Plantlets from mesophyll protoplasts of Solanum xanthocarpum

Young leaves of Solanum xanthocarpum from axenic shoot cultures released viable protoplasts when treated with appropriate enzymes. The protoplasts on culture in modified Murashige and Skoog (1962) medium supplemented with 2,4-dichlorophenoxy-acetic acid (0.5 mg/l), naphtha leneacetic acid (1 mg/l), kinetin (1 mg/l) and organic nutrients of KM (Kao and Michayluk 1975) regenerated to form callus tissue as a result of repeated divisions. Protoplast-derived calli differentiated into shoots on MS medium enriched with kinetin (0.5 mg/l) and rooting could be initiated by transferring the shoot-buds to basal medium.     

Production of haploid plantlets in anther cultures of Albizzia lebbeck L.

Anthers of Albizzia lebbeck on B₅ medium (BM) supplemented with kinetin (2 mg/l) and 2, 4-D (0.5 mg/l) showed callus initiation from microspores. Differentiation of embryoids and shoots was obtained on BM + BAP (1 mg/l) + IAA (0.5 mg/l) and of roots on BM. Root tip squashes of the regenerated plantlets showed the haploid chromosome number (n=13), confirming the microspore origin of the regenerants.     

Micropropagation of Cephaelis ipecacuanha rich

Shoot cultures of ipecac, Cephaelis ipecacuanha Rich. were established by inoculating seedling nodal explants onto modified Murashige and Skoog's medium supplemented with 8 mg/l kinetin, 0.05 mg/l NAA and 200 mg/l adenine. Upto 12 new axillary shoots per explant were induced after 12 weeks incubation. Shoot cultures were also established by placing shoot tips on medium containing 0.1–0.25 mg/l NAA with 8 mg/l kinetin for 4 weeks and then to shoot multiplication medium for 8 weeks. The multiplication was maintained over several passages. Shoots were rooted using 2 mg/l IBA and normal plants were re-established.     

In vitro clonal multiplication of turmeric (Curcuma spp.) and ginger (Zingiber officinale Rosc.)

Rhizome buds, excised from threeCurcuma spp., and ginger, inoculated aseptically on MS medium with varying levels of BAP and kinetin, produced multiple shoots. For shoot multiplication, a concentration of 3.0 mg/l BAP was found to be optimum for all the species.In vitro plants were successfully established in the field and were morphologically uniform. A simple method to extend the subculture interval was used and its relevance to germplasm conservation is discussed.Abbreviations BAP 6-benzylaminopurine - kinetin 6-furfurylaminopurine - MS Murashige and Skoog (1962)     

Clonal propagation of Picrorhiza kurroa royle ex benth. by shoot tip culture

A procedure has been developed for the clonal propagation of Picrorhiza kurroa Royle ex Benth. through shoot tip culture. Murashige and Skoog's medium (1962) supplemented with kinetin (3.0 to 5.0 mg/l) supported rapid proliferation of multiple shoots from the explants. Addition of indole-3-acetic acid (1.0 mg/l) to the kinetin containing medium showed marked improvement in the growth of regenerated shoots. However, presence of IAA in the medium did not alter the frequency of shoot multiplication. Rooting was readily achieved upon transferring shoots onto MS medium containing -naphthaleneacetic acid (1.0 mg/l). Plantlets were successfully transferred to soil.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kn Kinetin - MS Murashige and Skoog's (1962) medium - NAA -naphthaleneacetic acid     

High efficiency plant regeneration from cotyledons of watermelon (Citrullus vulgaris Schrad)

Cotyledons of various ages from seedlings of eight watermelon (Citrullus vulgaris) cultivars were cultured on MS medium supplemented with different combinations of phytohormones. High frequency shoot regeneration (60.0–92.0%) was induced from 5-day-old cotyledons of cultivars cultured on MS medium containing 5.0 mg/l 6-benzylaminopurine (BA) and 0.5 mg/l indole-3-acetic acid (IAA). Multiple shoot buds elongated on MS medium containing 0.2 mg/l kinetin (KT) and 5–10 shoots per expiant could be recovered depending on the cultivars. Elongated shoots rooted on MS medium with 0.1 mg/l -naphthalene acetic acid (NAA). Zeatin riboside (ZT) had a similar efficiency as BA in shoot induction, and it was significantly more functional than 2-isopentenyladenine (2iP) or kinetin (KT). Cotyledons from 5-day-old seedlings were the most responsive to shoot induction.Abbreviation BA 6-benzylaminopurine - GA₃ gibberellic acid - IAA indole-3-acetic acid - 2iP 2-isopentenyladenine - KT kinetin - MS Murashige and Skoog (1962) - NAA -naphthalene acetic acid - ZT zeatin riboside     

Propagation of Indian Rhubarh (Rheum emodi Wall.) using shoot-tip and leaf explant culture

Shoot-tip explants of Rheum emodi Wall. (Polygonaceae) gave rise to multiple shoots when cultured on a Murashige and Skoog (1962) medium (MS) with 2.0 mg/l 6-benzylaminopurine (BAP) and 1.0 mg/l indole-3-butyric acid (IBA). Also, shoot buds developed from leaf explants using MS medium with 2.0 mg/l BAP and 0.25 to 1.0 mg/l indole-3-acetic acid (IAA) or IBA. Roots were induced when the resulting shoots were placed on MS medium with 1.0 mg/l IBA. Both regeneration procedures gave rise to healthy plantlets that were established in soil under glasshouse conditions at 80% frequency after hardening phase of two weeks. Regenerated plants showed a constant chromosome number of 2n=2x=22, same as the parent plant. The use of liquid shake cultures minimized the time and culture medium requirements for propagation. This procedure can be applied for the conservation and utilization of elite clones of R. emodi.Abbreviations BAP 6-benzylaminopurine - Dd H₂O Double glass distilled water - IAA indole-3-acetic acid - IBA indole-3-butyric acid - K Kinetin - MS Murashige and Skoog's (1962) medium - RH Relative humidity CIMAP Publication No. 876     

Regeneration of plants from callus tissue of the pasture legume Centrosema brasilianum

Plants were regenerated from leaf explants of Centrosema brasilianum cultured in vitro. Callus and buds were produced on Murashige and Skoog medium (MS), 0.8% agar, 0.1 mg/l NAA and 1 mg/l BAP. Regeneration of multiple shoots was achieved by transferring callus onto fresh medium containing 0.01 and 1 mg/l of NAA and BAP, respectively. Shoots formed roots upon transfer to MS with 0.01 mg/l NAA. Plantlets were succesfully transferred to soil. Leaf-derived calli of Centrosema arenarium, C. macrocarpum, C. pascuorum, C. pubescens, and C. virginianum did not produce shoots when cultured in vitro.     

ORIGIN OF ADVENTITIOUS SHOOTS REGENERATED FROM CULTURED TOBACCO LEAF TISSUE

Leaf discs from Nicotiana tabacum L., N. glauca Grahm., and a series of interspecific periclinal chimeras were cultured on Murashige and Skoog (MS) medium containing either 2.5 mg/1 6-benzylaminopurine(BA) or 3.0 mg/16-furfurylaminopurine (kinetin). Most shoots regenerated from chimeral leaf discs were non-chimeral but 51 of 658 shoots were chimeral. The histogenic composition of plants regenerated from leaf discs of periclinal chimeras indicated that any cell layer in a leaf can be the ultimate source of shoots, and that shoots can be multicellular in origin. Certain periclinal arrangements were recovered more frequently and their chimeral nature was more stable during subsequent shoot growth. While N. tabacum leaf discs regenerated shoots on MS medium supplemented with either BA or with kinetin, N. glauca leaf discs did not form shoots on the medium containing kinetin. However, chimeras which possessed cells of both species arose on medium containing BA or kinetin, indicating that the morphogenetically competent (i.e., able to produce shoots in culture), N. tabacum cells either interacted with N. glauca cells leading to their competence or incorporated non-competent N. glauca cells into nascent shoot apical meristems.     

Morphogenesis and plant regeneration from cultured endosperm of Emblica officinalis Gaertn

Mature endosperm of Emblica Officinalis (Euphorbiaceae) formed a continously growing callus on MS medium supplemented with an auxin (2,4-D or IAA) and a cytokinin (K or BAP). Subculture of callus on MS with BAP (0.2 mg/l) and IAA (0.1 mg/l) resulted in formation of shoots and embryo-like structures in 50 and 8 per cent cultures, respectively. Regeneration of shoots was more frequent when both BAP (0.2 mg/l) and IAA (0.1 mg/l) were present than on BAP (0.2 mg/l) alone. The embryo-like structures produced plantlets.Abbreviations BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indoleacetic acid - K kinetin - NAA naphthaleneacetic acid - PDB para-dichlorobenzene (née Arora)     

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     

Somatic embryogenesis and shoot organogenesis from interspecific hybrid embryos of Vigna glabrescens and V. radiata

Somatic embryogenesis occurred on cotyledons of morphologically abnormal embryos derived from Vigna glabrescens x V. radiata crosses and cultured on Murashige and Skoog (MS) medium without growth regulators. The frequency of 15–17 day old embryos that gave rise to somatic embryos increased from 8% to 29% by application a mixture of 100 mg/l gibberelllc acid, 25 mg/l -naphthaleneacetic acid (NAA) and 5 mg/l kinetin daily to the pedicels of the developing pods. However, only callus formed on immature hybrid embryos of the reciprocal cross. These callus tissues occasionally gave rise to shoots via organogenesis when transferred to MS medium with 2 mg/l N⁶-benzyladenine and 0.05 mg/l NAA. Treatment of pods with growth regulators did not influence the frequency of organogenic callus. Selfed embryos of the parents did not form somatic embryos in culture, nor did callus derived from the selfed embryos produce shoots. Thus, the ability to redifferentiate appears to be associated with interspecific hybridity.     

Genetic transformation of NERICA,interspecific hybrid rice between <Emphasis Type="Italic">Oryza glaberrima</Emphasis> and <Emphasis Type="Italic">O. sativa</Emphasis>, mediated by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

We developed an efficient gene transfer method mediated by Agrobacterium tumefaciens for introgression of new rice for Africa (NERICA) cultivars, which are derivatives of interspecific hybrids between Oryza glaberrima Steud. and O. sativa L. Freshly isolated immature embryos were inoculated with A. tumefaciens LBA4404 that harbored binary vector pBIG-ubi::GUS or pIG121Hm, which each carried a hygromycin-resistance gene and a GUS gene. Growth medium supplemented with 500 mg/l cefotaxime and 20 mg/l hygromycin was suitable for elimination of bacteria and selection of transformed cells. Shoots regenerated from the selected cells on MS medium containing 20 g/l sucrose, 30 g/l sorbitol, 2 g/l casamino acids, 0.25 mg/l naphthaleneacetic acid, 2.5 mg/l kinetin, 250 mg/l cefotaxime, and 20 mg/l hygromycin. The shoots developed roots on hormone-free MS medium containing 30 mg/l hygromycin. Integration and expression of the transgenes were confirmed by PCR, Southern blot analysis, and histochemical GUS assay. Stable integration, expression, inheritance, and segregation of the transgenes were demonstrated by molecular and genetic analyses in the T₀ and T₁ generations. Most plants were normal in morphology and fertile. The transformation protocol produced stable transformants from 16 NERICA cultivars. We also obtained transformed plants by inoculation of calluses derived from mature seeds, but the frequency of transformation was lower and sterility was more frequent.     

Shoot regeneration and somatic embryogenesis from different explants of Brahmi [Bacopa monniera (L.) Wettst.]

The morphogenetic potential of node, internode and leaf explants of Brahmi [Bacopa monniera (L.) Wettst.] was investigated to develop reliable protocols for shoot regeneration and somatic embryogenesis. The explants were excised from shoots raised from axillary buds of nodal explants cultured on Murashige and Skoog (MS) basal medium. Presence of 6-benzylaminopurine (BA) or kinetin influenced the degree of callus formation, from which a large number of shoot buds regenerated. Leaf explants gave the largest number of shoot buds followed by node and internode explants. BA was superior to kinetin; BA at 1.5 – 2.0 mg/l appeared to be optimum for inducing the maximum number of shoot buds. MS + 0.1 mg/l BA + 0.2 mg/l indole-3-acetic acid was the most suitable for shoot elongation. Elongated shoots were rooted on full- or half-strength MS medium with or without 0.5 – 1.0 mg/l indole-3-butyric acid or 0.5 – 1.0 mg/l α-naphthaleneacetic acid. The rooted plants were successfully established in soil. Calli derived from nodal explants cultured on MS medium containing 0.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), when subcultured on MS medium containing 0.1 or 0.5 mg/l BA or 0.2 mg/l 2,4-D + 0.1 or 0.5 mg/l kinetin, developed somatic embryos. The somatic embryos germinated either on the same media or on MS basal medium, and the resulting plantlets were successfully transplanted to soil. Received: 25 September 1996 / Revision received: 23 October 1997 / Accepted: 12 November 1997     

In vitro plantlet formation by organogenesis in E. camaldulensis and by somatic embryogenesis in Eucalyptus citriodora

Adventitious shoots were formed through callus on leaf explants of Eucalyptus camaldulensis Dehnh. (River red gum) taken from shoot cultures of mature trees. Callus formed in dark on a medium containing 1 g/l casein hydrolysate, 3 mg/l 1-naphthaleneacetic acid, 0.1 mg/l 6-benzyladenine and 50 g/l sucrose. Shoot initiation occurred in 4 weeks on calli shifted to light on a regeneration medium containing 10% coconut milk, 0.5 mg/l 6-benzyladenine and 20 g/l sucrose. Rooting occured in dark on a liquid medium containing 4 mg/l 1-naphthaleneacetic acid. Zygotic embryos of Eucalyptus citriodora Hook f. (Lemon scented gum) cultured in dark on a medium containing 3 mg/l 1-naphthaleneacetic acid and 50 g/l sucrose formed somatic embryoids which grew to normal plantlets on the same regeneration medium used for organogenesis.Abbreviations BAP 6-benzyladenine - CH Casein hydrolysate - CM Coconut Milk - NAA 1-naphthaleneacetic acid NCL Communication no. 4162     

In vitro plant regeneration in lateral bud explants of Cryptanthus bromelioides Var. Tricolor M. B. Foster

Lateral buds of Cryptanthus bromelioides var. tricolor variegated ornamental bromeliad, were cultured on Murashige and Skoog's basal medium (MS) supplemented with napthaleneacetic acid, indolebutyric acid, and kinetin, each at 2 mg/l. The basal part of the cultured buds proliferated and produced adventitious shoots. The shoots developed into plants of three different phenotypes: (a) entirely green, (b) striated (green and yellowish-white) and (c) yellowish-white. Rooting could be induced in (a) and (b) types but successful transplantation was possible only with green plants some of which underwent color changes after establishment in soil.Abbreviations BA benzyladenine; 2,4-D, 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - IBA indolebutyric acid; 2,i-P, 6 -dimethylallylaminopurine; Kn, kinetin - NAA naphthaleneacetic acid, Z, zeatin     

A standard procedure for the micropropagation of the neem tree (Azadirachta indica A. Juss)

Micropropagated shoots were initiated from leaf explants of the neem tree, Azadirachta indica A. Juss. Regardless of their origin, shoots were successfully produced by culturing leaf explants on Murashige and Skoog medium containing benzylaminopurine (1 mg l^–1), kinetin (0.8 mg l^–1) and adenine sulphate (6 mg l^–1) in complete darkness. These shoots were further multiplied on Murashige and Skoog medium containing benzylaminopurine (0.1 mg l^–1), kinetin (0.08 g l^–l) and adenine sulphate (0.6 mg l^–1). Within 32 weeks, 80 shoots could be produced from a single leaf explant (10 mm×10 mm). Fifty-five percent of these shoots rooted on Murashige and Skoog medium containing indolebutyric acid (1 mg l^–1) and all of these grew on transfer to soil. Received: 5 May 1996 / Revision received: 23 August 1996 / Accepted: 5 October 1996     

Tissue,cell culture and micropropagation of Mandevilla velutina,a natural source of a bradykinin antagonist

Leaf, stem and root explants of Mandevilla velutina were cultured in vitro and produced vigorous callus in LS basal medium containing one auxin (2,4-D or NAA) plus BAP. Calli can be subcultured indefinitely with vigorous growth. Subculture of calli to NAA (1.0 mg/l) plus BAP (5.0 mg/l) caused profuse regeneration of shoots. Isolated shoots were rooted in basal medium plus NAA (5.0 mg/l) or IBA (8.0 mg/l). Rapidly growing cell suspensions can be easily obtained from friable callus cultured in liquid medium.Abbreviations LS Linsmaier & Skoog - 2,4-D 2,4 dichlorophenoxi-acetic acid - NAA -naphthalene-acetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - BAP 6-benzylaminopurine - IBA indole-3-butyric acid     

In vitro plant regeneration from leaf and cotyledon explants of Cucumis melo L.

Five different genotypes from in vitro as well as greenhouse grown melon plants were shown to be highly responsive for in vitro shoot formation from leaf explants when placed on basic MS medium supplemented with 1 mg/l 6-benzylaminopurine. In addition, a very suitable regeneration system was obtained when cotyledon pieces of mature seeds were incubated on the same culture medium. In this case, the first shoots already appeared after 10 days of incubation, and hundreds of shoots were formed on the cut surface 3 to 4 weeks later. Explants from mature cotyledons derived from seedlings did not lead to any shoot formation.Abbreviations MS Murashige and Skoog - IAA 3-indoleacetic acid - BA 6-benzylaminopurine     

Plant regeneration from hypocotyl-derived protoplasts of Brassica juncea (L.) Czern and Coss

Protoplasts isolated from etiolated hypocotyls of 6-day-old seedlings of Brassica juncea cv RLM 198 were cultured in a modified V₄₇ medium containing 7% mannitol, 2% sucrose, 1.0 mg/l 2,4-D, 0.1 mg/l NAA and 0.4 mg/l BAP, at a density of 5×10⁴ protoplasts per ml of medium. Cultures were incubated in the dark at 25+1°C. After 7 d of culture, cell colonies were diluted with 8_p medium containing 5% mannitol and a similar hormone combination as described earlier. After 14 d, cell colonies were embedded in 8_p medium containing agarose and 3.5% mannitol. Immediately upon gelling, liquid 8_p medium was added to each Petri dish as an overlayer, and cultures were incubated in the light. After a total of 3 to 4 weeks in culture, microcalli were obtained. A modified MS medium with 2% sucrose, 1.0 mg/l 2,4-D and 0.1 mg/l kinetin solidified with 0.5% agarose was used for growing microcalli into callus lines. On MS medium containing 2% sucrose, 0.1 mg/l IAA, 2.0 mg/l zeatin riboside and 2.0 mg/l BAP, solidified with 0.5% agarose, about 35% of the calli regenerated multiple shoots. The time required from culture of protoplasts to multiple shoot regeneration was about 10 weeks. Regenerated shoots were rooted and plants were re-established in a growth chamber at high frequency.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - IBA Indole-3-butyric acid