Plant regeneration from tissue cultures initiated from immature inflorescences of a grass,Echinochloa colonum (L.) link

Organised structures develop on a white and compact callus initiated from small segments of immature inflorescences ofEchinochloa colonum cultured on Murashige and Skoog's (MS) medium supplemented with 5.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 10% coconut milk. These develop into plantlets upon subculture onto MS medium containing 0 or 0.2 mg/l 2,4-D. Twelve out of 17 plantlets regenerated grew well on transfer to soil and eleven plants produced seeds.     

Development of secondary inflorescences and in vitro plantlets from inflorescence cultures of Amaranthus paniculatus

Immature inflorescences of Amaranthus paniculatus were used as explants for in vitro culture studies. When placed on a medium supplemented with 3–6 mg/l kinetin, explants developed into secondary inflorescences. Leaves and shoots developed following culture of inflorescence tissue on media containing 8–15 mg/l kinetin or 5–10 mg/l BAP. These shoots when subcultured on MS medium supplemented with 12 mg/l kinetin + 15% coconut milk, formed roots. These rooted plantlets later flowered in vitro.Abbreviations MS Murashige and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - BAP 6-benzylaminopurine - Kn 6-furfurylaminopurine - CM coconut milk     

The effect of explant material on somatic embryogenesis of Cyclamen persicum Mill

Somatic embryos of Cyclamen persicum Mill. could be produced through a callus phase from juvenile explant material including anthers, ovaries and zygotic embryos. The auxin 2,4-D (1.0–1.5 mg l^-1) and coconut milk (10% v/v) in MS medium were important factors for the induction of somatic embryogenesis. Somatic embryos germinated into plantlets in MS medium without growth regulators. The plants grew well in the greenhouse and flowered normally. The plants were phenotypically identical to the mother plants with a few exceptions.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthylacetic acid - IAA 3-indoleacetic acid - BA 6-benzyladenine - ABA abscisic acid - CM coconut milk     

Plantlets from somatic callus tissue of the woody legume Sesbania bispinosa (Jacq.) W. F. Wight

In vitro regeneration of plantlets and multiplication of Sesbania bispinosa (Jacq.) W.F. Wight plants from cultured callus tissue were demonstrated. Callus was established from both cotyledons and mature leaflets on Murashige and Skoog (MS) basal medium supplemented with BAP (0.5 mg/l) and 2,4-D (2 mg/l). Callus mediated shoot bud differentiation was studied under defined nutritional, hormonal and cultural conditions. Various concentrations of BAP or kinetin (Kn) with coconut milk (CM) in MS media induced different levels of shoot bud differentiation as well as multiplication. Multiple shoot bud differentiation occurred in most of the primary calli. The best medium for shoot bud differentiation from cotyledon derived callus, contained BAP (2 mg/l) and 15% CM (V/V). More efficient shoot bud organogenesis was recorded with BAP than Kn. Supplementation with CM in MS media accelerated shoot bud organogenesis in differentiating callus tissue. Rooting of differentiated shoots was achieved by a three step culture procedure involving (a) MS solid medium containing IBA (2 mg/l), (b) growth regulator free half strength MS medium with 1% charcoal, and (c) half strength MS liquid medium free of vitamins, growth regulators and charcoal.Abbreviations IAA indoleacetic acid - IBA indole-3-butyric acid - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - Kn kinetin - CM coconut milk - MS Murashige and Skoog's medium - SBI shoot bud inducing medium     

Studies on a drought resistant legume: The moth bean,Vigna aconitifolia (Jacq) marechal. I. Protoplast culture and organogenesis

High yields of viable protoplasts were obtained from callus cultures derived from shoot apices of Vigna aconitifolia (JACQ) Marechal. The protoplasts divided and formed cell clusters on modified MS medium. The protoplast-derived callus formed multiple shoot buds on MS and B5 basal media without supplements, on MS medium containing supplements and on B5 medium containing charcoal (0.25%). Shoot formation occurred.Abbreviations MS Murashige and Skoog (1962) - B5 B5 basal medium (Gamborg et al 1968) - CM coconut milk - NAA 1-napthaleneacetic acid - BA 6-benzyladenine - IAA indole-3-acetic acid - 2,4 D 2,4-dichlorophenoxyacetic acid - GA gibberellic acid - CB Cellulase from Penicillium funiculosum - C Cellulase from Trichoderma reesei - CRIO Cellulase R10 - MR10 Macerozyme R10 - PDS Potassium dextran sulphate NCL communication No. 3376     

Shoot regeneration from stem and leaf callus of Eucalyptus tereticornis

Adventitious shoots were obtained from leaf and stem callus of Eucalyptus tereticornis SM. Callus was induced on B5 medium with 0.1 mg/l benzyladenine (BA) and 3 or 5 mg/l naphthalene acetic acid in the dark. Shoot initiation occurred on modified Woody Plant medium (mWP) containing 0.5 mg/l BA, 500 mg/l polyvinylpyrrolidone and 10% (v/v) coconut milk. Multiple shoots were also regenerated directly from hypocotyl segments of 4 to 6 week old seedlings on B5 medium with 0.5 mg/l BA. Regenerated shoots could be rooted with 100% efficiency on mWP medium containing 0.5 mg/l indolebutyric acid and transferred to soil in the greenhouse. Suspension cultures were obtained from the callus using B5 medium with 0.5 mg/l 2,4-dichlorophenoxyacetic acid. Callus clumps grew from less than 1 mm to 4–6 mm in diameter within two weeks on transfer to shoot regeneration medium but failed to form shoots or somatic embryos.Abbreviations BA Benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA Indolebutyric acid - NAA Naphthaleneacetic acid - PVP Polyvinylpyrrolidone - mWP modified Woody Plant medium Scientific Contribution No. 1689 from New Hampshire Agricultural Experiment Station.     

Plant regeneration from callus cultures of Durum and emmer wheat

Callus cultures were initiated from isolated mature embryos of Triticum turgidum L. Thell ssps durum and dicoccum on a basal medium supplemented with 2,4-D, 2,4,5-Cl₃POP or 2,4-D+CM. Shoot bud regeneration was observed on 2,4,5-Cl₃POP medium. In both the cultivars of durum, further development of shoot buds occurred on transfer of tissues to basal medium whereas in dicoccum basal medium supplemented with coconut milk or coconut milk with NAA (0.2 mg/l) was necessary. The regenerated shoot buds were induced to root on basal medium supplemented with NAA. The in vitro obtained plants were transferred to soil and successfully grown to maturity. Chlorophyll variants were observed among the regenerated plants of dicoccum.Abbreviations BA benzyladenine - CM coconut milk - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,iP 6---dimethylallylamine purine - IAA indoleacetic acid - NAA -naphthalene acetic acid - Kn kinetin - 2,4,5-Cl₃POP 2,4,5-trichlorophenoxypropionic acid - MS modified Murashige and Skoog's medium - RH relative humidity - Z zeatin     

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     

In vitro Studies on Pinus

Hypocotyl tissue from Pinus gerardiana was established in culture on White's basal medium supplemented with 2 % sucrose, 10% (v/v) coconut milk, 500 mg/l casein hydrolysate and 1 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D). Various organic and inorganic nutrients were studied in order to determine the specific nutritional requirements of the tissue in vitro. Sucrose, glucose and maltose were equally effective as fixed carbon sources. The inorganic nutrient combination of White's medium was found to be better than that of Murashige and Skoog's medium. White's modified basal medium supplemented with coconut milk, casein hydrolysate and 2,4-D was the most successful nutrient combination. Glutamine was as effective as casein hydrolysate in promoting growth of the tissue.     

Cryptosin — a new cardenolide in tissue culture and intact plants of Cryptolepis buchanani Roem. & Schult

A steroidal glycoside of the digoxin type was isolated from fresh leaves and cultured cells of Cryptolepis buchanani Roem & Schult (Asclepiadaceae). Cultures were raised from leaf and shoot. Explants were cultured on MS medium containing 2,4-D, NAA, BA and coconut milk. Metabolically active calluses were proliferated and maintained on Gamborg's B₅ medium with 2,4-D and Kn. The new compound, named cryptosin, was characterised by infrared nmr and mass spectroscopy. Cryptosin was also screened for its pharmacological properties and its cardiotonic action was established.Abbreviations PVP Polyvinylpyrrolidone - 2,4-D 2,4-Dichlorophenoxyacetic acid - NAA Naphthaleneacetic acid - BA Benzyl adenine - Kn Kinetin - MS Murashige & Skoog - Na,K-ATPase Sodium, potassium — sensitive adenosine triphosphate     

Somatic embryogenesis and plant regeneration from leaf tissues of Panicum maximum Jacq.

Summary Somatic embryogenesis was induced in proliferating leaf segments of Panicum maximum Jacq., cultured on Murashige and Skoog's nutrient medium containing 2,4-dichlorophenoxyacetic acid and coconut milk. The embryoids gave rise to plants on a medium containing gibberellic acid. The plants were successfully transplanted to soil and grown to maturity. Histological examination of proliferating leaves showed that the embryogenic callus tissue was formed by divisions in cells of the lower epidermis as well as the mesophyll tissue. The regenerated plants showed the normal chromosome number of 2n=4x=32.Florida Agriculture Experiment Station Journal Series No. 2775     

High frequency somatic embryogenesis and plant regeneration in tissue cultures of Codonopsis lanceolata

Culture conditions for high frequency somatic embryogenesis and plant regeneration from cotyledonary explants of Codonopsis lanceolata are described. The maximum induction frequency of somatic embryos from cotyledonary explants was 80% on Murashige and Skoog (MS) medium containing 6% sucrose with 1 mg/l 2,4-dichlorophenoxyacetic acid and 10% coconut water. Upon transfer onto MS basal medium containing 3% sucrose, most somatic embryos developed into plantlets.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellin a₃ - MS Murashige and Skoog     

Morphogenic potential of mechanically isolated single cells from Digitalis obscura L. callus

Calli from hypocotyl and root explants of Digitalis obscura L. showed regeneration of adventitious shoots, roots and embryos when transferred to Murashige & Skoog medium supplemented with cytokinins alone or in combination with auxins. Optimum shoot-bud formation was achieved in the presence of IAA and BA, while roots mainly appeared either in absence of growth regulators or with IAA and Kn. Embryo formation took place only in those combinations that included Kn. Embryo development was influenced by the type of auxin, and precocious germination occurred in media with NAA. Mechanically isolated cells from hypocotyl- and root-derived calli were plated in MS medium supplemented with several IAA and BA combinations. Single cells were able to proliferate forming callus within 20–30 days in culture. In order to induce organogenesis, calli were transferred to various regeneration media. Shoot-bud differentiation efficiency depended on both callus origin and medium initially used for cell culture, best results being obtained in calli grown from hypocotyl-derived cells cultured in the presence of casein hydrolysate. A further subculture to medium containing coconut milk and lower concentrations of NH₄NO₃ and sucrose promoted shoot development. Rooting was readily achieved upon transferring shoots onto half-strength MS medium. Plantlets were ultimately established in soil.Abbreviations BA benzyladenine - BM basal medium - CH casein hydrolysate - CM coconut milk - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - Kn kinetin - MS Murashige & Skoog - NAA naphthaleneacetic acid     

Rapid multiplication of Sapium sebiferum Roxb. by tissue culture

Multiple shoots formation and elongation was induced from stem explants of Sapium seedlings on media containing cytokinins. Leaf explants produced callus on a medium containing cytokinins, auxin, casein hydrolysate and coconut milk, which could be induced to form multiple shoots on transfer to a medium lacking casein hydrolysate, coconut milk and auxin. Rooting of isolated shoots by treatment with an auxin mixture (indole-3-acetic acid, indole-3-butyric acid and indole-3-propionic acid) and transfer of the plantlets to field have also been successful.     

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     

Somatic embryogenesis from hypocotyl callus cultures of Digitalis obscura L.

Hypocotyl-derived calli obtained in agar solidified medium with several growth regulator combinations gave rise to proembryonal masses and globular embryos when transferred to liquid media with lower growth regulator and higher NH₄HO₃ levels. By transferring cultures from liquid media to different solidified media, new embryo formation took place, but further development of these embryos or those previously induced depended on the characteristics of these media. Normal development was only achieved on 8 g/l agar solidified medium without growth regulators. Typical cotyledonary embryos developed into whole plants when transferred to this same medium.Abbreviations BA 6-benzylaminopurine - CH casein hydrolysate - CM coconut milk - 2,4-D 2,4-dichlorophenoxyacetic acid - 2iP 2-isopentenyladenine - Kn kinetin - NAA naphthaleneacetic acid - IAA indoleacetic acid     

HISTOLOGY OF SOMATIC EMBRYOGENESIS IN PANICUM MAXIMUM (GUINEA GRASS)

Immature embryos of Panicum maximum Jacq. (Guinea grass) were cultured on Murashige and Skoog's medium containing 2,4-dichlorophenoxyacetic acid (5 mg/1) and coconut milk (5%). The epidermal and subepidermal cells of the scutellum at the coleorhizal end either formed somatic embryos directly or gave rise to embryogenic callus at several loci. Somatic embryos were formed later also at the periphery of these embryogenic calli. The embryoids originated from single, densely staining, nonvacuolated and starch-containing cells with thickened walls and developed through characteristic stages of grass embryogeny.     

Preliminary observations on organogenesis inTaraxacum officinale tissue cultures

Summary Tissue cultures ofTaraxacum officinale have been isolated from the secondary thickened root. Callus development and leaf and root formation occur on a basal medium supplemented with coconut milk and IAA or NAA, and the addition of kinetin to these media enhances callus growth and organogenesis. Cultures grown on the basal medium with coconut milk and 2,4-D show only callus growth, but organogenesis is induced by the substitution of IAA for 2,4-D. In the 2,4-D grown callus a layer of secondary meristematic tissue is present and organogenesis apparently occurs from localized regions of this tissue which have undergone de-differentiation to the primary meristematic condition.     

籼稻幼穗细胞悬浮培养物的胚胎发生和植株再生

籼稻幼穗诱导的愈伤组织,培养在MS基本成分并附加1—7 mg/l 2,4-D和5％椰子汁的液体培养基中,得到胚性小细胞团。这些细胞的特征是:形状小而圆,细胞壁薄,细胞内为稠密的胞质所充满,没有或具小的液泡,具淀粉粒。把它们转移到含1 mg/l BA的琼脂培养基中,在扫描电镜下观察到球形、心形、梨形和成熟胚,两周后萌发成具根、芽的完整植株。10—14天龄的悬浮培养物具有较高的再生植株能力。     

Somatic embryogenesis and plants from zygotic embryos of coconut (Cocos nucifera L.) in vitro

Complete plants were grown from zygotic embryos cultured on Y3 basal liquid medium supplemented with coconut milk, BA and NAA. Explants from stem, leaf and rachilla of mature coconut trees turned green and swelled on Y3 semi-solid basal media supplemented with 2,4-D, K, NAA, BA and activated charcoal. Callus was initiated in explants from the subapical regions of the stem on Y3 basal medium supplemented with 2,4-D (4.52×10²M). Globular embryo-like structures were obtained when this callus was subcultured to auxinless medium. Root formation was obtained from leaf explants on Y3 basal medium containing citric acid, ascorbic acid and 2,4-D (4.52×10² M). Globular embryo-like structures were also obtained directly from leaf explants on a Y3 basal medium supplemented with 2,4-D (2.26×10² M). Callus isolated from rachilla explants on Y3 basal medium containing 2,4-D(4.52×10² M), formed nodular structures when transferred to medium with 2,4-D (2.3×10¹ M). These nodules developed roots from the base of the nodular growth whereas from the upper portion shoots were observed on Y3 basal liquid medium.Abbreviations K kinetin - BA Benzyl adenine - 2,4-D 2,4-Dichlorophenoxyacetic acid - NAA Naphthalene acetic acid - CM Coconut milk - IAA Indole acetic acid - 2iP N⁶-r-r-dimethyl allyl amino purine NCL Communication No. 3471