Plant regeneration from somatic embryogenic suspension cultures of cotton (Gossypium hirsutum L.)

Maintainable, highly embryogenic suspension cultures of cotton (Gossypium hirsutum L. cv. Coker 310) have been obtained. Callus cultures were initiated from cotyledonary tissues from aseptically-germinated seedlings. To establish the suspension cultures, callus tissue was placed in a liquid medium containing either 0.5 mg/l picloram or 0.1 mg/l 2,4-dichlorophenoxyacetic acid. For proliferation of the embryogenic suspension, 5 mg/l of 2,4-dichlorophenoxyacetic acid was used. Embryo development took place when the embryogenic tissue was transferred to an auxin-free liquid medium containing 15 mM glutamine. Early embryo development was fairly synchronous and large numbers of somatic embryos were produced. Regenerated plants were fertile and smaller than seed-derived plants.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid     

Somatic embryogenesis in <Emphasis Type="Italic">Schisandra chinensis</Emphasis> (Turcz.) Baill.

Summary Regeneration of plants via somatic embryogenesis was achieved from zygotic embryo explants isolated from mature seeds of Schisandra chinensis. Merkle and Sommer's medium, fortified with 2,4-dichlorophenoxyacetic acid (2,4-D; 9.04 μM) and zeatin (0.09 μM), was effective for induction of embryogenic callus. The development of a proembryogenic mass and somatic embryos occurred on Murashige and Skoog medium (MS) free of plant growth regulators. The embryogenic callus induced on Merkle and Sommer's medium supplemented with 2,4-D (9.04 μM) and zeatin (0.09 μM) showed development of the maximum number of somatic embryos when transferred to MS medium free of plant growth regulators. The maximum maturation and germination of cotyledonary somatic embryos (46.3%) occurred on MS medium supplemented with 2,4-D (0.45 μM) and N⁶-benzyladenine (1.11 μM). The somatic embryo-derived plants were successfully hardned, with a survival rate of approximately 67%, and established in the field.     

Initiation of embryogenic callus and suspension cultures of eastern white pine (Pinus strobus L.)

Embryogenic callus and suspension cultures of eastern white pine (Pinus strobus) have been obtained. The whole female gametophyte was plated on a medium containing 50 mg/l glutamine, 500 mg/l casein hydrolysate, 3% sucrose, 2 mg/1 2,4-D, 1 mg/1 BA and 0.2% Gelrite as a solidifying agent. Embryogenic calli could be seen as early as 5 days following culture. Histological studies indicate proliferation of pre-existing embryogenic tissue in the corrosion cavity followed by extrusion of embryogenic callus through the micropylar end of the gametophyte. Embryogenic suspension cultures were obtained by placing embryogenic callus into liquid medium. Embryogenic suspension cultures were subcultured weekly and proliferated as early-stage embryos with attached suspensors. Embryo development was obtained following transfer of the embryogenic tissue to an auxin-free medium containing 50 mM glutamine, 38 M abscisic acid, and 6% sucrose. Although embryo development could be consistently obtained, whole plants have not yet been recovered from these somatic embryos.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - ABA Abscisic acid - BA 6-Benzyladenine Salaries and research support were provided by State and Federal funds appropriated to OSU/OARDC. Journal Article No. 62–89     

Influence of culture conditions on embryo formation and maturation in auxin-induced embryogenic cultures of Digitalis obscura

Somatic embryogenesis was induced in hypocotyls of Digitalis obscura using indoleacetic acid or 2,4-dichlorophenoxyacetic acid with different culture and subculture conditions. Indoleacetic acid-induced embryogenic cultures were used to investigate the effects of amino acids, polyamines and growth regulators on embryo differentiation and maturation. Supplementation of the media with amino acids, polyamines or abscisic acid did not influence or had an adverse effect on embryogenic response. Gibberellic acid at 1.4 M in either culture (30 days) or subculture medium was effective in promoting both differentiation and normal embryo development. The efficiency of somatic embryogenesis was greatly enhanced when isolated indoleacetic acid-induced proembryogenic masses were subcultured in liquid medium with reduced auxin content.Abbreviations ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA gibberellic acid - IAA indoleacetic acid - Ptr putrescine - Spd spermidine - Spn spermine     

Induction of somatic embryogenesis in cashewnut (Anacardium occidentale L.)

Summary Somatic embryogenesis was induced in callus cultures derived from nucellar tissue of cashewnut (Anacardium occidentale L.). Callus was obtained from nucellar tissue after 3 wk of culture on semisolid Murashige and Skoog (MS) basal medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D, 5 μM)+gibberellic acid (GA₃, 15 μM)+N⁶-benzyladenine (BA, 5 μM). This callus gave rise to an embryogenic mass after 9 wk on maintenance medium containing 2,4-D (10 μM)+GA₃ (15 μM)+4% sucrose +0.5% activated charcoal +10% coconut water (CW) +0.05% casein hydrolysate (CH). The embryogenic mass, after transfer to medium supplemented with 2,4-D (5 μM)+GA₃ (30 μM)+4% sucrose +0.5% activated charcoal +10% CW +0.05% CH, gave rise to somatic embryos. The developmental stages of somatic embryos were observed using light and stereo microscopes. Histological study of somatic embryo development was also carried out. The present study would be useful for clonal propagation, and variety improvement in cashewnut, which is essential due to its increasing demand and export potential.     

Factors affecting somatic embryogenesis in <Emphasis Type="Italic">Prunus incisa</Emphasis> cv. February Pink

Factors affecting somatic embryogenesis from root explants of Prunus incisa Thunb. cv. February Pink were investigated. Using a medium containing Murashige and Skoog salts and vitamins supplemented with 10 M 2,4-dichlorophenoxyacetic (2,4-D), we evaluated the effects of light, growth regulators, amino acids, carbohydrate source, and root induction medium. Explants cultured under light or dark conditions both resulted in the formation of embryos. Embryogenesis was inhibited by the addition of 6-benzyladenine, thidiazuron, or gibberellic acid to the medium. Amino acids were not effective in promoting embryogenesis, with high levels of amino acids actually inhibiting it. Sucrose and glucose effectively induced embryogenesis, while sorbitol and mannitol completely inhibited it. Sucrose and glucose also promoted secondary embryogenesis. Embryos that formed in medium containing 4% or 5% sucrose were abnormally shaped and did not fully develop, while those that formed in medium with sucrose concentrations of 2% or 3% were much more vigorous. Root explants that were induced on medium containing 1.0 M indole-3-butyric acid (IBA) produced more somatic embryos than explants induced on medium without IBA. Approximately 50% of the roots induced on medium containing 1.0 M IBA produced somatic embryos on medium containing 10 M 2,4-D and 3% sucrose.Abbreviations BA 6-Benzyladenine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - IBA Indole-3-butyric acid - NAA -Naphthaleneacetic acid - TDZ Thidiazuron     

Epigenetics of long-term somatic embryogenesis in <Emphasis Type="Italic">Theobroma cacao</Emphasis> L.: DNA methylation and recovery of embryogenic potential

In Theobroma cacao L., declined embryogenic potential was observed in regenerated somatic embryos from long-term secondary somatic embryogenesis (SE). In order to explore the relationship between DNA methylation and the long-term secondary SE, the embryogenic potential and global DNA methylation levels in young (12 months-old), aged (36 months-old) and extra somatic embryogenesis (39 months-old) subjected to different 5-Azacytidine (5-azaC) treatments were comparatively assessed. Global DNA methylation levels increased in aged somatic embryos with long-term in vitro culture, but 5-azaC-supplemented treatments resulted in unaltered levels. In addition, DNA methylation pattern during SE was not affected by 5-azaC. DNA methylation increased during SE expression. Interestingly, the extra SE induction showed that aged somatic embryos can recovery the embryogenic potential in treatment supplemented with 5-azaC at specific concentration. The outcome of this study suggested that the long-term SE in cacao induced the decline on embryogenic potential, which can be reversible trough 5-azaC supplementation. Besides, increased DNA methylation levels might be a response to the stress conditions that plant cells were exposed to during SE.     

Somatic embryogenesis and plant regeneration from leaves of<Emphasis Type="Italic"> Ulmus minor</Emphasis> Mill.

Somatic embryogenesis from mature elm (Ulmus minor Mill.) in vitro-cloned material is possible. Embryogenic callus was obtained from leaves inoculated on two different MS-based media—one supplemented with 2.3 M 2,4-dichlorophenoxyacetic acid (I2) and the other supplemented with 1.1 M kinetin (I6). However, only leaves cultured on medium I6 produced somatic embryos, at the globular stage, when embryogenic callus was maintained in induction media. When embryogenic callus from medium I6 was transferred to basal medium, somatic embryos with green cotyledons were obtained. An average of 35.9% of these embryos converted easily into normal plants in conversion medium with 1% sucrose. Acclimatisation reached 39.7%, and this was not significantly different from a control group consisting of plants propagated by axillary buds. No morphological differences were observed between plants derived from somatic embryos and control plants. Also, no differences in ploidy were detected between the somatic embryo-derived plants and the mother plants.Abbreviations BA: Benzyladenine - C1, C2: Conversion media - 2,4-D: 2,4-Dichlorophenoxyacetic acid - Kn: Kinetin - NAA: -Naphthaleneacetic acid - PI: Propidium iodide - I2, I6: Induction media Communicated by D. Bartels     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration via somatic embryogenesis through cell suspension cultures of horsegram [<Emphasis Type="Italic">Macrotyloma uniflorum</Emphasis> (Lam.) verdc.]

Summary In vitro regeneration of plants via somatic embryogenesis through cell suspension culture was achieved in horsegram. Embryogenic calluses were induced on leaf segments on solid Murashige and Skoog (MS) medium with 9.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Differentiation of somatic embryos occurred when the embryogenic calluses were transferred to liquid MS medium containing 2,4-D. Maximum frequency (33.2%) of somatic embryos was observed on MS medium supplemented with 7.9 μM 2,4-D. Cotyledonary-torpedo-shaped embryos were transferred to liquid MS medium without growth regulators for maturation and germination. About 5% of the embryos germinated into plants, which grew further on solid MS medium. The plants were hardened and established in soil. Effects of various auxins, cytokinins, carbohydrates, amino acids, and other additives on induction and germination of somatic embryos were also studied. A medium supplemented with 7.9 μM 2,4-D, 3.0% sucrose, 40 mg l⁻¹ L-glutamine, and 1.0 μM abscisic acid was effective to achieve a high frequency of somatic embryo induction, maturation, and further development.     

Efficient plant regeneration from embryogenic suspension cultures of sweetpotato

Summary Using 15 Chinese and Japanese cultivars of sweetpotato, Ipomoea batatas (L.) Lam., we succeeded in developing an efficient plant regeneration system from embryogenic suspension cultures. The embryogenic callus derived from shoot apices of the 15 cultivars was used to initiate embryogenic suspension cultures in Murashige and Skoog (MS) medium containing 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Rapidly proliferating and well-dispersed embryogenic suspension cultures were established. Cell aggregates 0.7–1.1 mm in size from embryogenic suspension cultures were transferred to solid MS medium supplemented with 9.05 μM of 2,4-D and formed embryogenic callus with somatic embryos. The embryogenic callus with somatic embryos was further transferred to MS medium supplemented with 3.78 μM of abscisic acid, resulting in the germination of somatic embryos. Within 20 wk after the initiation, the frequencies of cell aggregates forming plantlets reached approximately 100% for the 15 tested cultivars. These plantlets, when transferred to soil, showed 100% survival. No morphological variations were observed.     

Variation in endoplasmic-reticulum-associated glycoproteins of carrot cells cultured in vitro

Glycoproteins extracted from microsomes of in-vitro-cultured cells of Daucus carota L. cv. US-Harumakigosun were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detected by peroxidase-conjugated concanavalin A. The appearance of a glycoprotein with M_r 31 000 (GP 31) was correlated with the ability of cells to form somatic embryos. GP 31 appeared in embryogenic cells cultured in 2,4-dichlorophenoxyacetic acid (2,4-D)-containing medium, but not in somatic embryos and non-embryogenic cells; it disappeared when the cultures were transferred to auxin-free medium. Another glycoprotein with M_r 32 000 (GP 32) was detected only in non-embryogenic cells, regardless of the presence or absence of 2,4-D. Both glycoproteins, GP 31 and GP 32, were associated with the rough endoplasmic reticulum and were extractable with 0.05% deoxycholate.Abbreviations Con A concanavalin A - 2,4-D 2,4-dichlorophenoxyacetic acid - ER endoplasmic reticulum - GP 31, GP 32 a glycoprotein with an apparent molecular mass of 31 or 32 kdalton - kDa kilodalton - MS Murashige and Skoog - M_r apparent molecular mass - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

Embryogenesis and plant regeneration from anther culture of bamboo (Sinocalamus latiflora (Munro) McClure)

Embryogenic callus was initiated from bamboo (Sinocalumus satiflora (Munro) McClure) anthers cultured on N₆ medium supplemented with 1 mg/l 2,4-D, 1 mg/l BA, 2 g/l charcoal, 0.8% agar (Sigma) and 9% sucrose. Anthers with microspores at miduninucleate to early-binucleate stages showed better rate of response for callus induction. Prolonged culture of these embryogenic calli on the original medium or subculture to an auxin-free medium resulted in embryoid formation and their subsequent germination to form rooted plantlets. Chromosome counts from root-tip cells of anther-derived plant indicated that they were haploid (N=36).Abbreviations N₆ Chu et al. (1975) - MS Murashige and Skoog (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - BA 6-benzylaminopurine     

High frequency somatic embryogenesis and plant regeneration in zygotic embryo cultures of Japanese honeysuckle

Japanese honeysuckle plant (Lonicera japonica Thunb.) is rich in iridoid secologanin and is a potentially useful model for the study of secologanin biosynthesis. Culture conditions for high frequency plant regeneration via somatic embryogenesis from zygotic embryo cultures and zygotic embryo-derived embryogenic cell suspension cultures of this species are described. Mature zygotic embryos formed embryogenic calluses at a frequency of 46.7% when cultured on Murashige and Skoog (MS) medium supplemented with 4.52 M 2,4-dichloro-phenoxyacetic acid (2,4-D). Cell suspension cultures were established with embryogenic calluses using liquid MS medium with 4.52 M 2,4-D. Upon plating onto MS basal medium, embryogenic cell suspension cultures produced numerous somatic embryos, which subsequently developed into plantlets at a frequency of 68%. Regenerated plantlets were transplanted to potting soil and grown to maturity in a greenhouse.     

Effect of abscisic acid and cytokinins on the development of somatic embryos in Hevea brasiliensis

Addition of liquid medium, conditioned by an embryogenic suspension, to MH1 solid medium (3,4-dichlorophenoxyacetic acid 9 M, 6-benzyladenine 9 M) permitted the frequent induction of highly embryogenic calli from slices of internal integument of immature seeds of Hevea brasiliensis Müll. Arg. The proliferation of embryogenic cell clusters was achieved in MH1 liquid medium. Abscisic acid (ABA), cytokinins and adenine were tested for their ability to affect development of somatic embryos to plantlets. The transfer of embryogenic cell clusters on auxin-free solid medium with 10^-5M ABA for 2 months stimulated embryo development. When torpedo-shaped embryos were transferred to medium with adenine or cytokinins they turned green in 1 month. Green embryos produced secondary embryos when they were collected and placed on medium without growth regulators.Abbreviations ABA abscisic acid - BA 6-benzyladenine - IBA indole-3-butyric acid - NOA -naphthoxyacetic acid - 2iP 2-iso-pentenyladenine - 3,4-D 3,4-dichlorophenoxyacetic acid     

High-frequency plant regeneration from coleoptile tissue of indica rice (Oryza sativa L.)

Summary An efficient method was established for high-frequency embryogenic callus induction and plant regeneration from 3-,4-, 5- and 7-d-old coleoptile segments of Indica rice (Oryza sativa L. cv. Kasturi), Compact and friable callus developed from the cut ends and also on the entire length of the coleoptile segments cultured on Murashige and Skoog (MS) basal medium (1962) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D, 4.50–18.0 μM), kinetin (2.32 μM) and sucrose (3%, w/v). High frequency embryogenic callus induction and somatic embryo development was achieved when embryogenic calluses were transferred to MS medium supplemented with 2.25 μM 2,4-D, 2.32 μM kinetin, 490 μM L-tryptophan and 3% (w/v) sucrose. Plant regeneration was achieved by transferring clumps of embryogenic callus onto MS medium containing 2.85 μM indole-3-acetic acid (IAA), 17.77 μM 6-benzylaminopurine (BA) and 3% (w/v) sucrose. Histological observations of embryogenic calluses revealed the presence of somatic embryos and also plant regeneration via multiple shoot bud formation. Three, 4- and 5-d-old coleoptile segments showed a significantly (P<0.05) higher frequency of plant regeneration and mean number of plantlets per explant in comparison to 7-d-old coleoptile segments. The highest frequency (73.5%) of plant regeneration and mean number of plantlets (11.9±1.0) was obtained from 4-d-old coleoptile segments. Regenerated shoots were rooted on MS basal medium containing 4.92 μM indole-3-butyric acid (IBA) and plants were successfully transferred to soil and grown to maturity.     

Somatic embryogenesis inGnetum ula Brongn. (Gnetum edule) (Willd) Blume

Summary Somatic embryos ofGnetum ula (Gnetum edule) an endangered gymnosperm closely related to the angiosperms have been induced in vitro. Megagametophyte tissue with immature embryos was cultured on Murashige and Skoog medium. A mucilaginous, translucent embryogenic callus was obtained with 5 mg/l BA. Callus induced with 2,4-D was non-embryogenic. The embryogenic callus in liquid half strength Murashige and Skoog medium without inorganic nitrates supplemented with 2.5 g/l casein hydrolysate and 0.5 g/l L-glutamine gave rise to immature embryos. The embryos matured when treated with 60 g/l sucrose and 10 mg/l abscisic acid.Abbreviations MS Murashige and Skoog - BA 6-benzylaminopurine - 2,4-D 2,4 - dichlorophenoxyacetic acid - ABA abscisic acid     

Plant regeneration protocol of medicinally important <Emphasis Type="Italic">Andrographis paniculata</Emphasis> (Burm. F.) Wallich <Emphasis Type="Italic">ex</Emphasis> Nees via somatic embryogenesis

Summary In vitro propagation of Andrographis paniculata (Burm. f.) Wallich ex Nees through somatic embryogenesis, and influence of 2,4-dichlorophenoxyacetic acid (2,4-1) on induction, maturation, and conversion of somatic embryos were investigated. The concentration of 2,4-D in callus induction medium determined the induction, efficacy of somatic embryogenesis, embryo maturation, and conversion. Friable callus initiated from leaf and internode explants grown on Murashige and Skoog (MS) medium supplemented with 2.26, 4.52, 6.78, and 9.05μM 2,4-D started to form embryos at 135, 105, 150, and 185d, respectively, after explant establishment. Callus initiated at 13.56μM 2,4-D did not induce embryos even after 240 d, whereas those initiated on MS medium with 4.52μM 2,4-D was most favorable for the formation and maturation of somatic embryos. Callus subcultured on the medium with reduced concentration of 2,4-D (2.26μM) became embryogenic. This embryogenic callus gave rise to the highest number of embryos (mean of 312 embryos) after being transferred to half-strength MS basal liquid medium. The embryos were grown only up to the torpedo stage. A higher frequency of embryos developed from callus initiated on 2.26 or 4.52 μM 2,4-D underwent maturation compared to that initiated on higher concentrations of 2.4-D. The addition of 11.7μM silver nitrate to half-strength MS liquid medium resulted in 71% of embryos undergoing maturation, while 83% of embryos developed into plantlets after being transferred to agar inedium with 0.44 μMN⁶-benzyladenine and 1.44 μM gibberellic acid. Most plantlets (88%) survived under field conditions and were morphologically identical to the parent plant.     

Embryogenic cell lines from somatic embryos of grape (Vitis vinifera L.)

Somatic embryo formation occurred on leaf callus of grape (Vitis vinifera cv. Koshusanjaku). An embryogenic callus was induced from somatic embryo clusters cultured on vitamin-, inositol- and glycine-free Nitsch and Nitsch (1969) medium supplemented with 1.0M 2,4-D. This callus has retained a high embryogenic activity after repeated subculture on the same medium for over two years, and has produced numerous embryos after transfer to a hormone-free medium. The effect of cytokinin treatment on somatic embryogenesis from leaf callus was also examined. N-(2-chloro-4-pyridyl)-N-phenylurea (KT-30) and N-(1,2,3-thiadiazol-5-yl)-N-phenylurea (TAG), both synthetic cytokinins, were found to be effective for the induction of somatic embryogenesis. When leaf callus was induced by these cytokinins combined with 2,4-D at either 5.0 or 10.0M, somatic embryos were produced.Abbreviations B₅ Basal medium, Gamborg et al. (1968) - BA 6-benzylaminopurine - 2,4-D 2,4- dichlorophenoxyacetic acid - IAA indole-3-acetic acid - 2iP (2-isopentenyl)adenine - KIN kinetin - KT-30 N-(2-chloro-4-pyridyl)-N'-phenylurea, also called 4PU-30, Kyowa Hakko Kogyo Co., Japan - NAA 1-naphthaleneacetic acid - NN Basal medium, Nitsch and Nitsch (1969) - MS Basal medium, Murashige and Skoog (1962) - TAG N-(1,2,3-thiadiazol-5-yl)-N'-phenylurea, also called thidiazuron or TDZ, Tomono Noyaku Co., Japan - ZEA zeatin     

A partially disarmed<Emphasis Type="Italic"> vir</Emphasis> helper plasmid,pKYRT1, in conjunction with 2,4-dichlorophenoxyactic acid promotes emergence of regenerable transgenic somatic embryos from immature cotyledons of soybean

Agrobacterium tumefaciens strain KYRT1 harboring the virulence helper plasmid pKYRT1 induces transgenic somatic embryos (SEs) at high frequency from infected immature soybean cotyledons. KYRT1 is derived from the highly oncogenic strain Chry5. However, pKYRT1 is not completely disarmed and still contains an entire T-right (T_R) and a portion of T-left (T_L). In this report, binary strains, each carrying fully disarmed vir helper plasmids including pKPSF2, which is a fully disarmed version of pKYRT1, were compared to strain KYRT1 for their ability to induce transgenic SEs on immature cotyledons of soybean. Six weeks following cocultivation, histochemical GUS assays of cultured explants indicated that all fully disarmed vir helper plasmids transferred their binary T-DNA, containing a GUS-intron gene, into soybean tissues. However, none of these transformed tissues developed SEs on medium with or without 2,4-dichlorophenoxyactic acid (2,4-D). On the other hand, immature cotyledons cocultivated with strain KYRT1 exhibited high induction of transgenic SEs, but only on medium supplemented with 2,4-D. Derivatives of strain Chry5 harboring other vir helper plasmids did not induce transgenic SEs under any conditions tested, thus suggesting that the chromosomal background of KYRT1 alone was not sufficient to promote somatic embryogenesis. PCR analysis indicated that 55% of transgenic embryogenic cultures and 29% of transgenic T₀ soybean plants derived by transformation using strain KYRT1 contained T_R from pKYRT1 in addition to the uidA gene from the binary construct. None of the transgenic tissues or T₀ plants contained T_L DNA. These results suggest that some function coded for by T_R of pKYRT1 influences somatic embryogenesis in conjunction with exposure of the plant tissues to 2,4-D. Since the co-transformation frequency of the undesirable T-DNA sequences from the vir helper plasmid was relatively low, the partially disarmed strain KYRT1 will likely be very useful for the production of normal transgenic plants of diverse soybean cultivars.Abbreviations 2,4-D 2,4-Dichlorophenoxyactic acid - GUS -Glucuronidase - hpt Hygromycin phosphotransferase gene - SE Somatic embryo - uidA -Glucuronidase gene     

In vitro somatic embryogenesis from cell suspension cultures of cowpea [Vigna unguiculata (L.) Walp]

We report, an efficient protocol for plantlet regeneration from the cell suspension cultures of cowpea through somatic embryogenesis. Primary leaf-derived, embryogenic calli initiated in MMS [MS salts (Murashige and Skoog 1962) with B5 (Gamborg et al. 1968) vitamins] medium containing 2,4-Dichlorophenoxyacetic acid (2,4-D), casein hydrolysate (CH), and l-Glutamic acid-5-amide (Gln). Fast-growing embryogenic cell suspensions were established in 0.5 mg l^–1 2,4-D, which resulted in the highest recovery of early stages of somatic embryos in liquid MMS medium. Embryo development was asynchronous and strongly influenced by the 2,4-D concentration. Mature monocotyledonary-stage somatic embryos were induced in liquid B5 medium containing 0.1 mg l^–1 2,4-D, 20 mg l^–1 l-Proline (Pro), 5 M Abscisic acid (ABA), and 2% mannitol. B5 medium was found superior for the maturation of somatic embryos compared to MS and MMS media. The importance of duration (5 d) for effective maturation of somatic embryos is demonstrated. A reduction in the 2,4-D level in suspensions increased the somatic embryo induction and maturation with decreased abnormalities. Sucrose was found to be the best carbon source for callus induction while mannitol for embryo maturation and maltose for embryo germination. Extension of hypocotyls and complete development of plantlet was achieved in half-strength B5 medium supplemented with 3% maltose, 2500 mg l^–1 potassium nitrate, and 0.05 mg l^–1 thidiazuron (TDZ) with 32% regeneration frequency. Field-established plants were morphologically normal and fertile. This regeneration protocol assures a high frequency of embryo induction, maturation, and plantlet conversion.