frequency of germination (46.66±0.88) of somatic embryos was obtained on MS basal medium containing benzyladenine (0.5 mg/l) with 30 g/l sucrose.     

An efficient regeneration system via somatic embryogenesis in mango ginger (Curcuma amada Roxb.)

A reproducible protocol for somatic embryogenesis was established for mango ginger (Curcuma amada Roxb.)—an important horticultural aromatic rhizomatous plant. Embryogenic callus induction was obtained from leaf sheath explants of in vitro raised plants on Murashige and Skoog (MS) agar medium containing 2.0 mg/L 2,4-dichlorophenoxyacetic acid and 0.5 mg/L 6-benzyladenine (BA). Embryogenic callus proliferation, somatic embryo (SE) formation and subsequent plantlet conversion occurred under optimal culture conditions. The effects of MS medium strength, sucrose and BA on SE formation were also evaluated. Half strength MS liquid medium necessary for SE formation and optimal sucrose concentration was found to be 3.0 %. BA at 0.3 mg/L produced the highest number (84.71 %) of SEs from leaf sheath explants. Secondary somatic embryos originated from primary somatic embryos on the same medium supplemented with 0.4–0.6 mg/L BA. Stereo microscopic and scanning electron microscopic observation revealed that the globular and torpedo shaped somatic embryos resulted in suspension culture during development. Mature somatic embryos germinated readily and developed into normal plantlets after 3 weeks on half strength MS basal agar medium under dark condition. Well rooted plantlets were successfully acclimatized at the survival rate of 70 %.     

Somatic embryogenesis of the banana hybrid cultivar FHIA-18 (AAAB) in liquid medium and scaled-up in a bioreactor

The development of somatic embryos in liquid culture medium has a number of advantages for large-scale propagation of plants. This paper describes an improved system for the mass propagation via somatic embryogenesis of the banana hybrid cultivar FHIA-18 (AAAB). Explants from immature male flowers were used to form high frequency embryogenic tissue, this tissue was then used to establish embryogenic cell suspensions in a basic MS medium plus 1.0 mg l^–1 biotin, 100 mg l^–1 glutamine, 100 mg l^–1 malt extract (Sigma), 1.0 mg l^–1 2,4-D and 45 g l^–1 sucrose. Secondary multiplication of somatic embryos was achieved in liquid media in rotary shaker and in bioreactors. The number of embryos per litre obtained with 80.0% DO₂ and effects of pH were also studied. A high regeneration percentage of plants were obtained (89.3%) in only 1 month of culture, somatic embryos were then placed to germinate in temporary immersion systems and field testing of somaclonal variation.     

Regeneration of pineapple (Ananas comosus L.) plant through somatic embryogenesis

An efficient protocol for a complete plant regeneration by somatic embryogenesis was developed with Smooth Cayenne pineapple (Ananas comosus L.). Previous works showed that this species is responsive to somatic embryogenesis. In the present work the influence of components of culture medium in the induction, development and conversion of somatic embryos was investigate in order to establish a somatic embryogenesis protocol. Nodular callus (83.67%) was initiated from leaf explants of young plants on CIM₃ medium. The highest frequency (37.6%) of embryogenic callus induction was obtained from 4-week-old calluses on EIM₃ medium supplemented with 3.0 mg/l picloram. The highly organized callus induction and the development of somatic embryos were achieved after the transfer of callus clumps onto EIM₃ medium containing 1.0 mg/l BAP + 0.1 mg/l NAA. The frequency of somatic embryo formation was of 39.5?±?2.45 embryos per callus. Up to 97% of the somatic embryos were converted into complete plants within 4 week on MSB medium with 1.0 mg/l BAP + 0.05 mg/l GA₃ + 500 mg/l glutamine. The continuation of the elongation of the shoots occurred on this medium). Shoots obtained from all the above methods were rooted in MSB medium with activated charcoal. Complete plantlets were transferred onto specially made polyethylene bags containing soil mixture and transferred to the greenhouse. Survival rate of the plantlets under ex vitro conditions was 98% and maximum average number of plantlets (80?±?0.6). The well-developed plantlets were transferred to an open field where the plants produced normal fruits.     

Plant regeneration via somatic embryogenesis in chickpea (Cicer arietinum L.)

Efficient plant regeneration via somatic embryogenesis has been developed in chickpea cultivar C235. Leaf explants, on MS medium supplemented with 1.25 mg/l 2,4-D and 0.25 mg/l kinetin, yielded somatic embryos with high efficiency during dark incubation. MS medium supplemented with B₅ vitamins, 0.125 mg/l IBA and 2 mg/l BAP was found suitable for embryo maturation. The well formed embryos germinated into plantlets on basal B₅ medium supplemented with 0.25 mg/l BAP. Further development into healthy plantlets was obtained on basal B₅ medium. Hardened plantlets produced normal, fertile plants upon transfer to soil.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-Benzyl-aminopurine - IAA IndoIe-3-acetic acid - IBA Indole-3-butyric acid - NAA 1-Naphthalene acetic acid - Kinetin 6-furfuryl aminopurine - Zeatin 6-(4-hydroxy-3-methylbut-2-enylamino)-purine     

Recovery of avocado (<Emphasis Type="Italic">Persea americana</Emphasis> Mill.) plants transformed with the antifungal plant defensin gene PDF1.2

Embryogenic avocado cultures derived from ‘Hass’ protoplasts were genetically transformed with the plant defensin gene (pdf1.2) driven by the CaMV 35S promoter in pGPTV with uidA as a reporter gene and bar, the gene for resistance to phosphinothricin, the active ingredient of the herbicide Finale® (Basta) (Bayer Environmental Science, Research Triangle Park, Durham, NC ). Transformation was mediated by Agrobacterium tumefaciens strain EHA105. Transformed cultures were selected in the presence of 3.0 mg l^?1 phosphinothricin in liquid maintenance medium for 3–4 mo. Liquid maintenance medium consisted of modified MS medium containing (per liter) 12 mg NH₄NO₃ and 30.3 mg KNO₃ and supplemented with 0.1 mg l^?1 thiamine HCl, 100 mg l^?1 myo-inositol, 30 g l^?1 sucrose, 3.0 mg l^?1 phosphinothricin, and 0.41 μM picloram. Somatic embryo development from transformed cultures was initiated on MS medium supplemented with 45 g l^?1 sucrose, 4 mg l^?1 thiamine HCl, 100 mg l^?1 myo-inositol, 10% (v/v) filter-sterilized coconut water, 3.0 mg l^?1 phosphinothricin, and 6.0 g l^?1 gellan gum. Limited plant recovery occurred from somatic embryos on semi-solid MS medium supplemented with 3.0 mg l^?1 phosphinothricin, 4.44 μM 6-benzylaminopurine (BA), and 2.89 μM GA₃; transformed shoots were micrografted on in vitro-grown seedling rootstocks. Approximately 1 yr after acclimatization in the greenhouse, transformed shoots were air-layered to recover transformed roots. Genetic transformation of embryogenic cultures, somatic embryos, and regenerated plants was confirmed by polymerase chain reaction (PCR), Southern blot hybridization, the XGLUC reaction for uidA, and application of the herbicide Finale® to regenerated plants.     

A rapid protocol for somatic embryogenesis from immature leaflets of groundnut (Arachis hypogaea L.)

Summary Plant regeneration via somatic embryogenesis was developed in two groundnut varieties. Somatic embryogenesis was induced from immature leaflets on MS medium with different concentrations of the auxins 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthaleneacetic acid (NAA) in combination with 0.5 mg/l of the cytokinin BA. The highest frequency of somatic embryo formation occurred on MS medium fortified with 20 mg 2,4-D per l. Of the two auxins tested individually 2,4-D was more effective for induction of embryogenesis as well as production of embryos. Embryo development and maturation was achieved on MS medium supplemented with N⁶-benzyladenine (BA) (0.5–2.0 mg/l) and 2,4-D (0.5 mg/l). Plant conversion frequency from somatic embryos was highest in presence of 2.0 mg BA per l and 0.5 mg NAA per l. The frequency of embryogenesis and plant regeneration was higher in the VRI-2 cultivar than in the other cultivar tested. Regenerated plants were transferred to soil, grown to maturity, and produced viable seeds.     

Somatic embryogenesis from mature zygotic embryos of Distylium chinense (Fr.) Diels and assessment of genetic fidelity of regenerated plants by SRAP markers

The objective was to establish an efficient regeneration protocol for Distylium chinense based on somatic embryogenesis and evaluate the genetic stability of plants regenerated in vitro. To induce callus mature zygotic embryos were cultured on Murashige and Skoog’s (MS) medium that was supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and N⁶-benzyladenine (BA). After 20 days, the highest rate of callus formation (88.9 %) occurred on MS medium supplemented with 0.5 mg l^?1 2,4-D and 0.1 mg l^?1 BA. It was observed that light-yellow, compact, dry, nodular embryogenic calli had formed. These calli were then subcultured on fresh MS medium supplemented with 0.1 mg l^?1 BA and 0.5 mg l^?1 α-naphthaleneacetic acid (NAA) for proliferation for an additional 30 days. To induce somatic embryos and plant regeneration, the embryogenic callus was transferred to fresh MS medium that was supplemented with different concentrations of BA and NAA. After 30 days, 0.5 mg l^?1 BA in combination with 0.5 mg l^?1 NAA produced the best result in terms of somatic embryogenesis (%), shoot differentiation (%), number of shoots per callus and shoot length. Next, the plantlets were transferred to the field for 5 weeks and a 95 % survival rate was observed. The sequence-related amplified polymorphism markers confirmed genetic stability of plants regenerated in vitro. To our knowledge, this is the first report that describes a plant regeneration protocol for D. chinense via somatic embryogenesis to be used for germplasm conservation and commercial cultivation.     

Somatic embryogenesis and organogenesis in Dendrocalamus hamiltonii

In this study, mature zygotic embryos, plant growth regulators, and various media were tested with the aim of developing an efficient regeneration system for plantlets of the bamboo species Dendrocalamus hamiltonii. Callus formation was induced in explants cultured in Murashige and Skoog (MS) medium supplemented with 1.0–3.0 mg/l 2,4-dichlorophenoxyacetic acid. Optimal shoot differentiation and subsequent shoot growth were also obtained in MS medium supplemented with 2 mg/l benzyladenine, 1 mg/l kinetin, and 1 mg/l naphthaleneacetic acid. Root induction was enhanced by the addition of 5 mg/l indole-3-butyric acid to the culture medium. Histological analysis revealed that both somatic embryogenesis and organogenesis were induced during callus initiation, shoot differentiation, and the development of plantlets from the mature zygotic embryos. Our data provide a useful basis for developing culture protocols for the regeneration of bamboo plants.     

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.     

High-efficiency somatic embryogenesis and plant regeneration from suspension cultures of grapevine

Embryogenic suspensions of grapevine (Vitis vinifera L.) were initiated from somatic embryos of `Thompson Seedless' and `Chardonnay'. Suspension cultures consisted of proembryonic masses (PEM) that proliferated without differentiation in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D). `Chardonnay' somatic embryos developed fully from PEMs following subculture in medium without 2,4-D; however, somatic embryo development did not advance beyond the heart stage in `Thompson Seedless' suspension cultures. Highly synchronized development of somatic embryos was obtained by inoculating <960-μm PEMs into liquid medium without 2,4-D. Somatic embryos were also produced in large numbers from suspension-derived PEMs of both cultivars on semisolid medium lacking 2,4-D. Somatic embryos matured and regenerated into plants in MS basal medium containing 3% sucrose. Using this method more than 60% of the somatic embryos regenerated plants. More than 90% of the regenerated plants were successfully transferred to the greenhouse. Received: 27 July 1998 / Revision received: 15 October 1998 / Accepted: 27 October 1998