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     

Plant regeneration through somatic embryogenesis from immature and mature zygotic embryos of Musa acuminata ssp. burmannica

A simple and efficient protocol has been developed for in vitro regeneration of M. acuminata ssp. burmannica (AA) plants. Somatic embryos were produced when immature and mature zygotic embryo explants were cultured on Murashige and Skoog medium supplemented with plant growth regulators 2,4-dichlorophenoxyacetic acid; (2,4-D), picloram or benzyl adenine and indole acetic acid. In general, immature embryos responded better than mature embryos. Callus proliferation was highest in medium supplemented with 2,4-D (4.5???M). Subsequent transfer of callus to fresh medium produced rapidly proliferating embryogenic calli. Embryogenic calli were maintained in complete darkness for 15?d followed by cycles of 8?h dark and 16?h light, under white fluorescent lamps with a light intensity of 3,000?lm/m² and at temperature of 28?±?2°C. Regeneration of embryogenic calli into plantlets was higher for immature embryos (76.6%) than for mature embryos (50.6%). This plant regeneration protocol using mature or immature zygotic embryos, via somatic embryogenesis, has significant potential to improve germination efficiencies of hybrid progenies used in conventional breeding strategies. Furthermore, tests on seed storage showed that seed viability rapidly decline after harvesting and was negligible after 9?mo of storage. This indicates using freshly harvested seeds as explant material is necessary for maximizing the tissue culture response.     

Plant regeneration through direct somatic embryogenesis from immature zygotic embryos of the medicinal plant,Paris polyphylla Sm.

A protocol for induction of direct somatic embryogenesis and subsequent plant regeneration for the medicinally important and endangered plant Paris polyphylla Sm. has been developed for the first time. Immature zygotic embryos (IZEs) were cultured on different media namely Gamborg (B5), ½ B5, Murashige and Skoog (MS), ½ MS, Chu et al. (N6), ½ N6, Schenk and Hildebrandt (SH) and ½ SH. Highest frequency of somatic embryogenesis (32.6 %) and mean number of somatic embryos (SEs) per explant (28.7 ± 1.7) were obtained on ½ MS medium directly without an intermediate callus phase. The frequency of SE induction was significantly increased to 40.7 % when ½ MS medium was solidified with gelrite compared to agar (32.6 %). Secondary somatic embryos (SSEs) appeared on the primary SEs in a repetitive way on plant growth regulator-free ½ MS medium but with a gradual decrease in embryogenic potential during subsequent subcultures. Plasmolyzing pre-treatment of SSEs with 1.0 M mannitol for 12 h effectively maintains its embryogenic capacity. Primary embryos at the elongated dimpled and early cotyledonary stage displayed the highest embryo forming capacity of 26.94 and 27.87, respectively. High frequency of SE germination (94.0 %) occurred on ½ MS medium with 0.5 mg/l gibberellic acid. Highest percentage of seedling to plantlet conversion was observed in the medium supplemented with 0.05 mg/l 6-benzylaminopurine and 0.1 mg/l α-naphthalene acetic acid. Regenerated plants displayed morphological characteristics similar to that of the wild plants. Flow cytometry analysis showed ploidy stability of the regenerated plants.     

Plant regeneration through somatic embryogenesis from spear callus culture of Asparagus cooperi Baker

Somatic embryogenesis and plantlet formation were obtained from callus derived from the subapical region of spears of Asparagus cooperi Baker. Callus was obtained in Murashige and Skoog's medium supplemented with 1-naphthaleneacetic acid and kinetin. Increase in the concentration of potassium nitrate in subsequent subcultures resulted in the formation of embryos. Rapid multiplication of embryos was secured on transfer to a medium containing a different source of nitrogen and a low level (0.01 mg/1) of gibberellic acid. Media containing zeatin or gibberellic acid led to the formation of complete plantlets from embryos. Regenerated plants were cytologically and phenotypically stable.Abbreviations IBA Indole-3-butyric acid - NAA 1-Naphthaleneacetic acid - 2ip (2-Isopentenyl) adenine - BAP 6-Benzylaminopurine, GA,, Gibberellin - ABA Abscisic acid - MS Murashige and Skoog (1962) medium     

Plant regeneration from cultured immature embryos and inflorescences ofTriticum aestivum L. (wheat): Evidence for somatic embryogenesis

Summary Tissue cultures ofTriticum aestivum L. (wheat) initiated from young inflorescences and immature embryos possessed the potential for regeneration of whole plants. Both a friable and a compact type of callus were produced on Murashige and Skoog's medium with 2 mg/l 2,4-dichlorophenoxyacetic acid. The friable callus contained meristematic centers in which the peripheral cells ceased dividing, elongated, and could be easily separated. Roots were frequently formed in this type of callus. The compact, yellowish, and nodular callus arose from the epithelial and sub-epithelial cells of the embryo scutellum, and the rachis and glumes of the young inflorescence. Such callus had a smooth surface and characteristic chlorophyllous areas. Plants were regenerated only from the compact callus. The first sign of differentiation in the compact callus was the formation of a cleft or notch on the smooth surface, followed by the appearance of trichomes and the direct development of leafy structures which were not associated initially with any shoot meristems. Multiple shoots subsequently arose at the bases of the leafy structures, which are considered modifications of the scutellum, a definitive part of the cereal embryo. Accordingly, we suggest that while typical bipolar embryos are generally not formed, plant regeneration nevertheless takes place through embryogenesis and the precocious germination of the embryoids. Plants regenerated from immature embryo and inflorescence cultures were grown to maturity in soil, and were shown to have the normal chromosome number of 2n=6x=42.     

Cytokinin induced somatic embryogenesis from immature embryos of Abies nordmanniana Lk.

Summary Abies nordmanniana Lk. is used in short intensive rotations for Christmas tree production. Thus there is a high demand for development of advanced propagation and breeding methods. Somatic embryogenesis was easily induced from immature (precotyledonary) embryos collected in July 1989 with cytokinin as the sole plant growth regulator. The proliferating embryogenic cell masses were characteristic of conifer somatic embryogenesis and could be maintained on a simple basal medium containing 5 M benzylaminopurine. Auxin inhibited induction as well as proliferation. Proliferation was improved by up to 30 % by addition of L-glutamine and/or casein hydrolysate. Neither cytokinin concentration nor culture on 3 different basal media, differing markedly in their nitrogen composition, affected the proliferation rate. Embryos matured using a 4 week subculture on medium containing 10 M abscisic acid and subsequent transfer to medium devoid of plant growth regulators.Abbreviations TDZ thidiazuron (Schering) - BAP benzylaminopurine (Sigma) - KIN kinetin (Sigma) - 2,4-D 2,4-dichlorophenoxyacetic acid (Sigma) - ABA +/2-cis-4-trans-abscisic acid (Sigma) - CH casein hydrolysate (Sigma Type 1, acidic) - L-gln L-glutamine (Sigma) - EDTA ethylene diamine tetra acetic acid (Sigma)     

Plant regeneration by somatic embryogenesis from callus cultures of sweet sorghum

Tissue culture methods were developed for the induction, maintenance, and regeneration of embryogenic callus in sweet sorghum (Sorghum bicolor) cultivars Keller, Rio, and Wray. No significant differences were observed in production of embryogenic callus in cultures established from developmentally immature or mature embryo explants cultured on LS medium with 2 mg/1 2,4-D plus 0.5 mg/1 kinetin. Prolific callus production did not occur until the third four-week culture period. Long-term maintenance of embryogenic callus was dependent upon the selective transfer of embryogenic callus, with other callus types discarded. High-frequency plant regeneration was achieved and quantified on a fresh weight basis of embryogenic callus.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA benzyladenine - IAA indoleacetic acid - IBA indolebutyric acid - LS Linsmaier and Skoog basal medium (Linsmaier and Skoog, 1965)     

Plant regeneration through somatic embryogenesis from suspension culture-derived protoplasts of Paspalum scrobiculatum L.

Protoplasts were released from embryogenic suspension culture of Paspalum scrobiculatum and cultured in either liquid or semisolid KM medium supplemented with 2,4-D in the dark at 24°C with or without a feeder layer. Cell wall formation was observed in 75% of the plated protoplasts. Microcolonies developed after 10 d of culture, which in turn formed callus upon transfer to M-2 medium (Nayak and Sen, 1989). The highest plating effeciency (ca 7%) was obtained in thin-layer liquid culture. The macrocalli formed somatic embryos which regenerated to plantlets. The plantlets were grown to flowering plants upon transfer to soil.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA Fluoresceine diacetate - MES 2-(N-Morpholino) ethanesulfonic acid - MS Murashige & Skoog medium (1962)     

Plant regeneration through direct somatic embryogenesis from protoplasts of banana (Musa spp.)

Summary We report the isolation and regeneration of protoplasts from an embryogenic banana (Musa spp.) cell suspension culture initiated from in vitro proliferating meristems. A high yielding isolation method (up to 6×10⁷ protoplasts.ml^–1 packed cells) is discussed. Optimal regeneration, with more than 50% of the protoplasts showing initial cell division, occurred when high inoculation densities (10⁶ protoplasts.ml^–1) or nurse cultures were applied. Under these conditions, the frequency of microcolony formation was 20–40%. These microcolonies developed directly, without an intervening callus phase, into somatic embryos which later germinated and formed plantlets.     

Direct somatic embryogenesis induced from cotyledons of mango immature zygotic embryos

Summary For the first time, regenerated plantlets were obtained from immature zygotic embryos of mango (Mangifera indica L.) through direct somatic embryogenesis. Pro-embryogenic mass (PEM)-like structures, which are differentiated as clusters of globular structures, were easily induced directly from the abaxial side of cotyledons from immature fruits, 2.0–3.5 cm diameter by a 2-wk culture period on a modified Murashige and Skoog medium with 5 mgl⁻¹ (25μM) indole-3-butyric acid (IBA). Conversion of somatic embryos into plantlets was achieved after 4 wk of culture on the conversion medium containing 5mgl⁻¹ (23 μM) kinetin. Secondary somatic embryogenesis could also be obtained directly from the hypocotyls of mature primary somatic embryos cultured on the conversion medium. In our experimental system, only minor problems were noted with browning of cultures.     

Plant regeneration through organogenesis from callus induced from mature zygotic embryos of loblolly pine

Mature zygotic embryos from three seed sources of loblolly pine were cultured on callus induction medium containing 10 mg l^–1 α-naphthaleneacetic acid, 4 mg l^–1 benzyladenine (BA), 400 mg l^–1 casein hydrolysate, and 400 mg l^–1 glutamine for 6 weeks. Light-yellow, loose, glossy, globular callus was formed, and the highest frequency was 35.7%. The highest differentiation frequency of callus on adventitious bud induction medium was 62.1%. After culture of calli with adventitious buds on elongation medium for 6 weeks, adventitious shoots more than 1.0 cm in height were selected for rooting. On rooting medium supplemented with 0.1 mg l^–1 indole-3-butyric acid, 1 mg l^–1 BA, and 0.5 mg l^–1 gibberellic acid, the highest rooting frequency of adventitious shoots was 46% in a culture period of 6 weeks. Established plants survived following transfer to soil at a frequency of 71%. Received: 14 May 1997 / Revision received: 25 September 1997 / Accepted: 11 October 1997     

Plant regeneration from callus and protoplasts of Brassica nigra (IC 257) through somatic embryogenesis

A method to obtain plants from embryogenic callus of Brassica nigra and protoplasts of hypocotyl expiants is described. Callus was initiated on Murashige and Skoog medium containing kinetin (kn) and 2,4-dichlorophenoxy acetic acid (2,4-D). Lowering of auxin induced embryo formation. Supplementation with gibberellic acid (GA₃) enhanced embryogenic response tenfold. Passage through liquid medium devoid of growth regulators was essential for the growth of embryos. Secondary embryos were produced on transfer to solid basal medium. Embryogenic callus retained its morphogenic ability even after 12 subcultures. Both primary and secondary embryos produced fertile plants. Hypocotyl-derived protoplasts were also regenerated to plants following the same protocol. The survival of plants on transfer to soil was about 80%. The seeds from plants derived from callus and protoplasts were viable.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - NAA naphthalene acetic acid - IAA indole acetic acid - kn kinetin - GA₃ gibberellic acid     

Plant regeneration of Sapindus trifoliatus L. (soapnut) through somatic embryogenesis

Somatic embryogenesis and subsequent formation of plantlets was obtained from callus cultures derived from leaves of mature (over 60years old) Soapnut (Sapindus trifoliatus L.) tree. Callus was induced from leaf explants and grown on Murashige and Skoog's medium supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D) and kinetin. Reduction of 2,4-D concentration during subsequent subcultures resulted in formation of embryoids. These embryoids developed further when transferred to a medium containing benzylaminopurine and kinetin and then to a hormone-free medium. Unless 5-methyl tryptophan was added and the level of sucrose raised, the embryoids began to recallus and failed to form plantlets.     

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     

Plant regeneration from encapsulated somatic embryos of Carica papaya L.

Carica papaya L. (papaya) single somatic embryos (2.0 mm diameter) produced in a high-frequency liquid production system were encapsulated in two different synthetic encapsulation compounds. The frequency of regeneration from encapsulated embryos was significantly affected by (1) the concentration of sodium alginate, (2) the presence or absence of nutrient salts in the capsule, and (3) the duration of exposure to calcium chloride. A 2.5% sodium alginate concentration in a half-strength MS salts base resulted in significantly higher germination frequencies than other treatments. A relatively short (10 min) exposure to CaCl₂ provided uniform encapsulation of embryos and the highest frequencies of successful germination (77.5%). Germinated artificial seeds produced normal plantlets. Received: 12 March 1997 / Revision recieved: 24 June 1997 / Accepted: 18 July 1997     

Plant regeneration from protoplasts of mango (Mangifera indica L.) through somatic embryogenesis

 This report describes a protocol for the regeneration of plants from protoplasts isolated from proembryogenic masses (PEMs) in a suspension culture derived from the nucellar callus of mango (Mangifera indica L. cv 'Amrapali'). The maximum yield (24.6±1.1×10⁶), with 81.04±4.1% viable protoplasts per gram PEMs, was obtained with an enzyme mixture containing 1.2% cellulase, 1.0% hemicellulase and 0.6% pectinase. An optimum density of 5×10⁴ cultured protoplasts per milliliter culture medium was required for the highest frequency (88.89±5.40%) of division. Dividing protoplasts developed into microcalli that proliferated on medium supplemented with growth regulators (auxins or kinetin alone, or auxins with kinetin) and produced somatic embryos after transfer to a growth regulator-free medium. The protocallus on 2,4-D-containing medium produced the maximum number (102.50±6.93) of somatic embryos. Maturation of somatic embryos depended upon the presence, and the nature and combination of growth regulators in the medium during proliferation of the callus. The mature somatic embryos germinated and developed into plants that were transferred to soil. Received: 1 April 1999 / Revision received: 27 July 1999 / Accepted: 23 August 1999     

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.     

Plant regeneration through somatic embryogenesis in peanut (Arachis hypogaea L.)

Somatic embryos were induced from immature cotyledons and immature embryonal axis ofArachis hypogaea L. on L-6 basal medium supplemented with NAA, picloram or 2,4-D at 5–50 mg 1^-1. Immature embryonal axis produced a higher number of somatic embryos in comparison with immature cotyledons. The highest number of responding cultures was produced on medium supplemented with NAA (50 mg 1^-1), while the highest average number of somatic embryos per culture was produced on medium with 2,4-D (10 or 20 mg 1^-1) and picloram (30 mg 1^-1) from cotyledons. The somatic embryos developed into plants on basal medium supplemented with activated charcoal and about 100 plants were successfully transferred to the field. Acknowledgement: The authors wish to thank Nuclear Agriculture Division, BARC for supplyingA. hypogaea seeds and Mr. R.M. Mudliar for photography.     

Plantlet regeneration via somatic embryogenesis in anther callus of Vitis latifolia L.

Anthers of Vitis latifolia L. (wild grape) cultured on Nitsch and Nitsch medium supplemented with 20 μM 2,4-D and 9 μM BAP produced callus after 4–6 weeks. Subculture of callus onto Nitsch and Nitsch medium containing 10 μM NAA produced somatic embryos within 6 weeks. On growth regulator-free Nitsch and Nitsch basal medium somatic embryos converted to plantlets in 6–8 weeks. One gram of callus produced more than 400 somatic embryos with 13.7% being converted to complete plantlets, which were subsequently established in soil. Regenerated plants were found to have mixoploid populations of cells, 2n = 38 and n = 19. Received: 23 May 1998 / Revision received: 21 September 1998 / Accepted: 10 October 1998     

Plant regeneration by somatic embryogenesis from cultured immature embryos of oak (Querem robur L.) and linden (Tilia cordata Mill.)

Embryogenic cultures and somatic embryos were obtained from immature zygotic embryos of oak (Quercus robur L.) cultured on a modified MS medium and WPM containing BAP (1 mg·l^–1) and GA₃ (1 mg·l^–1) or BAP and IBA. Germination and conversion of oak somatic embryos into plantlets was achieved on WPM containing a reduced concentration of cytokinin. Linden (Tilia cordata Mill.) somatic embryos developed in embryogenic tissues initiated from immature zygotic embryos cultured on a modified MS medium supplemented with 2,4-D (0.3-2.0 mg·l^–1). Germination of linden somatic embryos and plantlet formation occurred on MS medium containing a low concentration of IBA. Oak and linden plantlets produced from somatic embryos were successfully established in soil. Somatic embryos and plantlets were also regenerated from embryogenic cultures of Quercus petraea and Tilia platyphyllos.Abbreviations BAP 6-benzyIaminopurine - GA₃ gibberellic acid - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) - WPM woody plant medium