Somatic embryogenesis from pea embryos and shoot apices

Conditions were defined for plant regeneration via somatic embryogenesis in pea, using explants from immature zygotic embryos or from shoot apices. For the induction of somatic embryos, an auxin (picloram or 2,4-dichlorophenoxyacetic acid) was required. Embryogenic callus originated from embryonic axis tissue of immature embryos and from the axillary-bud region and the plumula of shoot apices. A clear effect of embryo size on somatic embryogenesis was shown. There were differences in frequency of somatic embryogenesis among the five genotypes used in the study. Additions of BA to auxin-containing medium reduced embryo production. Histological examinations confirmed the embryogenic nature of the immature embryo cultures and revealed that somatic embryos originated from the meristematic areas near the callus surface.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Somatic embryogenesis in peach palm using the thin cell layer technique: induction, morpho-histological aspects and AFLP analysis of somaclonal variation

BACKGROUND AND AIMS: The thin cell layer (TCL) technique is based on the use of very small explants and has allowed enhanced in vitro morphogenesis in several plant species. The present study evaluated the TCL technique as a procedure for somatic embryo production and plantlet regeneration of peach palm. METHODS: TCL explants from different positions in the shoot apex and leaf sheath of peach palm were cultivated in MS culture medium supplemented with 0-600 microM Picloram in the presence of activated charcoal. The production of primary calli and embryogenic calli was evaluated in these different conditions. Histological and amplified fragment length polymorphism (AFLP) analyses were conducted to study in vitro morphogenetic responses and genetic stability, respectively, of the regenerated plantlets. KEY RESULTS: Abundant primary callus induction was observed from TCLs of the shoot meristem in culture media supplemented with 150-600 microM Picloram (83-97%, respectively). The production of embryogenic calli depends on Picloram concentration and explant position. The best response observed was 43% embryogenic callus production from shoot meristem TCL on 300 microM Picloram. In maturation conditions, 34+/-4 somatic embryos per embryogenic callus were obtained, and 45.0+/-3.4% of these fully developed somatic embryos were converted, resulting in plantlets ready for acclimatization, of which 80% survived. Histological studies revealed that the first cellular division events occurred in cells adjacent to vascular tissue, resulting in primary calli, whose growth was ensured by a meristematic zone. A multicellular origin of the resulting somatic embryos arising from the meristematic zone is suggested. During maturation, histological analyses revealed bipolarization of the somatic embryos, as well as the development of new somatic embryos. AFLP analyses revealed that 92% of the regenerated plantlets were true to type. The use of TCL explants considerably improves the number of calli and somatic embryos produced in comparison with previously described protocols for in vitro regeneration of peach palm. CONCLUSIONS: The present study suggests that the TCL somatic embryogenesis protocol developed is feasible, although it still requires further optimization for in vitro multiplication of peach palm, especially the use of similar explants obtained from adult palm trees.     

Stimulatory Effect of Partial Desiccation on Plant Regeneration in Indica Rice (Oryza sativa L)

A simple in vitro protocol was established for high frequency plant regeneration via organogenesis and somatic embryogenesis from the callus cultures derived from immature inflorescence segments of indica rice (Oryza sativa L cvs Safari-17 and Kasturi). Embryogenic and nodular calli were initiated on MSB medium supplemented with 2, 4-D and sucrose (3.0%, w/v). Somatic embryogenesis occurred after transfer of embryogenic calli to MSB medium containing 2.25 μM 2,4-D, 2.2 μM BAP, 2.9 μM thiamine HCl and 244.86 μM L-tryptophan. Plantlet/shoot regeneration occurred after transfer of embryogenic calli to MSB medium containing 17.6 μM BAP and 1.12 μM 2,4-D. Partial desiccation (up to 12, 24, 48, 72 and 96 h) of embryogenic calli prior to transfer to regeneration medium stimulated regeneration frequency. Highly significant (P<0.001) difference was observed for regeneration frequency and average number of plantlets/shoots regenerated per callus in partially desiccated calli in comparison to non-dehydrated calli. Regeneration frequency increased from 33.3% to 80% after 24 h of desiccation treatment to callus cultures of cv. Safari-17, and from 46.7% to 93.3% after 48 h of desiccation treatment to callus tissues of cv. Kasturi. Regenerated shoots were rooted on MSB medium supplemented with 4.9 μM IBA. Plants with well-developed roots were transferred to pots where they grew well and attained maturity.     

Somatic embryogenesis and regeneration of Cenchrus ciliaris genotypes from immature inflorescence explants

An efficient, highly reproducible system for plant regeneration via somatic embryogenesis was developed for Cenchrus ciliaris genotypes IG-3108 and IG-74. Explants such as seeds, shoot tip segments and immature inflorescences were cultured on Murashige and Skoog (MS) medium supplemented with 2.0–5.0 mg dm^?3 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg dm^?3 N⁶-benzyladenine (BA) for induction of callus. Callus could be successfully induced from all the three explants of both the genotypes. But the high frequency of embryogenic callus could be induced only from immature inflorescence explants. Somatic embryos were formed from nodular, hard and compact embryogenic calli when 2,4-D concentration was gradually reduced and BA concentration increased. Histological studies of somatic embryos indicated the presence of shoot apical meristem with leaf primordia. Ultrastructural details of globular and scutellar somatic embryos further validated successful induction and progression of somatic embryogenesis. Shoots were differentiated upon germination of somatic embryos on MS medium containing 2,4-D (0.25 mg dm^?3) and BA or kinetin (1–5 mg dm^?3). Roots were induced on ½ MS medium containing charcoal (0.8 %), and the regenerated plants transferred to pots and established in the soil showed normal growth and fertility.     

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.     

High frequency somatic embryogenesis and plant regeneration from zygotic embryo-derived callus cultures of three Allium species

The plant regeneration ability of zygotic embryo-derived callus cultures was studied for 12 A. cepa varieties and accessions, two A. fistulosum varieties, one A. fistulosum x A. cepa interspecific hybrid and two A. porrum varieties. Compact embryogenic callus was induced on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid. The embryogenic calluses of all three Allium species were similar in appearance. For all accessions tested plants could be regenerated at a high frequency from this compact callus through somatic embryogenesis, when using kinetin supplemented MS medium (regeneration medium). Addition of abscisic acid to the regeneration medium stimulated the formation of both somatic embryos and shoots for a number of varieties. Concerning shoot regeneration from callus cultures, significant differences existed between genotypes of all accessions except one.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - VDH Van Der Have Seed company     

Efficacious somatic embryogenesis and fertile plant recovery from shoot apex explants of onion (Allium cepa. L.)

An efficient somatic embryogenesis and regeneration system was developed for the first time in onion using shoot apex explants. These explants were used to initiate callus in Murashige and Skoog (MS) medium supplemented with 4.0 mg l^?1 2,4-dichlorophenoxyacetic acid. The induction frequency of primary callus in this medium was 85.3%. The primary calli were then transferred onto medium supplemented with 2.0 mg l^?1 2,4-dichlorophenoxyacetic acid. Following two biweekly subcultures, embryogenic callus formed. Inclusion of a low concentration of 6-benzylaminopurine in the subculture medium promoted the formation of embryogenic callus. The addition of 2.0 mg l^?1 glycine, 690 mg l^?1 proline, and 1.0 g l^?1 casein hydrolysate also increased the frequency of callus induction and embryogenic callus formation. The highest frequency of embryogenic callus (86.9%) and greatest number of somatic embryos (26.3 per callus) were obtained by the further addition of 8.0 mg l^?1 silver nitrate. Somatic embryos formed plantlets on regeneration medium supplemented with 1.5 mg l^?1 6-benzylaminopurine; addition of 2.0 mg l^?1 glycine to the regeneration medium promoted a high frequency of regeneration (78.1%) and plantlet formation (28.7 plants per callus). The regenerated plantlets were transferred to half-strength MS medium supplemented with 1.5 mg l^?1 indole-3-butyric acid for root development; the maximum frequency of root formation was 87.7% and the average number of roots was 7.6 per shoot. The regenerated plantlets were successfully grown to maturity after hardening in the soil. This is the first report of somatic embryogenesis and regeneration from shoot apex explants of onion.     

Efficient somatic embryogenesis in sugar beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.) breeding lines

Efficient regeneration via somatic embryogenesis (SE) would be a valuable system for the micropropagation and genetic transformation of sugar beet. This study evaluated the effects of basic culture media (MS and PGo), plant growth regulators, sugars and the starting plant material on somatic embryogenesis in nine sugar beet breeding lines. Somatic embryos were induced from seedlings of several genotypes via an intervening callus phase on PGo medium containing N⁶-benzylaminopurine (BAP). Calli were mainly induced from cotyledons. Maltose was more effective for the induction of somatic embryogenesis than was sucrose. There were significant differences between genotypes. HB 526 and SDM 3, which produced embryogenic calli at frequencies of 25–50%, performed better than SDM 2, 8, 9 and 11. The embryogenic calli and embryos produced by this method were multiplied by repeated subculture. Histological analysis of embryogenic callus cultures indicated that somatic embryos were derived from single- or a small number of cells. 2,4-dichlorophenoxyacetic acid (2,4-D) was ineffective for the induction of somatic embryogenesis from seedlings but induced direct somatic embryogenesis from immature zygotic embryos (IEs). Somatic embryos were mainly initiated from hypocotyls derived from the cultured IEs in line HB 526. Rapid and efficient regeneration of plants via somatic embryogenesis may provide a system for studying the molecular mechanism of SE and a route for the genetic transformation of sugar beet.     

Long-term optimized embryogenic cultures in durum wheat (Triticum durum Desf.)

Five varieties of durum wheat: Appulo, Ofanto, Latino, Creso, and Castello (Triticum durum Desf.) adapted to the semi-arid mediterranean environment have been tested for their in vitro response. Compact, embryogenic, highly regenerable calli originated from primary callus derived through proliferation of the scutellum of immature embryos explanted in the presence of 2,4 dichlorophenoxyacetic acid. Selective subculture of the white, compact, embryogenic sectors led to the establishment of long-term cultures. Regeneration occurred on hormone-free medium either via germination of somatic embryos, or via multiple-shoot formation probably due to precocious germination of somatic embryos. The three varieties, Ofanto, Creso and Appulo, were the best responding genotypes. Callus fragmentation and two subsequent transfers onto fresh medium at 7-day intervals yielded a frequency of plant regeneration of some 25–40 plantlets per gram fresh weight callus in 21 days on Murashige and Skoog's hormone-free medium. Plantlets could be efficiently established in soil, thus confirming the possibility of biotechnological approaches with varieties of this crop species.Abbreviations E embryogenic - NE non embryogenic - MS Murashige and Skoog's (1962) medium - 2,4-D 2,4 dichlorophenoxyacetic acid - DAA days after anthesis - FWT fresh weight tissue     

CHARACTERIZATION OF TISSUE CULTURE INITIATION AND PLANT REGENERATION IN SPOROBOLUS VIRGINICUS (GRAMINEAE)

Tissue cultures of the halophytic saltmarsh grass Sporobolus virginicus were initiated from unemerged immature inflorescence tissue. Typical graminaceous embryogenic and nonembryogenic callus and cell types were noted. Embryogenic callus was compact golden yellow. Histological evidence indicated that proliferation of the ovary tissue of the immature pistil was the source for embryogenic callus. Plants regenerated after first reducing and then eliminating auxin from the culture medium. Regeneration was observed both through the concerted development of bipolar meristems from somatic embryos and by the formation of multiple shoot meristems that were either connected through callus tissue to root meristems or which later adventitiously rooted. The main mode of regeneration appeared to be somatic embryogenesis with additional multiple shoot formation probably due to precocious germination of somatic embryos. Plants recovered from culture were acclimated to soil, grown up in a greenhouse, and planted in field plots with saline irrigation to ensure stability of salt tolerance.     

Plant Regeneration Through Somatic Embryogenesis in Taxus wallichiana

We describe an efficient process for regeneration of Taxus wallichiana (Zucc) plants from callus cultures derived from zygotic embryos. Zygotic embryos cultured on half strength Lloyd and McCown’s basal medium supplemented with SH vitamin (1/2 WPMSH), 0.5 mg I^?1 6-benzyladenine (BA) and 1.0–2.0 mg I^?1 á-Napthaleneacetic acid (NAA) produced compact yellow (CY) callus after 4 weeks of culture. The 8-week-old CY call! (lines CY-A and CY-B) were initially slow growing but proliferated on transfer to WPM basal medium supplemented with 8.0 mg I^?1 2,4-D, 0.1–0.9 mg^?1 NAA and 0.3–1.0 mg^?1 BA after 4 weeks. Four morphologically distinct calli lines were obtained, of which only two call! lines, CY-B-FW and CY-B-FY were embryogenic. The 12-week-old callus line CY-B-FW developed globular somatic embryos on transfer to secondary medium after 8 weeks and matured in maturation medium after 4 weeks. Only 10% of the mature somatic embryos regenerated into complete plantlets after 4 weeks on conversion medium. Although the frequency of conversion was low, complete regenerated plantlets via somatic embryogenesis were obtained after 7–8 months of initiation of culture. Taxane analysis showed that the paclitaxel accumulation was higher in embryogenic callus than in non-embryogenic callus.     

Efficient regeneration system from wheat leaf base segments

Efficient plant regeneration system from leaf base segments of wheat (Triticum aestivum L.) was developed. The factors affecting the callus formation and regeneration capacity of leaf segments of two genotypes; Bobwhite and Pavon 76, were investigated. The highest number of somatic embryos (SE) was obtained on Murashige and Skoog medium supplemented with 2 mg dm⁻³ 2,4-dichlorophenoxyacetic acid + 1 mg dm⁻³ naphthalenacetic acid (14.7 SE per segment). Highest frequency of embryogenic callus (96 %) and somatic embryo formation (24.3 SE per segment) were achieved in the first segments. The highest plantlet regeneration was obtained after transfer of embryogenic calli to regeneration medium supplemented with 1 mg dm⁻³ kinetin (6.3 plantlets per segment).     

Genotype effects on proliferative embryogenesis and plant regeneration of soybean

Summary Proliferative somatic embryogenesis is a regeneration system suitable for mass propagation and genetic transformation of soybean [Glycine max (L.) Merr.]. The objective of this study was to examine genotypic effects on induction and maintenance of proliferative embryogenic cultures, and on yield, germination, and conversion of mature somatic embryos. Somatic embryos were induced from eight genotypes by explanting 100 immature cotyledons per genotype on induction medium. Differences in frequency of induction were observed among genotypes. However, this step was not limiting for plant regeneration because induction frequency in the least responding genotype was sufficient to initiate and maintain proliferative embryogenic cultures. Six genotypes selected for further study were used to initiate embryogenic cultures in liquid medium. Cultures were evaluated for propagation of globular-stage tissue in liquid medium, yield of cotyledon-stage somatic embryos on differentiation medium, and plant recovery of cotyledon-stage embryos. Genotypes also differed for weight and volume increase of embryogenic tissue in liquid cultures, for yield of cotyledon-stage embryos on differentiation medium, and for plant recovery from cotyledon-stage embryos. Rigorous selection for a proliferative culture phenotype consisting of nodular, compact, green spheres increased embryo yield over that of unselected cultures, but did not affect the relative ranking of genotypes. In summary, the genotypes used in this study differed at each stage of plant regeneration from proliferative embryogenic cultures, but genotypic effects were partially overcome by protocol modifications.     

Plant regeneration via somatic embryogenesis and shoot organogenesis from immature cotyledons of Camellia nitidissima Chi

Camellia nitidissima Chi (Theaceae) is a world-famous economic and ornamental plant with golden-yellow flowers. It has been classified as one of the rarest and most endangered plants in China. Our objective was to induce somatic embryogenesis, shoot organogenesis and plant regeneration for C. nitidissima. Three types of callus (whitish, reddish and yellowish) were induced from immature cotyledons on improved woody plant medium (WPM) with different plant growth regulators (PGRs). Among the callus, whitish callus was induced by 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and reddish and yellowish callus were induced by strongly active cytokinins, thidiazuron (TDZ) or 6-benzylaminopurine (BAP), singly or combined with weakly active auxin, α-naphthaleneacetic acid (NAA). The embryogenic callus could differentiate into somatic embryos, nodular embryogenic structures (large embryo-like structures) or adventitious shoots depending on the PGR used in WPM. BAP was best for adventitious buds and zeatin was best for somatic embryogenesis while kinetin (Kt) was best for the formation of nodular embryogenic structures. The three regeneration pathways often occurred in the same embryogenic callus clumps. Most shoots (80.0%) developed roots in WPM supplemented with 24.6 μM IBA and 0.3 μM NAA while 47.5% of somatic embryos could germinate directly and develop into plantlets on induction medium supplemented with 0.9 μM BAP and 0.1 μM NAA. The nodular embryogenic structures could be sub-cultured and cyclically developed in one of two differentiation pathways: shoot organogenesis or somatic embryogenesis. Plantlets derived from shoot buds rooted and somatic embryos germinated when transplanted into soil in a greenhouse; 66.7% of plantlets from shoot culture and 78.6% of plantlets from somatic embryos survived after 8 weeks’ acclimatization.     

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.     

Embryogenic culture initiation and somatic embryo development in hybrid firs (Abies alba×Abies cephalonica,and Abies alba×Abies numidica)

Summary Embryogenic cultures were established from silver fir (Abies alba Mill.) female megagametliophytes with developing embryos and from excised mature embryos after pollination with Abies cephalonica Lond. or Abies numidica DeLann pollea The frequency of embryogenic callus formation was dependent on genotype, collection time, medium and explants used. The embryogenic callus initiation potential of megagamethophytes with developing embryos in both hybrids was higher in early July and dropped as the zygotic embryos matured. Excised cotyledonary embryos were less suitable for induction of embryogenic cultures. SH medium supplemented with 1mg/l BAP was the most efficient for callus induction and maintenance. Cultures were composed of early somatic embryos with an embryonal mass formed of highly cytoplasmic cells, rich in cell organelles and a suspensor built up by vacuolated, strongly elongated cells. Maturation of embryos was detected with the formation of bipolar structures with shoot and root apices. Nutrition reserves were observed in cells of embryos cultured on DCR medium containing 1 or 10 mg/l ABA. Cotyledon formation, hypocotyl elongation and low frequency germination occured following transfer of the embryos to the same medium without ABA.     

贡蕉胚性细胞悬浮系的建立和植株再生

鲜食蕉品种的高度不育性和多倍性制约了用传统育种方法培育生产实践中所需的新品种 ,建立稳定的胚性细胞悬浮系是香蕉生物技术育种的前提。以目前国内尚未建立该体系的鲜食蕉品种贡蕉 (AA)未成熟雄花序的第 1～ 15位花梳为外植体 ,对胚性细胞悬浮系的建立和植株再生体系进行了优化。结果表明 ,5～ 6个月的培养后可获得分生小球体和浅黄色、松散易碎的胚性愈伤组织。 9μmol/L 2,4 D对外植体愈伤组织的诱导效果最好 ,诱导率为 40.96 % ,胚性愈伤组织诱导率可达7.45 % ,其中5.79%的胚性愈伤组织来源于第 6～12号位置的花梳。胚性愈伤组织悬浮培养后 ,通过 3个月的筛选和继代培养 ,可得到均质的胚性细胞悬浮系。该培养体系合适继代周期为 15d ,继代时合适的起始接种量为每 30mL培养基加 2mLPCVECS。培养 6个月的胚性细胞在体细胞胚诱导培养基中培养15d后可见到白色半透明体细胞胚的发生 ,体细胞胚诱导率为 2 80× 10³个 mLPCV。成熟体细胞胚的萌发率为 17 2 8% ,其中发育成正常的再生植株的百分率为 14 16 %。     

Establishing japonica rice suspensions retaining a high regeneration potential after 14 months of culture

We report here a method of ‘two cycles of selection’ for rapidly establishing rice embryogenic cell suspensions which had high regeneration potential even after long-term culture (up to 14 months). Embryogenic calli were induced from immature embryos of 17 genotypes of cultivated japonica rice (Oryza sativa L.) and tested for their regeneration potential. Five of the 17 genotypes showed relatively high regeneration frequencies (27%–45%) and were selected. This process was the first cycle selection, namely, the selection for responding genotypes. The second cycle of selection, or the selection for responding individual callus, was then conducted. Calli from different immature embryos of the same genotype were found to differ in regeneration potential, therefore calli with high regeneration potential were selected again in each of the five genotypes. Five finely-dispersed and rapidly-growing embryogenic cell suspensions, one for each of the genotypes, were established from the calli chosen by the two cycles of selection. The regeneration potential of these cell suspensions was tested every two months over a period of 14 months. All of the five genotypes retained high regeneration frequency (above 62%) over the whole period tested.     

Embryogenic callus formation,growth and regeneration in callus and suspension cultures of Miscanthus x ogiformis Honda Giganteus' as affected by proline

The effects of proline additions to culture systems of Miscanthus x ogiformis Honda Giganteus' were investigated. Proline was added in concentrations of 0, 12.5, 25, 50, 100 or 300 mM to the callus induction and suspension culture media containing either Murashige and Skoog or N6 basal salts and 22.6 μM 2,4-dichlorophenoxyacetic acid. Shoot apices and leaves from in vitro-propagated shoots, and immature inflorescences from greenhouse-grown plants were used as explants for callus induction and formation. Suspension cultures initiated from embryogenic callus of immature inflorescences were used to test the effect of proline in suspension cultures. The proline additions affected the formation of embryogenic callus and the growth of suspension cultures. Improvements depended on the proline concentration and the basal salts of the medium. Addition of 12.5 to 50 mM proline to callus induction medium with Murashige and Skoog salts increased embryogenic callus formation on shoot apices and leaf explants while proline had no effect on embryogenic callus formation in medium with N6 salts. Increased growth with increasing proline concentration was obtained in suspension aggregates grown in medium with N6 salts, whereas proline only increased growth of suspension aggregates grown in medium with Murashige and Skoog salts at concentrations of 12.5 or 25 mM. A stimulating effect of proline on plant regeneration was observed in short-term cultures of callus as well as in long-term cultures of suspension aggregates. An optimum proline concentration for plant regeneration was found at 12.5 mM. This revised version was published online in June 2006 with corrections to the Cover Date.     

Somatic embryogenesis from immature and mature embryos of a minor millet Paspalum scrobiculatum L.

Immature and mature zygotic embryos of Paspalum scrobiculatum L. cv. PSC 1 cultured on MS or N₆ nutrient medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), formed embryogenic callus. Induction of embryogenic callus and subsequent somatic embryogenesis was possible at a lower concentration of 2,4-D on N₆ than MS medium. Immature embryos were highly totipotent, forming somatic embryos at a higher frequency than mature embryos. Addition of amino acids (L-proline or L-tryptophan) to 2,4-D medium resulted in significant enhancement of embryogenesis on culture of mature embryos. Silver nitrate also supported an increased frequency of embryogenesis. Thus it is possible to have high frequency of somatic embryogenesis on culture of mature embryos, which are available in abundance and with ease than immature embryos. The somatic embryos readily germinated and formed plantlets on hormone-free regeneration medium. The regenerated plantlets were successful on transfer to soil and set seed.