Plant regeneration via somatic embryogenesis in many cultivars of cotton (Gossypium hirsutum L.)

Summary Embryogenic callus was formed from several cultivars of cotton (Gossypium hirsutum L.) when sections of hypocotyl and cotyledon were cultured on medium supplemented with 5 mg/liter 6-(γ, γ-dimethylallyl-amino)-purine (2iP) and 0.1 mg/liter α-naphthaleneacetic acid (NAA) for callus initiation and proliferation, and subcultured on medium supplemented with 5 mg/liter NAA and 0.1 to 1 mg/liter 2iP for embryogenic callus induction. It seems that a high 2iP:auxin ratio is preferred for callus initiation and proliferation, but should be exchanged with a higher NAA:cytokinin ratio before differentiation will occur. Embryogenic calluses were recovered at a frequency of 2 to 85% depending on the cultivar used. Coker cultivars produced embryogenic callus faster and at higher frequencies than other cultivars. Embryogenic callus produced somatic embryos on phytohormone-free medium. This medium was used to maintain and proliferate embryogenic callus for a perid of 18 to 24 mo. Somatic embryos were converted to plants on a lower ionic strength medium supplemented with 0.1 mg/liter gibberellic acid (GA₃) and 0.01 mg/liter NAA. Glucose was the only carbohydrate used through all phases of tissue culture and was much better than sucrose, on which phenolic production was very high. High temperature (30° C) and low light intensity (9 μE · m⁻² · s⁻¹) were optimal conditions for callus initiation, embryogenic callus induction, and maintenance, whereas lower temperature (25° C) and high light intensity (90 μE · m⁻² s⁻¹) were the optimal conditions for somatic embryo maturation, germination, and plantlet development. Plants could be regenerated within 10 to 12 wk in Cokers or 7 to 8 mo. in others.     

Plant regeneration via somatic embryogenesis from protoplasts of six explants in Coker 201 (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>)

Fertile regenerated plants were obtained from protoplasts via somatic embryogenesis in Coker 201 (Gossypium hirsutum L.). Protoplasts were isolated from six different explantsleaves, hypocotyls, young roots, embryogenic callus, immature somatic embryos and suspension cultures and cultured in liquid thin layer KM8P medium. Callus-forming percentage of 20–50% was obtained in protoplast cultures from embryogenic callus, immature embryos and suspension cultures, and visible callus formed within 2 months. Callus-forming percentage of 5–20% in protoplast cultures from young roots, hypocotyls and leaves, and visible callus formed in 3 months. NAA 5.371 μM/kinetin 0.929 μM was effective to stimulate protoplast division and callus formation from six explants. Percentage of callus formation in the medium with 2,4-D 0.452 μM/kinetin 0.465 μM was over 40% from suspension cultures and immature embryos, 25% from embryogenic callus and 10% from hypocotyls. Callus from protoplasts developed into plantlets via somatic embryogenesis. Over 100 plantlets were obtained from protoplasts derived from 6 explants. Ten plants have been transferred to the soil, where they all have set seeds.     

Plant regeneration in cotton: A short-term inositol starvation promotes developmental synchrony in somatic embryogenesis

Summary Despite high commercial interest, the success of biotechnological applications in cotton (Gossypium hirsutum) has been limited due to difficulties in genetic transformation. Major problems have been genotype dependence and low frequency of somatic embryogenesis, making it difficult to regenerate plants from transgenic tissue. This study reports an increase in somatic embryogenesis efficiency and the induction of developmental synchrony in embryogenic callus cultures of cotton by a single cycle of myo-inositol depletion in liquid culture. Calluses were initiated on hypocotyl or cotyledon explants of cultivar Coker 312 by culturing these explants on callus-inducing solid medium [Murashige and Skoog salts plus vitamins of Gamborg's B₅ medium, 30 g l⁻¹ glucose, 100 mg l⁻¹ myo-inositol, 2.2 μM 2,4-dichlorophenoxyacetic acid, and 0.88 μM 6-benzyladenine]. The calluses were transferred to an identical liquid basal medium devoid of plant growth regulators. This induced the development of embryogenic cells. Friable clumps of cells formed after 20 d in the medium were selectively collected over filter mesh 40 subjected to one cycle of myo-inositol starvation. This induced a highly synchronized embryogenesis in the culture. The optimized protocol gave 100% embryos at the globular stage, out of which more than 80% developed into bipolar torpedo-stage embryos. About 68% of these were converted to plantlets by subculturing onto a simplified solid medium, and finally grown into healthy, fertile plants.     

Effects of kanamycin on tissue culture and somatic embryogenesis in cotton

The aminoglycoside antibiotic kanamycin was evaluated for its effects on callus initiation from hypocotyl and cotyledon explants, proliferation of non-embryogenic and embryogenic calli, initiation and development of somatic embryos in cotton (Gossypium hirsutum L.). On this basis, the potential use of kanamycin as a selective agent in genetic transformation with the neomycin phosphotransferase II gene as the selective marker gene was evaluated. Cotton cotyledon and hypocotyl explants, and embryogenic calluses were highly sensitive to kanamycin. Kanamycin at 10 mg/L or higher concentrations reduced callus formation, with complete inhibition at 60 mg/L. Kanamycin inhibited embryogenic callus growth and proliferation, as well as the initiation and development of cotton somatic embryos. The sensitivity of embryogenic callus and somatic embryos to kanamycin was different during the initiation and development stages. Kanamycin was considered as a suitable selective agent for transformed callus formation and growth of non-embryogenic callus. Forty to sixty mg/L was the optimal kanamycin concentration for the induction and proliferation of transformed callus. The concentration of kanamycin must be increased (from 50 to 200 mg/L) for the selection of transformation embryogenic callus and somatic embryos. A scheme for selection of transgenic cotton plants when kanamycin is used as the selection agent is discussed.     

Putrescine enhances somatic embryogenesis and plant regeneration in upland cotton

Improvement in somatic embryogenesis has been achieved in several cotton lines (Gossypium hirsutumL.) from the Georgia and Pee Dee germplasm with culture media containing various Putrescine concentrations. The best results were obtained with the -naphthalene acetic acid (NAA)-based treatments, S15 g.05 NAA and EMMS₂, as compared to the 2,4-dichlorophenoxyacetic acid (2,4-D)-based culture medium, EMMS₄. Inclusion of 0.5 mg l^–1 Putrescine improved somatic embryo (SE) induction for most treatments and lines tested. An 8-and 2-fold improvement was achieved in SE production on the EMMS₂-0.5 Putrescine treatment as compared to EMMS₂ alone for cotton lines PD 97019 and GA 98033, respectively. A significant increase in SE number (53-fold) was obtained with the addition of 0.5 mg l^–1Putrescine to EMMS₂ for PD 97021, which was essentially recalcitrant without Putrescine treatment. Conversion of SEs into plants was both genotype- and culture medium-dependent.     

植物激素对棉花体细胞胚胎发生的诱导及调节作用

选用11种激素研究了外源激素对棉花胚性愈伤组织增殖、胚胎发生和发育的调控作用。结果表明不同激素对棉花胚性愈伤组织增殖、胚胎发生与发育的影响不同。除2,4-D和BA对棉花胚性愈伤组织的增殖影响不大外,其他激素对棉花胚性愈伤组织的增殖均具有抑制作用,且具有一定的时间效应,同时还受基因型的影响。激素对棉花体细胞胚的形成和发育的影响极大,2,4-D既抑制了体细胞胚的形成,又抑制了体细胞胚的发育;TDZ的作用与2,4-D相似,显抑制了体细胞胚的形成,且诱导获得的体细胞胚均停留在球形胚阶段;GA也抑制了体细胞胚的形成,且不利于体细胞的成熟与萌发;BU-30对棉花体细胞胚形成与发育的影响不大。其他7类生长素类物质和细胞分裂素类物质对棉花体细胞胚的形成均具有促进作用,且依IBA、ABA、IAA、BA、KT、ZT、2iP序增强,其总胚数为对照的1．193—3．852倍;其中2iP的促进作用最大,可使产生的体细胞胚数提高2．852倍。     

Somatic embryogenesis and plant regeneration in wild cotton (Gossypium klotzschianum)

A simple and efficient method for high frequency somatic embryogenesis and plant regeneration from hypocotyl-derived cultures and suspension cultures of Gossypium klotzschianum Anderss, a wild, diploid species of cotton is described here. Embryogenic cultures were induced from hypocotyl sections on MSB medium with 0.9 M 2,4-D and 2.32 M kinetin. MSB medium containing 0.045 M 2,4-D, 0.93 M kinetin, 2.46 M IBA promoted embryogenic culture proliferation and embryo development. Suspension cultures with 0.23 M 2,4-D and 0.93 M kinetin also produced many embryos. Somatic embryos cultured on MSB medium with PGRs produced secondary embryos, and embryos developed into normal plantlets on PGR-free MSB medium. Regenerated plantlets were transferred onto the quarter-strength MSB medium with 0.5% active charcoal to avoid recallusing. Hypocotyls were better than cotyledons for culture induction and plant regeneration. 2,4-D and kinetin were essential for culture induction and maintenance.     

Plant regeneration from protoplasts of immature Vigna sinensis cotyledons via somatic embryogenesis

Summary Protoplasts were isolated from immature cotyledons of Vigna sinensis and cultured in a modified MS Liquid medium containing 0. 2 mg/l 2, 4-dichlorophenoxyacetic acid (2, 4-D), 1 mg/l naphthaleneacetic acid (NAA) and 0. 5 mg/l 6-benzylaminopurine (BAP) in the dark at a density of 1 × 10⁵/ml. The protoplasts began to divide in 3–5 days. Sustained cell division resulted in formation of cell clusters and small calli, with the cell division frequency and plating efficiency of cell colonies reaching 27. 7% and 1. 7% respectively. When calli of 2 mm in size were transferred onto MSB medium (MS salts and B5 vitamins) containing 500mg/l NaCl, 500 mg/ 1 casein hydrolysate (CH), 2 mg/l 2,4-D and 0. 5 mg/l BAP for further growth, approximately 5% of the calli developed embryogenically. The embryogenic calli were selected and subcultured on the same composition of MSB medium and were able to maintain somatic embryogenesis capacity in subculture for a long time. When the calli were moved to MSB medium with 0. 1 mg/l indole-3-acetic acid (IAA), 0. 5mg/l kinetin(KT), 3–5% mannitol and 2% sucrose in the light, many somatic embryos formed from the calli. Only part of the embryoids developed further to the cotyledonary stage, and the others died at the globular, heart-shaped or torpedo stages. Finally, some cotyledonary embryoids germinated and developed into plants or shoots. The shoots were readily rooted on 1/2 strength MS medium with 0. 1–0.3 mg/l indole-3-butyric acid (IBA). The plants grew well in soil and were fertile.Abbreviations 2, 4-D dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - BAP 6-benzylaminopurine - IAA indole-3-acetic acid - KT kinetin - IBA indole-3-butyric acid - CH casein hydrolysate - CM coconut milk - ZT zeatin     

In-vitro regeneration of olive tree by somatic embryogenesis

We induced somatic embryogenesis from the cotyledon segments ofOlea europaea (L) cvs. ‘Chetoui’, ‘Chemleli’, and ‘Arbequina’. Calli were established from all three cultvars on OMc media supplemented with IBA and 2i-R The greatest success was obtained with media that contained zero or low concentrations of growth regulators. High levels of hormones (i.e.,>0.5 mgL^-1 IBA and 2i-P) inhibited embryogenesis. Embryos at different maturation stages were observed with continuously proliferating secondary embryogenesis. Abnormally shaped embryos and teratoma were also noted. Four weeks was the optimal incubation period for inducing embryogenesis on the auxin-containing medium. In addition, 30 to 40 gL^-1 sucrose was more effective than glucose in stimulating the growth and maturation of somatic embryos. Embryogeic efficiency was also higher when multivariate combinations of nitrogen sources (inorganic and organic nitrogen forms) were used. The plantlets that were derived from our germinating somatic embryos were similar to those obtained from axillary buds.     

Somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L.)

Tissue culture methods for improvement of cotton has lagged seriously compared to other major crops. A method for regeneration of cotton which includes a morphogenetically competent cell suspension was needed to facilitate selection of stress-resistant variants and gene manipulation. Preliminary screening of eight strains of Gossypium hirsutum L. for embryogenic potential resulted in the production of somatic embryos in all strains. Coker 312 was selected for use in the development of a model regeneration system for G. hirsutum. Calli were initiated from hypocotyl tissues of 3-day-old-seedlings. Globular embryos were present after six weeks in culture. Calli were subcultured to liquid suspension in growth regulator-free medium. After three to four weeks, suspensions were sieved to collect globular and heart stage embryos. Collected embryos developed further when plated onto semi-solid medium. To induce germination and plantlet growth, mature embryos were placed on sterile vermiculite saturated with medium. Upon development of roots and two true leaves, plantlets were potted in peat and sand, and hardened. Mature plants and progeny have been obtained with this procedure. A high percentage of infertile plants was observed among the regenerants.Abbreviations NAA 1 naphthaleneacetic acid - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - MS Murashige and Skoog - BA 6 benzylamino purine - 2i P N⁶-(²-isopentenyladenine     

Somatic embryogenesis in cotton (Gossypium). II. Requirements for embryo development and plant regeneration

Calli of cotton (Gossypium hirsutum L.) initiated from seedling hypocotyl tissue were placed in liquid suspension and maintained by serial subculture in hormone-free Murashige and Skoog (MS) medium. Suspensions were sieved and globular embryos collected, washed, resuspended in basal medium and plated onto various semi-solid media. High inorganic salts (MS), low salt (2/3 MS), excess KNO₃, and the growth regulators napthaleneacetic acid (NAA), gibberellic acid (GA₃) and kinetin were tested for their effects on somatic embryo maturation. Long-term embryo proliferation and maturation were best on medium containing MS plus 1.9g/l KNO₃. Embryos 3 mm to 10 mm in size were removed from this plating medium and placed on sterile vermiculite saturated with Stewart and Hsu's medium plus 0.1 mg/l indoleacetic acid (IAA). Plants were recovered from 10.6% of the embryos. When 5 mm embryos were placed on this medium, 30% of the embryos formed plants within six weeks. Smaller embryos required a longer period of development on the vermiculite and the addition of fresh medium supplemented with 0.1 mg/l GA₃. Plants with an extensive root system and two true leaves were removed from sterile culture and potted in either one-to-one peat and sand, or vermiculite. Eighty percent of the regenerants were successfully hardened when glass beakers of increasing size (10 to 150 ml) were sequentially placed over the young plants during a two-week period.     

Indirect somatic embryogenesis and plant recovery from cotton (Gossypium hirsutum L.)

Summary We describe a tissue culture procedure for somatic embryogenesis and plantlet regeneration in cotton (Gossypium hirsutum L. cv. Coker 312). Callused explants or individual globular embryos were transferred to basal media to induce somatic embryogenesis. To determine characteristic early indicators of successful germination and conversion, we identified six types of embryos that developed on basal media. Two of the six embryo types, designated as tulip-shaped and trumpet-shaped, could undergo conversion in preliminary tests, whereas the others had little or no developmental potential. Several media treatments designed to enhance the maturation of globular somatic embryos failed to increase the fraction of embryos which matured to form recoverable types. In efforts to improve plantlet recovery, tulip-shaped embryos were used in limited trials to contrast the effects of chemical and physical desiccation treatments on germination and conversion. The selective use of tulip-shaped somatic embryos, coupled with partial desiccation, seems to have augmented plant recovery. Growth habit, flowering, seed set, and lint production of most of the regenerated plants were comparable to seed-derived plants grown under the same conditions. Partial research support was provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University.     

Plant regeneration via somatic embryogenesis from in vitro tissue culture in two Tunisian Cucumis melo cultivars Maazoun and Beji

Hypocotyl, cotyledon and zygotic embryo explants from two Tunisian Cucumis melo L. cultivars Beji and Maazoun, cultured on the MS medium added with 2,4-D (0.25–1 mg l⁻¹) and BA (0.10–0.50 mg l⁻¹), produce calluses with somatic embryos after 3 weeks of culture. For Beji c.v. the highest percentage (62.50%) of embryogenesis was observed for cotyledons. The average embryo number per callus was 10.40. Embryogenesis induction for zygotic embryos reached 33.50% with 29 embryos per callus. The embryogenesis ability of hypocotyls did not exceed 12.50% (2.50 embryos per callus). Somatic embryogenesis for Maazoun c.v. explants was less efficient. Embryos formation was observed only for cotyledons (29%) and zygotic embryos (25%). Cotyledonary staged embryos, when transferred to hormone free MS medium, germinated. The maximum germination rates were 51.50 and 44.50%, respectively for Maazoun and Beji c.v. The highest percentage (36.50%) of survival plants was noted for Beji c.v. Regenerants were diploids (2n = 2x = 24) and morphologically similar to their parents issued from seeds.     

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。     

Somatic embryogenesis in cotton (Gossypium) I. Effects of source of explant and hormone regime

Optimal media for induction of somatic embryogenesis from mature and immature tissues ofG. hirsutum L. cv Coker 312 were determined. Explants of three-day-old seedlings form somatic embryos in 100% of cultures when treated with 0.1 mg/1 2,4-dichlorophenoxyacetic acid plus 0.5 mg/1 kinetin. Mature tissues are more recalcitrant than immature tissues and formed somatic embryos on a limited number of hormone treatments. Stem tissue is most readily induced to form somatic embryos by 2 mg/1 napthaleneacetic acid plus 0.1 mg/1 kinetin, whereas leaf tissue formed embryos best when treated with 0.1 mg/1 2,4-dichlorophenoxyacetic acid plus 1.0 mg/1 (2-isopentyl)-adenine, or 1.0 mg/1 napthaleneacetic acid plus 0.5 mg/1 (2-isopentyl)-adenine.     

High frequency plant regeneration from anther-derived cell suspension cultures via somatic embryogenesis in Catharanthus roseus

Summary A system for high frequency plant regeneration from cell suspension cultures in Catharanthus roseus is described. Calli were obtained from anthers cultured on Murashige and Skoog's medium supplemented with 1 mgl^-1 -naphthaleneacetic acid and 0.1 mgl^-1 kinetin. After the second subculture on solid medium, embryogenic callus was identified and transferred to liquid medium to initiate suspension cultures. Cells dispersed finely in the medium were subcultured at 14-day intervals. Upon plating onto the basal medium, yellowish compact colonies proliferated from the cells and more than 80% of them gave rise to somatic embryos. Subsequently, plantlets developed from the embryos. Both the plantlets and the source plants showed the normal somatic chromosome number of 2n=2x=16.Abbreviations MS Murashige and Skoog - MSNK MS medium + 1 mgl^-1 NAA + 0.1 mgl^-1 kinetin - NAA -naphthaleneacetic acid     

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.     

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.     

Efficient plant regeneration through somatic embryogenesis from callus induced on mature rice embryos (Oryza sativa L.)

To obtain a reproducible efficient procedure for regeneration of rice plants through somatic embryogenesis from callus four published methods of callus induction and regeneration were compared. Callus was initiated from mature embryos of the Japonica cultivar Taipei 309 of rice (Oryza sativa L.). The number, mass and morphology of the callus formed on the scutellum were dependent on the medium used. A limited humidity and an optimal aeration of the culture vessels enhanced the frequency of embryogenesis and plant regeneration. A method described by Poonsapaya et al. (1989) was found to be the most efficient and was slightly modified. As a result 98% of the T309 embryos formed callus, of which 63% regenerated into plants. Each callus yielded an average of 6 plants. Plant morphology, fertility and seed set of the regenerants were found to be normal.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - IAA 3-indole-acetic acid - BA 6-benzyladeninepurine - S.E.M. standard error of mean     

High Frequency Plant Regeneration from Astragalus melilotoides hypocotyl and stem explants via somatic embryogenesis and organogenesis

An efficient and reproducible procedure is established for the plant regeneration from hypocotyl explants and hypocotyl-or stem-derived calli in Astragalus melilotoides. High frequency somatic embryo formation (98.3%) occurred direct on hypocotyls on Murashige and Skoog (MS) medium supplemented with 2.69 µM NAA and 4.44 µM BA within 5 weeks. Three types of calli were induced from the hypocotyl and stem segments on MS medium containing 9.05 µM 2,4-D and 2.22–4.44 µM BA. Both somatic embryos and adventitious buds were initiated from hypocotyl-derived calli while only adventitious buds were formed from stem-derived calli in MS medium supplemented with 2.69 µM NAA and 4.44–8.89 µM BA. Somatic embryos or adventitious buds developed into plantlets following being cultured for 3 weeks on MS medium without any growth regulators or with 14.78 µM IBA, respectively. All the regenerated plants were normal with respect to morphology and growth characters, and produced fertile seeds after planting in soil.