In vitro somatic embryogenesis and plant regeneration in Acacia arabica

In vitro somatic embryogenesis and subsequent plant regeneration was achieved in callus cultures derived from immature zygotic embryos of Acacia arabica on semi-solid Murashige and Skoog (MS) basal salts and vitamins supplemented with 8.88 MBA, 6.78 M2,4-D and 30 g l^–1 (w/v) sucrose. Somatic embryos proliferated rapidly by secondary somatic embryogenesis after transfer to MS medium supplemented with 6.66 M BA, 6.78 M 2,4-D. The maximum number of somatic embryos per callus was 72.6 after 8 weeks of culture on medium containing 6.66 M BA and 6.78 M 2,4-D. The isolated somatic embryos germinated on half-strength basal MS salts and vitamins supplemented with 0.04 M BA, 0.94 M ABA and 2% (w/v) sucrose. The embryo-derived plantlets were acclimatized in the greenhouse and subsequently showed normal growth.     

Direct somatic embryogenesis and plant regeneration from mature sugarbeet (Beta vulgaris L.) zygotic cotyledons

An in vitro protocol has been developed for direct somatic embryogenesis of zygotic cotyledons from mature sugarbeet (Beta vulgaris L.) embryos. Explants were sequentially cultured on modified Murashige and Skoog (MS) medium supplemented with different combinations of 2,4-D, NAA, BAP and TIBA. Somatic embryogenesis was induced within 4 weeks of culture on embryogenesis induction medium which contained MS medium supplemented with BAP and TIBA. Proliferation of somatic embryos was observed on embryo proliferation medium, which contained MS medium supplemented with BAP and NAA within 4 weeks of culture. Plants were regenerated on hormone free half; strength MS medium containing a low sucrose concentration. With some sugarbeet lines, high frequencies of plant regeneration in excess of 90percnt; were observed. The incorporation of TIBA in the media was essential for successful regeneration.     

Factors important for somatic embryogenesis in zygotic embryo explants ofCapsicum annuum L.

We used four cultivars ofCapsicum annuum L.—Sweet Banana, California Wonder, Yolo Wonder, and Ace—to reexamine the critical factors influencing somatic embryogenesis from zygotic embryo explants, as reported in the literature. When we followed the protocol of Buyukalaca and Mavituna (1996), which had induced somatic embryogenesis from mature zygotic embryos of cv. Ace, only callus was formed without embryogenesis from our mature zygotic embryo expiants. Using the procedures of Harini and Lakshmi Sita (1993) and Binzel et al. (1996), with some modifications, we were able to induce somatic embryogenesis in all four cultivars. Rates of conversion were significantly reduced, from 75% and 65% to 40% and 28% in ’Sweet Banana’ and ’California Wonder’, respectively, when the immature zygotic embryo expiants were held on the induction medium for longer than two weeks. Likewise, somatic embryogenesis of ’Yolo Wonder’ was not observed if the induction medium was supplemented with 10% glucose or fructose, or without 10% sucrose. For somatic embryo induction and eventual plantlet conversion in Yolo Wonder’, maltose could adequately replace sucrose. In all four cultivars, somatic embryos were initiated from immature zygotic explants on media with or without coconut water, under both light and dark conditions.     

Cyclic somatic embryogenesis and efficient plant regeneration from callus of safflower

Efficient plant regeneration through somatic embryogenesis was established for safflower (Carthamus tinctorius L.) cv. NARI-6. Embryogenic calli were induced from 10 to 17-d-old cotyledon and leaf explants from in vitro seedlings. High frequency (94.3 %) embryogenic callus was obtained from cotyledon explants cultured on Murashige and Skoog’s germination (MSG) basal medium supplemented with thidiazuron, 2-isopentenyladenine and indole-3-butyric acid. Primary, secondary and cyclic somatic embryos were formed from embryogenic calli in a different media free of plant growth regulators, however, 100 % cyclic somatic embryogenesis was obtained from cotyledon derived embryogenic calli cultured on MSG. Somatic embryos matured and germinated in quarter-strength MSG medium supplemented with gibberellic acid. Cotyledons with root poles or non root poles were converted to normal plantlets and produced adventitious roots in rooting medium. Rooted plants were acclimatized and successfully transferred to the field.     

Secondary somatic embryogenesis and plant regeneration in cassava

Somatic embryos isolated from mature seed-derived cotyledon cultures of cassava (Mannihot esculenta Crantz) underwent direct secondary somatic embryogenesis or plant development under appropriate incubation conditions. Isolated somatic embryos were subjected to a two-stage culture procedure similar to that which induced their development on cotyledon explants. This involved incubation for 24–30 days on Murashige and Skoog basal medium supplemented with 2–8 mgl^-1 2,4-dichlorophenoxyacetic acid (2,4-D) (Stage I medium) before transfer to medium supplemented with 0.01 mgl^-1 2,4-D and 0.1 mgl^-1 6-benzylamino purine (BAP) (Stage II medium). Under these conditions, secondary somatic embryos developed directly from the cotyledons and shoot-tip region of primary somatic embryos by a developmental process morphologically very similar to that occurring on zygotic cotyledon explants. Apical shoot extension and adventitious root formation occurred when somatic embryos were isolated from parental cultures and incubated on Stage II medium. Somatic embryo-derived plants growing in greenhouse conditions appeared morphologically normal when compared with non-regenerated plants.     

In vitro plant regeneration of Aster scaber via somatic embryogenesis

We established an in vitro plant regeneration system via somatic embryogenesis of Aster scaber, an important source of various biologically active phytochemicals. We examined the callus induction and embryogenic capacities of three explants, including leaves, petioles, and roots, on 25 different media containing different combinations of α-naphthalene acetic acid (NAA) and 6-benzyladenine (BA). The optimum concentrations of NAA and BA for the production of embryogenic calli were 5.0 μM and 0.05 μM, respectively. Media containing higher concentrations of auxin and cytokinin (such as 25 μM NAA and 25 μM BA) were suitable for shoot regeneration, especially for leaf-derived calli, which are the most readily available calli and are highly competent. For root induction from regenerated shoots, supplemental auxin and/or cytokinin did not improve rooting, but instead caused unwanted callus induction or retarded growth of regenerated plants. Therefore, plant growth regulator-free medium was preferable for root induction. Normal plants were successfully obtained from calli under the optimized conditions described above. This is the first report of the complete process of in vitro plant regeneration of A. scaber via somatic embryogenesis.     

Somatic embryogenesis and plant regeneration through cell suspensions inMusa acuminata

Summary Cell suspensions ofMusa acuminata sspburmannicoides andMusa acuminata sspmalaccensis were obtained by culturing embryogenic callus initiated from immature zygotic embryos in liquid medium. Plant regeneration was then achieved through somatic embryogenesis. Germination of these embryos occurred in a modified MS medium containing auxin and cytokinin. Plant recovery frequencies were 20 to 36%. This method may allow a better utilization of biotechnologies in genetic improvement of theMusa diploid species, essential for banana and plantain breeding.     

Cytokinins induce direc somatic embryogenesis of <Emphasis Type="Italic">Dendrobium</Emphasis> chiengmai pink and subsequent plant regeneration

Summary Four auxins (2,4-dichlorophenoxyacetic acid [2,4-D], indole-3-acetic acid [IAA], indole-3-butyric acid [IBA], and naphthaleneacetic acid [NAA]), and five cytokinins (N ⁶-[2-isopentenyl]-adenine [2iP], N ⁶-benzyladenine [BA], 6-furfurylaminopurine [kinetin], 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea [TDZ], and 6-[4-hydroxy-3-methylbut-2-enylamino]purine [zeatin]) were examined for their effects on direct embryo induction from leaf explants of Dendrobium cv. Chiengmai Pink cultured on 1/2 Murashige and Skoog (MS) medium. Whether in light or darkness, explants easily became necrotic and no embryos were obtained on growth regulator-free or auxin-containing media after 60 d of culture. By contrast, five cytokinins tested induced direct embryo formation from leaf explants, and explants cultured in light had a higher embryogenic response compared with those cultured in darkness. The best condition for direct embryo induction was at 18.16 μM TDZ cultured in light for 60 d, where 33% of explants formed a mean number of 33.6 embryos per explant. During subculture on growth regulator-free 1/2 MS medium, embryos gradually developed into plantlets. Secondary embryogenesis was occasionally found on sheath leaves of embryos. Regenerated plantlets were successfully transplanted and grown in a greenhouse environment.     

Somatic embryogenesis and plant regeneration in Cyphomandra betacea (Cav.) Sendt

Callus cultures with globular proembryogenic structures were induced from zygotic embryos and hypocotyl segments of Cyphomandra betacea on MS medium supplemented with 2,4-D. Proembryogenic structures produced somatic embryos and plantlets on regulator-free basal medium. Pieces of embryogenic callus subcultured on medium with the same original composition gave rise to new globular structures and the potential for plantlet regeneration has been maintained for over a year. The histological examination of these proembryogenic structures suggested that somatic embryos arise from single cells. Regenerated plants are phenotypically normal, having diploid chromosome numbers (2n = 24).     

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     

湿地松体细胞胚胎发生和植株再生

以湿地松的未成熟合子胚为外植体,在附加８ｍｇ／Ｌ,２,４－Ｄ和４ｍｇ／Ｌ ＢＡ的ＬＰ培养基上诱导出胚性愈伤组织。在含１ｍｇ／Ｌ,２,４－Ｄ和０．５ｍｇ／Ｌ ＢＡ的培养基上保持并增殖。提高培养基的渗透压后,愈伤组织内大量的胚性胚柄细胞团和早期原胚。     

High-frequency plant regeneration through cyclic secondary somatic embryogenesis in black pepper (Piper nigrum L.)

A high-frequency plantlet regeneration protocol was developed for black pepper (Piper nigrum L.) through cyclic secondary somatic embryogenesis. Secondary embryos formed from the radicular end of the primary somatic embryos which were originally derived from micropylar tissues of germinating seeds on growth regulator-free SH medium in the absence of light. The process of secondary embryogenesis continued in a cyclic manner from the root pole of newly formed embryos resulting in clumps of somatic embryos. Strength of the medium and sucrose concentration influenced the process of secondary embryogenesis and fresh weight of somatic embryo clumps. Full-strength SH medium supplemented with 1.5% sucrose produced significantly higher fresh weight and numbers of secondary somatic embryos while 3.0 and 4.5% sucrose in the medium favored further development of proliferated embryos into plantlets. Ontogeny of secondary embryos was established by histological analysis. Secondary embryogenic potential was influenced by the developmental stage of the explanted somatic embryo and stages up to “torpedo” were more suitable. A single-flask system was standardized for proliferation, maturation, germination and conversion of secondary somatic embryos in suspension cultures. The system of cyclic secondary somatic embryogenesis in black pepper described here represents a permanent source of embryogenic material that can be used for genetic manipulations of this crop species.     

Spaceflight reduces somatic embryogenesis in orchardgrass (Poaceae)

Somatic embryos initiate and develop from single mesophyll cells in in vitro cultured leaf segments of orchardgrass ( Dactylis glomerata L.). Segments were plated at time periods ranging from 21 to 0·9 d (21 h) prior to launch on an 11 d spaceflight (STS-64). Using a paired t -test, there was no significant difference in embryogenesis from preplating periods of 14 d and 21 d. However, embryogenesis was reduced by 70% in segments plated 21 h before launch and this treatment was significant at P = 0·0001. The initial cell divisions leading to embryo formation would be taking place during flight in this treatment. A higher ratio of anticlinal:periclinal first cell divisions observed in the flight compared to the control tissue suggests that microgravity affects axis determination and embryo polarity at a very early stage. A similar reduction in zygotic embryogenesis would reduce seed formation and have important implications for long-term space flight or colonization where seeds would be needed either for direct consumption or to grow another generation of plants.     

Induction of direct somatic embryogenesis and plant regeneration in pepper (Capsicum annuum L.)

Summary Pepper (cv. New Mexico — 6 and Rajur Hirapur) plants were regenerated from immature zygotic embryos via direct somatic embryogenesis. Somatic embryos were formed directly, without any intervening callus, on the zygotic embryo apex, embryo axis and cotyledons on Murashige and Skoog's (MS) medium containing 2,4-D (418 M), thidiazuron (10 M) and a high concentration of sucrose (6–10%). The best response was observed on MS medium containing 2,4-D (9 M), coconut water (10%) and high sucrose (8%). The entire process of induction and maturation of the embryos was completed on the same medium. Histological examination indicated that secondary embryogenesis also occurred directly from the primary somatic embryos. Differentiation of embryos was nonsynchronous, and some embryos were swollen and distorted with fasciation. More than 70% of the mature normal somatic embryos germinated readily on MS medium containing GA₃ or TDZ, alone and in combination, and following transfer to pots developed into normal plants.Abbreviations CM Coconut milk - 2,4-D 2,4-dichlorophonoxyacetic acid - GA₃ gibberellic acid - MS Murashige and Skoog (1962) medium - NAA napthaleneacetic acid - TDZ thidiazuron     

Plant regeneration through somatic embryogenesis in Quercus suber

Cork oak ( Quercus suber L.) zygotic embryos, endosperm and ovules were treated with different concentrations of 2,4-D for induction of somatic embryos. Plant material was collected during the embryo development season, from June to September. Immature embryos proved to be the most reactive initial explant. Callus and somatic embryos developed a few weeks after the beginning of the 2,4-D treatment. For embryo development experiments, different growth regulators and cold and desiccation treatments were tested. Cold storage of somatic embryos matured in vitro at 5°C was the best treatment for breaking dormancy.     

Somatic embryogenesis and in vitro flowering inBrassica nigra

Summary This report describes a protocol for regeneration ofBrassica nigra in vitro from unorganized callus to a highly differentiated stage of flowering. Callus is initiated from seedling hypocotyl, and root explants and plantlets are obtained via somatic embryogenesis. Shoot cultures can be established from these plantlets. These shoots can either be induced to flower in vitro or rooted to produce plants which flower ex vitro. Each stage of development is marked with a specific growth regulator requirement. This has potential as a model system to understand the cellular and molecular mechanisms involved in morphogenesis, and it can be used to understand the mechanism of change of phase from vegetative to reproductive. An advantage of this system is that in vitro flowering can be obtained repeatedly in the shoots raised from the axillary buds of the flowering shoots. The protocol can also be used to procureB. nigra gametes under aseptic condition.     

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.     

Plant regeneration via somatic embryogenesis in cotton

An efficient in vitro plant regeneration system characterized by rapid and continuous production of somatic embryos using leaf and stem explants of abnormal seedling as an explant have been developed in Gossypium hirsutum L. Embryogenic callus and somatic embryos have been obtained directly from the explants of cotton abnormal seedlings. Plant growth regulators influenced the induction of cotton somatic embryogenesis. The optimal medium for direct somatic embryogenesis was modified MS medium supplemented with 0.1 mg l^-1 ZT and 2 g l^-1 activated carbon. On this medium, an average of 28.0 and 28.1 matured somatic embryos formed from per leaf and stem explants respectively. The highest frequency of somatic embryogenesis was 100%. The somatic embryos were converted into normal plantlets when cultured on modified MS medium supplemented with 0.1 mg l^-1 ZT. Upon transfer to soil, plants grew well and appeared normal. Plants could be regenerated within 60–80 days. The system of cotton somatic embryogenesis and plant regeneration described here will facilitate the application of plant tissue culture and genetic engineering on cotton genetic improvement. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of mutagens on somatic embryogenesis and plant regeneration in groundnut

The embryogenic calli (EC) were obtained from hypocotyl explants of groundnut (Arachis hypogaea L.) cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) in combination with 0.5 mg dm⁻³ 6-benzylaminopurine (BAP). The EC were exposed to γ-radiation (10–50 Gy) or treated with 1–5 mM of ethyl methane sulphonate (EMS) or sodium azide (SA). The mutated EC were subcultured on embryo induction medium containing 20 mg dm⁻³ 2,4-D. Somatic embryos (SE) developed from these calli were transferred to MS medium supplemented with BAP (2.0 mg dm⁻³) and 0.5 mg dm⁻³ 2,4-D for maturation. The well-developed embryos were cultured on germination medium consisting of MS salts with 2.0 mg dm⁻³ BAP and 0.25 mg dm⁻³ naphthaleneacetic acid (NAA). Well-developed plantlets were transferred for hardening and hardened plants produced normal flowers and set viable seeds. The fresh mass of the EC, mean number of SE per explant and regeneration percentage were higher at lower concentrations of mutagens (up to 30 Gy/3 mM). Some abnormalities in regenerated plants were observed, especially variations in leaf shape.