Direct somatic embryogenesis and plantlet regeneration from immature leaflets in chickpea

Summary Direct somatic embryo formation and plantlet regeneration was achieved from immature leaflets of chickpea (Cicer arietinum L.). Optimal somatic embryogenesis was obtained when immature leaflets were exposed to media supplemented with 15 μM 2,4-dichlorophenoxyacetic acid (2,4-D) for 7 d, to 2000 μM 2,4-D for 3 d, and to 50 μM 2,4-D for 10 d, followed by transfer onto Murashige and Skoog (MS) basal medium. Exposure of explants to high 2,4-D levels (200–2000 μM) for 3 d produced bottle-shaped embryos, while exposure to low 2,4-D levels (<50 μM) and 50–2000 μM for 10 d produced spherical-shaped embryos. Two percent of embryos converted into plants upon culture on MS medium containing 15 μM gibberellic acid and 1 μM 3-indolebutyric acid. All regenerated plants were phenotypically normal.     

Direct somatic embryogenesis and plantlet regeneration from cotyledonary leaves of safflower

Somatic embryos were induced directly on adaxial surface of cotyledonary leaves within 8–10 days of culture on Murashige and Skoog medium containing 5.37 to 10.74 M 1 — napthaleneacetic acid and 2.22 M benzyl adenine. Germinated embryos with shoot axes developed into complete plants after transfer onto half stength Murashige and Skoog medium containing 1.07 M 1 — napthaleneacetic acid. Histological studies suggested direct origin of somatic embryos with broad-base attachment.Abbreviations BA benzyl adenine - MS Murashige and Skoog - NAA 1-napthaleneacetic acid - RH relative humidity     

番木瓜体细胞胚胎发育与植株再生

以番木瓜(穗中红-48)漏斗型体细胞胚胎为材料,探讨体细胞胚胎发育及植株再生的适宜条件。研究结果表明,附加2%椰乳、0.1mg/L ABA及40g/L蔗糖的MS固体培养基较适合番木瓜漏斗型胚状体的发育及成熟。充分成熟的子叶型胚状体在大量元素减半、蔗糖含量30g/L的MS培养基上,配合15001x光照可再生健康小植株;再生率为78%。     

白杄体细胞胚胎发生及其植株再生条件的优化研究

为建立白木千体细胞胚胎发生及其植株再生的高频率实验系统 ,对聚乙二醇及干化处理的影响进行了系统研究。结果表明 ,在分化培养基中附加 5 0 g/L聚乙二醇可显著提高愈伤组织的分化频率和每块愈伤组织产生的体细胞胚个数 ,而干化处理又能使体细胞胚的萌发率大幅度上升     

Direct somatic embryogenesis and plantlet regeneration from seedling leaves of winged bean,Psophocarpus tetragonolobus (L.) DC

Excised seedling leaf segments of winged bean [Psophocarpus tetragonolobus (L.) DC.] underwent direct somatic embryogenesis under appropriate incubation conditions. Initiation and development of the somatic embryos occurred using a two-step culture method. The culture procedure involved incubation for 28 days on MS basal medium supplemented with 0.1–0.5 mg/l NAA and 1.0–2.0 mg/l BA (induction medium) before transfer to MS medium supplemented with 0.1 mg/l IAA and 2.0 mg/l BA (embryo development medium). The initial exposure to low levels of NAA coincident with high levels of BA in the induction medium was essential for embryogenic induction. Maximum embryogenesis (43.3%) was obtained with 0.2 mg/l NAA and 2.0 mg/l BA, and at least 14 days on induction medium were required prior to transfer to the embryo development medium. The conversion frequency of cotyledonary embryos was 53.3% upon culture on MS medium containing 0.1 mg/l ABA for 7 days followed by transfer to MS medium supplemented with 0.1 mg/l IBA and 0.2 mg/l BA. Following conversion, the regenerated plantlets were transferred to soil and showed normal morphological characteristics.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BA 6-benzylaminopurine - ABA abscisic acid     

High frequency somatic embryogenesis and plantlet regeneration from hypocotyl protoplast cultures of Brassica napus

A simple protocol has been developed for high frequency protoplast regeneration via somatic embryogenesis in B. napus. Protoplasts isolated from hypocotyl tissue of 8–12 day old seedlings of Brassica napus ISN706 (AACC) when cultured in KM(A) medium resulted in divisions with a, frequency ranging from 30–35%. Regeneration of plantlets was possible by both organogenesis and embryogenesis. Nearly 80% of the call transferred on to MS medium supplemented with 5.0 mg l^-1 2iP, 0.1 mg l^-1 NAA, 0.001 mg l^-1 GA₃, 0.5 g l^-1 PVP and 0.5 g l^-1 MES displayed somatic embryogenesis. The somatic embryos developed into normal plantlets, and also displayed secondary, repetitive embryogenesis.     

Direct somatic embryogenesis and plantlet regeneration from leaf explants of niger, Guizotia abyssinica (L.f.) Cass

Somatic embryogenesis was induced in niger (Guizotia abyssinica (L.f.) Cass.) using a simple one step method. Leaf explants were cultured on MS medium with 2,4-D and BAP/Kn individually and in combination. Somatic embryogenesis occurred directly without an intervening callus proliferation phase from subepidermal regions of leaf explants on MS medium supplemented with 2,4-D (2.0 and 5.0 microM) plus Kn/BAP (1.0 and 2.0 microM). Regenerated somatic embryos were successfully grown into whole plants.     

Somatic embryogenesis and plantlet regeneration in Cornus florida

Somatic embryos were initiated from 12 to 15 weeks postanthesis (WPA) zygotic embryos of Cornus florida L. (flowering dogwood) cultured on Murashige-Skoog (MS) or Schenk and Hildebrandt (SH) medium amended with either 3 mg/L 2,4-D or 5 mg/L 2,4-D and 1 mg/L kinetin. White, opaque globular and early cotyledonary stage embryos were formed directly on detached cotyledons from 2 of the 5 trees sampled after 7 weeks of culture. Morphologically mature embryos developed after an additional 4 weeks incubation on medium without growth regulators; however, many of the embryos were fused in pairs along the entire length of the hypocotyl-radicle axis. Indirect embryogenesis was observed from callus cultures initiated from 9 to 15 WPA zygotic embryos. These cultures have continued to produce embryos for 16 months. Many of the embryos formed roots on germination medium, but only 12% formed plantlets and none developed past the first true leaf stage.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - FPA Formalin-propionic acid-ethanol (50%) - WPA weeks post-anthesis     

Inheritance of somatic embryogenesis and plantlet regeneration from primary (type 1) callus in maize

Summary Genetic factors controlling the differential expression of somatic embryogenesis and plant regeneration of maize from tissue culture were studied in two crosses. Inbred, hybrid, F2 and backcross generations developed from crossing maize inbred A188 with two commercially important inbred maize lines (B73 and Mo17) demonstrated genetic and environmental effects on somatic embryogenesis and plant regeneration when immature zygotic embryos were cultured on MS medium. Additive gene effects were more important in both crosses than dominant gene effects for precent somatic embryogenesis and percent or number of plants regenerated per embryo when generation means were analyzed. In backcross generations of each cross, cytoplasmic, maternal and/or paternal effects were significant for frequency of somatic embryos three weeks after culture as well as frequency, or number of plants regenerated per embryo, nine weeks after culture. Analysis of genetic variances suggests at least one gene (or block of genes) controls the expression of the frequency of somatic embryogenesis in these crosses. Differences in somatic embryogenesis and plant regeneration between B73 and Mo17 are discussed. This is Journal Paper No. 11,435 of the Purdue University Agricultural Experiment Station.     

Direct somatic embryogenesis and plant regeneration from ray florets of chrysanthemum

Direct somatic embryogenesis from ray floret explants of five chrysanthemum cultivars has been obtained within 12 – 15 d on Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA). Scanning electron microscopic observation also confirmed the direct origin of somatic embryos from explants. Somatic embryos developed asynchronously on the adaxial surface of explants. Among the five cultivars tested, Birbal Sahani was best responding (40 % explants responded on 4 mg dm^–3 2,4-D and 2 mg dm^–3 BA supplemented medium). Precocious germination of somatic embryos was noticed on the same medium. The best sucrose concentration in the medium was found to be 60 g dm^–3 where 70 % explants responded while 55 % embryogenic response was obtained on medium supplemented with 400 mg dm^–3 inositol. Plants developed from somatic embryos were transferred to soil and produced true-to-type flowers.     

Direct somatic embryogenesis and plant regeneration from immature explants of chickpea

A protocol for plant regeneration via somatic embryogenesis was developed in two chickpea (Cicer arietinum L.) cultivars ICCV-10 and Annigeri. Somatic embryos were induced from immature cotyledons on Murashige and Skoog’s (MS) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), α-naphthaleneacetic acid (NAA) and picloram alone or in combination with 0.5 — 2.0 mg dm⁻³ N⁶-benzylaminopurine (BA) or kinetin (KIN). NAA was better for somatic embryo induction compared to other auxins. The well formed, cotyledonary shaped embryos germinated into plantlets with 36.6 % frequency on MS medium supplemented with 2.0 mg dm⁻³ BA + 0.5 mg dm⁻³ abscisic acid (ABA). The frequency of embryogenesis and plantlet regeneration was higher in cv. ICCV-10 as compared to cv. Annigeri. Regenerated plants were transferred to soil (40 % survival) and grown to maturity. Histological studies of explants at various developmental stages of somatic embryogenesis reveled that somatic embryos developed directly from the cotyledon cells and they were single cell origin.     

Somatic embryogenesis and plantlet regeneration in the genus Secale

Summary A tissue culture of five wild species of the Secale genus, i.e., S. africanum (Stapf.), S. ancestrale (Zhuk.), S. kuprianovii (Grossh), S. segetale (Rosher.), and S. vavilovii (Grossh), from immature embryos of sizes (stages) varying between 1.0 mm to 3.0mm, cultured on MS (1962) mineral nutrient medium supplemented with 0.62 mg/1–5.0 mg/1 of 2,4-D, was established. Initially various types of callus were observed and a correlation between genotype, size of explant and 2,4-D concentration was found. The best embryogenic response was observed when explants were smaller than 1.0 mm. Induction of somatic embryogenesis of 2.0 mm–3.0 mm explants required a higher concentration of 2,4-D. Most embryoids were formed in the presence of 5.0 mg/l of 2,4-D. Secale africanum and S. kuprianovii appeared to have the highest embryogenic capacity among the five investigated species. For embryoids germination to plantlets the MS medium supplemented with GA₃ and cytokinins was used. Ultimately, out of the 932 regenerants obtained 364 originated from somatic embryogenesis.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA3 deGibberellic acid - BAP Benzylaminopurine     

Direct somatic embryogenesis and plant regeneration from leaf explants of Phalaenopsis amabilis

Leaf explants of Phalaenopsis amabilis var. formosa formed clusters of somatic embryos directly from epidermal cells without an intervening callus within 20 – 30 d when cultured on 1/2-strength modified Murashige and Skoog medium supplemented with 0.1, 1 and 3 mg dm⁻³ TDZ. Repetitive production of embryos involved secondary embryogenesis could be obtained by culturing segments of embryogenic masses on TDZ-containing media. Plantlet conversion from embryos was successfully achieved on regulator-free growth medium.     

Direct somatic embryogenesis and plant regeneration from protoplasts of Bupleurum scorzonerifolium Willd

Protolasts of Bupleurum scorzonerifolium were prepared from stem node-derived embryogenic calli with an enzyeme mixture, in which snailase was a necessary component. Follolwing cell wall regeneration protoplasts divided and directly formed somatic embryos which developed into plantlets. The conditions favorable to direct embryo formation were investigated, and the nature of the callus used for protoplast preparation was found to be a critical factor. The osmotic concentration and the composition of the culture medium including the phytohormone combinations were also important.     

Somatic embryogenesis and plantlet regeneration in the soybean Glycine max

A tissue culture procedure for the regeneration of somatic embryos and plantlets from somatic cells of the soybean Glycine max is described. Bean pods of soybean cv. TGM119 were immersed in liquid nitrogen for 20 minutes. Young embryos were excised from the immature seeds and cultured to form calli. Calli grown from the young embryos were incubated in liquid culture for two weeks. The liquid suspension culture was filtered to obtain single cells. The soybean cells were cultured for one month in a liquid medium in hanging drop cultures for development into proembryoids. The proembryoids were maintained on a solid growth medium for 40 days. The resultant callus tissue was transferred into MS media containing selected combinations and concentrations of 2,4-Dichlorophenoxyacetic acid, Naphthaleneacetic acid, Kinetin, Benzyladenine and Indoleacetic acid. In the presence of Benzyladenine (0.2 mg/l) and Indoleacetic acid (0.01 mg/l), globular and heart shaped somatic embryos were formed on the surface of the calli. Calli containing somatic embryos were transferred into liquid medium and incubated under low light conditions. After six months further incubation, more than 1,000 plantlets and a large number of somatic embryoids at various developmental stages were obtained per flask.Abbreviations KT kinetin - CM coconut milk - BA benzyladenine - NAA napthalene acetic acid - IAA indole acetic acid - 2,4-D 2,4 dichlorophenoxy acetic acid - MS Murashige and Skoog medium     

水母雪莲体细胞胚胎发生及其植株再生

水母雪莲（Saussurea medusa Maxim.)茎和叶片的切段接种于MS＋2mg/L NAA 0.5mmg/L 6-BA的培养基上,20d后产生黄褐色的愈伤组织,经过几个月的继代培养,愈伤组织仍保持旺盛的增殖能力,但部分由黄褐色逐渐变为红色,将红色愈伤组织转到MS＋0．1mg/L NAA＋0．2mg/L 6-BA 5mg/L GA3的培养基上,30d后可分化出大量的体细胞胚,体细胞胚成熟后转到1／2MS＋0．2mg/L IAA 0.5%活性炭的培养基上,30d后可长出2－4cm的根,带根的小苗经锻炼后移栽到土壤中,成活率达76％,细胞组织学观察表明,发育成熟的体细胞胚具有胚根,胚轴和胚芽的完整结构,具有独立的维管系统。     

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.     

Influence of growth regulators on somatic embryogenesis,plantlet regeneration,and post-transplant survival of Echinochloa frumentacea

After placement on Murashige and Skoog's basal medium supplemented with 3–5 mg/l 2,4-D, immature inflorescence expiants of Echinochloa frumentacea gave rise to three distinct types of callus: a) loosely arranged and soft; b) compact and translucent; c) compact, sticky and mucilaginous. Somatic embryo formation occurred in type b callus in about 18–24 d. Callus types a and c did not produce somatic embryos. The highest percentage of cultures exhibiting somatic embryogenesis occurred on the medium containing 5 mg/l 2,4-D and 0.5 mg/l kinetin. Somatic embryos also formed directly on the inflorescence (without intervening callus formation) in about 15% of the expiants placed on this medium. The addition of paclobutrazol or uniconazole (0.25 or 1 mg/l) to the medium had no influence on the percentage of cultures exhibiting direct somatic embryogenesis, but paclobutrazol slightly increased the mean number of somatic embryos per culture. Many of the callus-derived somatic embryos germinated when subcultured on basal MS medium supplemented with kinetin. Addition of paclobutrazol or uniconazole to the culture medium at 0.25 or 1 mg/l decreased somatic embryo germination and shoot elongation but increased root length and leaf width. Both paclobutrazol and uniconazole increased survival of the plantlets following transplanting to soil. Increased post-transplant survival was accompanied by reduced water loss from plantlets produced on culture media containing triazoles.     

A temporary immersion system improves in vitro regeneration of peach palm through secondary somatic embryogenesis

Background and Aims

Secondary somatic embryogenesis has been postulated to occur during induction of peach palm somatic embryogenesis. In the present study this morphogenetic pathway is described and a protocol for the establishment of cycling cultures using a temporary immersion system (TIS) is presented.

Methods

Zygotic embryos were used as explants, and induction of somatic embryogenesis and plantlet growth were compared in TIS and solid culture medium. Light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to describe in vitro morphogenesis and accompany morpho-histological alterations during culture.

Key Results

The development of secondary somatic embryos occurs early during the induction of primary somatic embryos. Secondary somatic embryos were observed to develop continually in culture, resulting in non-synchronized development of these somatic embryos. Using these somatic embryos as explants allowed development of cycling cultures. Somatic embryos had high embryogenic potential (65·8 ± 3·0 to 86·2 ± 5·0 %) over the period tested. The use of a TIS greatly improved the number of somatic embryos obtained, as well as subsequent plantlet growth. Histological analyses showed that starch accumulation precedes the development of somatic embryos, and that these cells presented high nucleus/cytoplasm ratios and high mitotic indices, as evidenced by DAPI staining. Morphological and SEM observations revealed clusters of somatic embryos on one part of the explants, while other parts grew further, resulting in callus tissue. A multicellular origin of the secondary somatic embryos is hypothesized. Cells in the vicinity of callus accumulated large amounts of phenolic substances in their vacuoles. TEM revealed that these cells are metabolically very active, with the presence of numerous mitochondria and Golgi apparatuses. Light microscopy and TEM of the embryogenic sector revealed cells with numerous amyloplasts, large nuclei and nucleoli, and numerous plasmodesmata. Plantlets were obtained and after 3 months in culture their growth was significantly better in TIS than on solid culture medium. However, during acclimatization the survival rate of TIS-grown plantlets was lower.

Conclusions

The present study confirms the occurrence of secondary somatic embryos in peach palm and describes a feasible protocol for regeneration of peach palm in vitro. Further optimizations include the use of explants obtained from adult palms and improvement of somatic embryo conversion rates.     

Direct somatic embryogenesis, plant regeneration and in vitro flowering in rapid-cycling Brassica napus

 A simple method to induce somatic embryogenesis from seeds of rapid-cycling Brassica napus is described. Seedlings cultured on Murashige and Skoog (MS) basal medium produced somatic embryos directly on hypocotyls and cotyledons after 2 to 3 subcultures onto the same medium. A low pH of the medium (3.5–5) was more conducive to somatic embryogenesis than a higher pH (6 and 7). Embryogenic potential of the seeds was inversely correlated to seed age: about 41–68% of immature seeds between the ages of 14 and 28 days after pollination (DAP) formed somatic embryos compared to 0–11% of the seeds obtained 29–37 DAP. About 54% of the somatic embryos produced secondary embryos after subculturing onto the same medium. The embryogenic potential of the cultures has been maintained on MS basal medium for 2 years (12 generations) without diminution. Up to 75% of the secondary embryos developed into plantlets on MS medium enriched with 10^–6  M zeatin, and 40% of these produced flowers when transferred to an optimised flower-induction medium. Viable seeds were produced in self-pollinated in vitro flowers. Received: 15 February 2000 / Revision received: 18 July 2000 / Accepted: 19 July 2000