Somatic embryogenesis and plant regeneration in açaí palm (<Emphasis Type="Italic">Euterpe oleracea</Emphasis>)

An efficient and simple system for inducing somatic embryogenesis and regenerating plantlets from immature zygotic embryos of açaí palm (Euterpe oleracea) has been developed. Embryogenic calli (ECs) were induced from immature zygotic embryos of açaí palm on Murashige and Skoog (MS) modified medium with 2,4-dichlorophenoxyacetic acid and picloram. Embryogenic frequency was dependent on auxin type and concentration. The optimal concentration of picloram for the high-frequency induction of embryogenic calli (72%) was 225 μM. ECs were then subcultured on a differentiation and maturation medium composed of MS modified medium with 2-isopentenyladenine and naphthaleneacetic acid with subcultures at 4-week intervals. SEs were converted to plants on MS modified medium with half-strength macro- and micronutrients, 20 g l^?1 sucrose, and 2.5 g l^?1 activated charcoal and gelled with 2.5 g l^?1 Phytagel. Detailed morpho anatomical changes during the different stages of somatic embryogenesis were characterized. The development of SEs was asynchronous, and ontogenic studies confirmed that the initial cell divisions occur in the epidermal and subepidermal regions of the zygotic embryos. Broad base attachment of SEs to the epidermis indicates the presence of a suspensor.     

Differential responses to somatic embryogenesis of different genotypes of Brazilian oil palm (Elaeis guineensis Jacq.)

To assess the potential of different genotypes of Brazilian oil palm (Elaeis guineensis Jacq.) to somatic embryogenesis and somatic embryo proliferation, mature zygotic embryos of nine commercial genotypes of E. guineensis (BRSC2001, BRSC2328, BRSC2301, BRSC3701, BRSCM1115, BRSC7201, BRSC2528, BRSC2501, and BRSCN1637) were used. Explants were incubated on Murashige and Skoog (MS) supplemented with 450 μM picloram, 3.0 % sucrose, 500 mg l^?1 glutamine, and 2.5 g l^?1 activated charcoal, and gelled with 2.5 g l^?1 Phytagel. After induction, for differentiation and maturation, the embryogenic calli (ECs) were transferred into fresh medium supplemented with 0.6 μM naphthaleneacetic acid (NAA) and 12.30 μM 2-isopentenyladenine (2iP) or 40 μM picloram in combination with 0.3 g l^?1 activated charcoal, and 500 mg l^?1 glutamine. Somatic embryos were converted into plants on MS medium with macro- and micro-nutrients at half strength, 2 % sucrose, and 2.5 g l^?1 activated charcoal, and gelled with 2.5 g l^?1 Phytagel. In general, zygotic embryos swelled after 14 days. Primary calli, which were observed in all the genotypes after 45–60 days of culture, eventually progressed to ECs at 90 days. At this time, scanning electron microscopy (SEM) analysis showed cellular differences between compact and friable calli. After 150 days in the induction phase, the ECs with proembryos that were transferred to the medium for differentiation and maturation, differentiated asynchronically into somatic embryos at globular and torpedo stages. The results showed that BRSC2328 and BRSCM1115 had the highest potential for EC formation (90–100 %) and somatic embryo differentiation (40.7 and 52.5 somatic embryos per callus, respectively) when compared to other genotypes. After approximately 90 days of culture on MS basal medium without growth regulators, protrusion of the leaf primordia was observed, characterizing the onset of germination of the somatic embryos into plants.     

Plant regeneration through somatic embryogenesis from immature and mature zygotic embryos of Musa acuminata ssp. burmannica

A simple and efficient protocol has been developed for in vitro regeneration of M. acuminata ssp. burmannica (AA) plants. Somatic embryos were produced when immature and mature zygotic embryo explants were cultured on Murashige and Skoog medium supplemented with plant growth regulators 2,4-dichlorophenoxyacetic acid; (2,4-D), picloram or benzyl adenine and indole acetic acid. In general, immature embryos responded better than mature embryos. Callus proliferation was highest in medium supplemented with 2,4-D (4.5???M). Subsequent transfer of callus to fresh medium produced rapidly proliferating embryogenic calli. Embryogenic calli were maintained in complete darkness for 15?d followed by cycles of 8?h dark and 16?h light, under white fluorescent lamps with a light intensity of 3,000?lm/m² and at temperature of 28?±?2°C. Regeneration of embryogenic calli into plantlets was higher for immature embryos (76.6%) than for mature embryos (50.6%). This plant regeneration protocol using mature or immature zygotic embryos, via somatic embryogenesis, has significant potential to improve germination efficiencies of hybrid progenies used in conventional breeding strategies. Furthermore, tests on seed storage showed that seed viability rapidly decline after harvesting and was negligible after 9?mo of storage. This indicates using freshly harvested seeds as explant material is necessary for maximizing the tissue culture response.     

An improved protocol for somatic embryogenesis and plant regeneration in macaw palm (Acrocomia aculeata) from mature zygotic embryos

An improved protocol for plant regeneration via somatic embryogenesis was developed using mature macaw palm (Acrocomia aculeata) zygotic embryos as initial explant. For induction of the embryogenic callus (EC), two basic media (BM) were tested consisting of Murashige and Skoog and Eeuwens (Y3) salts with 30 g L^?1 sucrose, 0.5 g L^?1 glutamine and 2.5 g L^?1 Phytagel. The 3,6-dichloro-2-methoxybenzoic acid (dicamba), 4-amino-3,5,6-trichloro-picolinic acid (picloram) and 2,4-dichlorophenoxyacetic acid (2,4-D) auxins were added to the culture media at concentrations of 0, 1.5 or 3.0 mg L^?1. After 240 days, the embryogenic calli were transferred to the respective BM media with auxin concentrations reduced to 0.5 or 1.0 mg L^?1 in order to differentiate the somatic embryos (SEs). Plant regeneration was performed on the BM media without growth regulators. Embryogenic calli were observed after 180 days of culture and in all treatments with auxin. The Y3 medium showed the best EC formation results (60.8 %). These calli showed yellowish coloration, compact consistency and nodular aspect. After 60 days in differentiation medium, SEs were verified in different stages of development. Histological analysis showed that the SEs were formed from a nodular EC. The SEs generally presented unicellular origin with suspensor formation, and at the end of development, bipolar embryos were observed. The plant regeneration frequency reached levels up to 31.9 % when using induction medium consisting of Y3 associated to 1.5 mg L^?1 of 2,4-D and the subsequent auxin reduction to 0.5 mg L^?1 in the differentiation stage. Regenerated plants showed normal development, with root and aerial part growth.     

Somatic embryogenesis from peach palm zygotic embryos

The factors affecting the induction and development of somatic embryos and plantlet acclimatization of peach palm (Bactris gasipaes Kunth) were evaluated to establish an efficient regenerative protocol based on somatic embryogenesis. Mature zygotic embryos were cultured in Murashige and Skoog (MS) medium supplemented with 0–40 μM of picloram (4-amino-3,5,6-trichloropicolinic acid) and 0 or 5 μM of 2-isopentyladenine (6-dimethylaminopurine) (2-iP). After 5 mo. in culture embryogenic callus arose from primary calli. Picloram (10 μM) was effective in inducing embryogenic calli in 9.8% of the explants. The use of 1 μM of AgNO₃ enhanced embryogenic competence. Embryogenic calli showed an organized structure, a globular aspect, and were white to yellowish in color. Histological analyses showed that cell proliferation arose from subepidermal cells adjacent to vascular bundles, resulting in primary callus formed by a meristematic zone from which somatic embryos arose. Protein profile analyses revealed two high molecular mass bands in these embryogenic calli, but not in other tissues. Embryogenic calli were transferred to a culture medium containing 40 μM of 2,4-dichlorophenoxyacetic acid, 10 μM of 2-iP, plus 1 g l⁻¹ of glutamine, hydrolyzed 0.5 g l⁻¹ casein, and activated 1.5 g l⁻¹ of charcoal. Morphogenetic responses achieved in this medium were the development of somatic embryos, rooting, and loss of embryogenic capacity. Somatic embryos were converted to plantlets on MS medium plus 24.6 μM of 2-iP and 0.44 μM of naphthalene acetic acid. Plantlets were maintained in MS medium with activated charcoal (1.5 g l⁻¹) until they were 6 cm tall, and then acclimatized. After 16 wk, 84.2 ± 6.4% survival was observed. M. P. Guerra and C. R. Clement are Fellows of the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brasília, DF.     

Plant regeneration via somatic embryogenesis from solid and suspension cultures of Vitis vinifera L. cv. ‘Manicure Finger’

Vitis vinifera L. cv. ‘Manicure Finger’ is one of the major table grape varieties in China. To provide a strong foundation for genetic transformation with potential for crop improvement, we undertook plant regeneration via somatic embryogenesis. Anthers and gynoecia were harvested from immature flowers and used as explants to induce embryogenic calli. Explants cultured in MS1 medium (based on Murashige and Skoog basal salts), supplemented with 4.5-μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4-μM 6-benzylaminopurine (6-BA) showed the highest rates of embryogenic callus induction (3.7%?±?1.3% for anthers and 4.8%?±?2.5% for gynoecia). After several months, somatic embryos were produced from embryogenic calli cultured in plant growth regulator-free MS2 medium (with reduced sucrose). Somatic embryos (SE) at the cotyledonary stage were isolated and cultured on three different media (MS2, MS3, or B) for conversion into plantlets, the efficiency of which ranged from 63.9%?±?4.8% to 83.9%?±?8.4%. After 1 mo of in vitro culture, 80% of plants with at least six leaves were successfully transplanted into soil. SE was repeatedly induced from previously induced somatic embryos for up to 1.5 yr. Using embryogenic calli as starting material, suspension cultures containing embryogenic cell aggregates were also established in liquid MS medium supplemented with 4.5-μM 2,4-D. The embryogenic cell aggregates continued to proliferate without differentiating for successive subculture cycles. After transfer to 2,4-D-free liquid medium for 4 wk, an average of 63.7%?±?9.0% mature SEs were produced per 20 mL of liquid medium. More than 40% of somatic embryos at cotyledonary stage, derived from the suspension cultures, successfully germinated into plants using solid medium.     

In vitro plant regeneration from immature zygotic embryos and repetitive somatic embryogenesis in kohlrabi (Brassica oleracea var. gongylodes)

A simple and efficient protocol for direct somatic embryogenesis and plant regeneration of kohlrabi (Brassica oleracea var. gongylodes) was developed. Somatic embryos were induced from immature zygotic embryos at different developmental stages cultured on Murashige and Skoog medium supplemented with 0, 0.5, 1.0, or 1.5 mg/l 2,4-dichlorophenoxyacetic acid. Zygotic embryos at the early cotyledonary stage, which were cultured for 4 wk on plant growth regulator-free (PGR-free) medium, displayed the highest percentage of somatic embryogenesis (80.7%). Embryogenic tissue could be subcultured on the same medium for over 1 yr. Embryogenic lines derived from early cotyledonary stage zygotic embryos displayed the highest intensity of secondary embryogenesis (highest mean number of new somatic embryos per responsive somatic embryo explant). Histological analyses confirmed the direct origin of the secondary somatic embryos. Prolonged culturing of embryogenic tissue on PGR-free medium led to somatic embryo development into plantlets that were successfully acclimated in the greenhouse with a survival rate of 72.5%. Flow cytometry analysis showed no ploidy variation in 96.7% of the acclimated plants.     

Somatic embryogenesis and plant regeneration in oil palm using the thin cell layer technique

An efficient procedure has been developed for inducing somatic embryogenesis and regeneration of plants from tissue cultures of oil palm (Elaeis guineensis Jacq.). Thin transverse sections (thin cell layer explants) of different position in the shoot apex and leaf sheath of oil palm were cultivated in Murashige and Skoog (MS) (Physiol Plant 15:473–497, 1962) medium supplemented with 0–450 μM picloram and 2,4-D with 3.0% sucrose, 500 mg L⁻¹ glutamine, and 0.3 g L⁻¹ activated charcoal and gelled with 2.5 g L⁻¹ Phytagel. Embryogenic calluses were evaluated 12 wk after inoculation. Picloram (450 μM) was effective in inducing embryogenic calluses in 41.5% of the basal explants. Embryogenic calluses were maintained on a maturation medium composed of basal media, plus 0.6 μM NAA and 12.30 μM 2iP, 0.3 g L⁻¹ activated charcoal, and 500 mg L⁻¹ glutamine, with subcultures at 4-wk intervals. Somatic embryos were converted to plants on MS medium with macro- and micronutrients at half-strength, 2% sucrose, and 1.0 g L⁻¹ activated charcoal and gelled with 2.5 g L⁻¹ Phytagel.     

Somatic embryogenesis and plant regeneration through leaf culture in <Emphasis Type="Italic">Arachis glabrata (Leguminosae)</Emphasis>

Plants of two accessions of Arachis glabrata were regenerated via somatic embryogenesis. Embryogenic calli were initiated from leaflet explants on Murashige and Skoog medium supplemented with picloram alone or picloram in combination with 6-benzylaminopurine. Leaflets of accession A6138 induced the highest percentage of somatic embryos in media composed of 10 mg dm⁻³ and 15 mg dm⁻³ picloram. In contrast, 5 mg dm⁻³ picloram with 0.1 mg dm⁻³ 6-benzylaminopurine was one of the most effective combinations in accession AF385. MS medium supplemented with 2 g dm⁻³ activated charcoal (AC) used for 30 days was the most effective for embryo maturation. After 20 days of culture on MS medium devoid of growth regulators, 6 % of embryos converted into plantlets in accession A6138.     

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.     

Somatic embryogenesis and plantlet formation from a rare and endangered tree species,Oplopanax elatus

We tested the possibility of plantlet formation via somatic embryogenesis with leaf segments and mature zygotic embryos from a rare and endangered tree species,Oplopanax elatus. To induce calli, expiants were cultured under darkness in a solid MS medium containing 3% sucrose, 1g L^-1 glutamine, and 0.3% gelrite. Treatment supplements included 2,4-D alone or in combination with thidiazuron. Generally, callus induction and growth were good from leaf expiants, whereas embryogenic calli could be induced only from zygotic embryos. These embryogenic calli were white or pale yellow and very friable. ABA and activated charcoal appeared to be important factors when inducing somatic embryos, with optimum levels being 0.1 mg L^-1 and 0.02%, respectively. Many somatic embryos showed abnormalities during their development on the germination medium, but 35% could be converted if placed on a medium containing gibberellic acid (GA₃). The germinating embryos sometimes formed secondary embryos at the lower portion of the hypocotyls. Normal or converted plantlets were acclimatized in an artificial soil mixture; their survival was about 60% after two months. This culturing system provides a feasible approach for regenerating plants, via somatic embryogenesis, from mature zygotic embryos.     

High efficient somatic embryogenesis development from leaf cultures of <Emphasis Type="Italic">Citrullus colocynthis</Emphasis> (L.) Schrad for generating true type clones

We report an efficient somatic embryogenesis and plant regeneration system using leaf cultures of Citrullus colocynthis (L.) and assessed the effect of plant growth regulators on the regeneration process. Initially leaf explants were cultured on Murashige and Skoog medium supplemented with different concentrations of auxins viz., 2,4-dichlorophenoxyacetic acid, 1-naphthaleneacetic acid, gibberellic acid alone and along with combination of 6-benzylaminopurine. The different forms of calli such as compact, white friable, creamy friable, brownish nodular, green globular and green calli were induced from the leaf explants on MS medium containing different concentrations of auxins and gibberellins. Subsequently initial callus was subcultured at 1.5 mg L^?1 BAP + 1.0 mg L^?1 2,4-D which resulted in 25 % somatic embryos from 85 % nodular embryogenic nodular callus that is highest percentage. Similarly the lowest percentage of somatic embryos was recorded at 2.5 mg L^?1 BAP + 0.5 mg L^?1 NAA from 55 % embryogenic globular callus i.e., 16 %. High frequency of embryo development takes place at intermittent light when compared with continuous light in the individual subcultures. The cotyledonary embryos were developed into complete platelets on MS medium. In vitro regenerated plantlets were washed to remove the traces of agar and then transferred to sterile vermiculite and sand (2:1) containing pot.     

Somatic embryogenesis and plant regeneration in zygotic embryo explant cultures of rugosa rose

Rugosa rose (Rosa rugosa) is cultivated as a garden flower and an important genetic resource for the breeding of roses (R. hybrida). This study describes culture conditions for high frequency plant regeneration from zygotic embryo explants via somatic embryogenesis in rugosa rose. Mature zygotic embryo, cotyledon, and radicle explants formed embryogenic calluses at frequencies of 38, 6.7, and 8.8% when cultured on half-strength Murashige and Skoog medium (½MS) supplemented with 2.26, 9.05, and 9.05 μM 2,4-dichlorophenoxyacetic acid, respectively. Embryogenic calluses produced numerous somatic embryos, which then developed into plantlets on ½MS without growth regulators. Regenerated plantlets were grown to whole plants in a growth chamber.     

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.     

Development of an in vitro protocol for a difficult-to-propagate endemic Australian dryland sedge species Mesomelaena pseudostygia (Cyperaceae)

In vitro propagation for Mesomelaena pseudostygia a difficult-to-propagate dryland sedge species (Cyperaceae) endemic to Western Australia is described. Multiple avenues to in vitro propagation were investigated: shoot culture, organogenesis and somatic embryogenesis, with zygotic embryos as initiation material. The highest multiplication rate for shoots was 3.4?±?1.0 after 6 wk on basal medium (1/2 strength Murashige and Skoog) with 2.5 μM kinetin and 0.5 μM 6-benzylaminopurine. Shoots achieved peak rooting (83%) following a pulse treatment on basal medium containing 10 μM indolebutyric acid and 2 μM α-naphthaleneacetic acid for 7 wk, followed by transfer to medium (without growth regulators) for a further 7 wk. Alternatively, in vitro grown shoots were pulse treated on basal medium with both 100 μM indolebutyric acid and 20 μM α-naphthaleneacetic acid for 1 wk then placed in Rockwool plugs (under propagation house conditions) for another 7 wk resulting in 63% root induction. Rooted plantlets were also successfully transferred to potting mixture either in Rockwool plugs or bare rooted and maintained in propagation house conditions with ≥95% survival after 7 wk. These results indicate that micropropagation of M. pseudostygia is feasible for small to medium scale restoration purposes. The highest frequency of callus induction was from cultured zygotic embryos on basal medium with 5 μM α-naphthaleneacetic acid, whereas 2,4-dichlorophenoxacetic acid (2 or 5 μM) produced the largest callus sizes. A low frequency of shoot regeneration occurred in zygotic callus tissues in basal medium treatments containing cytokinin (kinetin or thidiazuron at 1 μM). A small proportion (<20%) of zygotic embryo callus explants from 2,4-dichlorophenoxyacetic acid treatments were found to be embryogenic, firstly developing embryo-like structures after 2 wk on basal medium (minus plant growth hormones), that continued to develop with approximately one in twenty germinating after a further 4 wk on basal medium to form small plantlets. Further optimisation is needed to improve somatic embryogenesis efficiency for mass propagation.     

Plant regeneration from embryogenic cultures initiated from mature loblolly pine zygotic embryos

Summary Mature zygotic embryos of eight (open-pollinated) families of loblolly pine (Pinus taeda L.) were cultured on eight different basal salt formulations, each supplemented with 36.2 μM 2,4-dichlorophenoxyacetic acid, 17.8 μM 6-benzyladenine, 18.6 μM kinetin, 500 mg l⁻¹ casein hydrolysate, and 500 mg l⁻¹ l-glutamine for 9 wk; embryogenic tissue was formed on cotyledons, hypocotyls, and radieles of mature zygotic embryos. Callus was subcultured on the callus proliferation medium, the same as the induction medium but with one-fifth concentration of auxin and cytokinin for 9 wk. On this medium a white to translucent, glossy, mucilaginous embryogenic callus containing embryogenic suspensor masses (ESMs) was obtained. The highest frequency of explants forming embryogenic tissue, 17%, occurred on a modified Murashige and Skoog salts basal medium containing the concentration of KNO₃, Ca(NO₃)₂·4H₂O, NH₄NO₃, KCl, ZnSO₄·7H₂O, and MnSO₄·H₂O, 720, 1900, 400, 250, 25.8, and 25.35 mg l⁻¹, respectively. Embryogenic suspension cultures were established by culturing embryogenic callus in liquid callus proliferation medium. Liquid cultures containing ESMs were transferred to medium containing abscisic acid, polyethylene glycols, and activated charcoal for stimulating the production of cotyledonary somatic embryos. Mature somatic embryos germinated for 4–12 wk on medium containing indole-butyric acid, gibberellic acid, 6-benzyladenine, activated charcoal, and reduced sucrose concentration (15 g l⁻¹). Two hundred and ninety-one regenerated plantlets were transferred to a perlite:peatmoss:vermiculite (1∶1∶1) mixture, then the plants were transplanted to soil in the earth, and 73 plantlets survived in the field.     

Effect of genotype on somatic embryogenesis from axes of mature peanut embryos

Genotypes representing the three botanical varieties of peanut (Arachis hypogaea L.) were assessed for somatic embryogenesis and subsequent plant conversion from mature zygotic embryo axes. Explants were initially cultured on Murashige and Skoog medium supplemented with 12.42 M 4-amino-3,5,6-trichloropicolinic acid. Individual somatic embryos wer isolated from explant tissue and used to initiate repetitive liquid cultures. There were significant differences among genotypes and varieties for somatic embryo formation and plant regeneration using a single media sequence. Botanical variety fastigiata had a lower embryogenic frequency and produced significantly fewer embryos than either hypogaea or vulgaris, which were similar in response.Abbreviations EA zygotic embryo axes - MS Murashige and Skoog (1962) medium - picloram 4-amino-3,5 - 6 trichloropicolinic acid     

Somatic embryogenesis and plant regeneration from cell suspension cultures of Rajeli (AAB), an endangered banana cultivar

Rajeli (AAB), a commercially valuable Indian banana cultivar, is presently under the threat of extinction due to various diseases, warranting development of the strategies for its conservation and incorporation of disease resistance. This elite genotype was successfully regenerated in vitro via establishment of cell suspensions followed by somatic embryogenesis. The immature male flower buds were cultured on gelled Murashige and Skoog medium supplemented with 4 mg/l 2,4-D, 1 mg/l each of IAA and NAA, D-Biotin, 100 mg/l Malt Extract, 100 mg/l L-Glutamine and 30 g/l sucrose. Embryogenic calli with translucent somatic embryos were observed after 4 months of male flower bud culturing. Fine embryogenic cell suspensions were established in the liquid medium of same composition but with 45 g/l sucrose. Of the various sugars tested, maltose in the maintenance medium yielded better growth of plated cells as compared to sucrose, fructose and dextrose. Embryos differentiated at a high frequency and mature somatic embryos developed into plantlets on MS medium supplemented with 0.5 mg/l BAP. Approximately 40 % of the torpedo stage somatic embryos germinated and developed into complete plants. The regenerated plants exhibited normal phenotype during acclimatization in the greenhouse. The technique can be employed for conservation of this endangered cultivar and its improvement via mutagenesis and genetic transformation.     

SOMATIC EMBRYOGENESIS AND PLANT REGENERATION FROM IMMATURE ZYGOTIC EMBRYOS OF MUSCADINE GRAPE (VITIS ROTUNDIFOLIA) CULTIVARS

Embryogenic cell lines of Vitis rotundifolia were produced from immature zygotic embryo explants obtained by culturing ovules, harvested at 20 d postanthesis, for 8 wk and then dissecting embryos from them. Ovules cultured on Nitsch and Nitsch medium with naphthoxyacetic acid and benzyladenine (BA) produced a brown exudate, necessitating three transfers to fresh medium at 2-wk intervals during the 8-wk culture cycle. Zygotic embryos that were subsequently isolated from cultured ovules and placed on the same medium produced a heterogenous callus from which eventually emerged embryogenic cell lines. A higher percentage of ovules from cultivars ‘Dixie’, ‘Fry’, ‘Nesbitt’, and ‘Welder’ produced zygotic embryos (31%–39%) than did those from ‘Carlos’ (3%). A higher percentage of ‘Fry’ ovules produced embryogenic lines from cultured zygotic embryos (6.3%) than did those of the other four cultivars (1%–1.6%). Embryogenic cell lines were white and composed of variably sized cell clusters, somatic embryos, and embryonic tissue embedded in a watery matrix. These lines were maintained for over 1 yr on modified Murashige and Skoog (MS) medium lacking growth regulators by transfer of selected cell clusters every 6 wk. White, opaque somatic embryos grew directly from cell clusters and passed through recognizable developmental stages. Germination was induced by transfer of somatic embryos to MS medium with BA. Although 80%–100% of embryos germinated, plant recovery was low due to poor shoot development.     

Plant regeneration through somatic embryogenesis from suspension cultures of a minor millet,Paspalum scrobiculatum

Compact, friable and embryogenic calli were initiated from immature inflorescences and young leaf bases of one week old seedlings of Paspalum scrobiculatum cultured on MS medium supplemented with 2,4-D. A stable, embryogenic suspension culture was initiated from these calli and maintained in a liquid version of the same MS medium. Embryogenic calli and somatic embryos were obtained by plating suspension culture cells onto semi-solid medium containing 2,4-D. Complete, normal plantlets developed on 2,4-D free medium at a high frequency from somatic embryos. NAA and BAP in the medium promoted plant development.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - BAP 6-benzylaminopurine - ABA Abscisic acid - MS Murashige and Skoog (1962) - CM Coconut milk