Ravinder Gill

Plant regeneration from mesophyll protoplasts of the tree legume Pithecellobium dulce benth

Abstract Conditions were developed for the isolation, culture and regeneration of mesophyll protoplasts of the tree legume, Pithecellobium dulce Benth. The presence of 2,4-dichlorophenoxyacetic acid (2,4-D) was essential to induce initial cell divisions and addition of naphthaleneacetic acid (NAA) improved the response. Sustained division and cell colony formation were achieved from the protoplasts cultured in a modified KM8P medium containing 2,4-D (2.3 μM), NAA (3 μM) and benzyladenine (BA) (2.3 μM). Dilution of the osmotica included in the protoplast culture medium was necessary to induce sustained proliferation of the protoplast-derived cells. Differentiation of shoots from the protoplast-derived calli occurred on Murashige and Skoog (MS) medium supplemented with BA (5 μM) and indole-3-acetic acid (1 μM). Omission of 2,4-D from the culture medium, after the initial 2 weeks of protoplast culture, was obligatory to induce shoot morphogenesis.

Morphogenic Studies of Cultured Cotyledons of Urd Bean (Vigna mungo L. Hepper)

Summary Excised cotyledons (with intact cotyledonary nodes) of Vigna mungo when cultured on moist filter paper or water agar or Murashige and Skoogs basal medium, developed into single plants. Addition of cytokinins to Murashige and Skoogs basal medium stimulated the formation of multiple buds. Histological examination of the cultured cotyledon revealed that multiple buds originated from cells adjacent to the existing bud. Callus cultures initiated from cotyledonary node on medium containing picloram, zeatin and indole-acetic acid developed embryoids on transfer to agitated liquid media. However, further development of embryoids was not observed.

Removal of browning and growth enhancement by polyvinylpolypyrrolidone in protoplast cultures ofCyamopsis tetragonoloba L.

The occurrence of browning in protoplast cultures ofCyamopsis tetragonoloba completely inhibited the growth of protoplast derived colonies. Of the various additives employed to counteract the problem of browning and subsequent necrosis, polyvinylpolypyrrolidone (PVPP) was found most effective. Simultaneous addition of polyvinylpyrrolidone (PVP) to the protoplast culture medium accentuated the effect of PVPP and also improved the frequency of protoplast division.

Callus induction from protoplasts of V. unguiculata, V. sublobata and V. mungo

SummaryProtoplasts were isolated from hypocotyl of V. mungo (L.) Hepper or hypocotyl-derived callus of V. sublobata (Phaseolus sublobata Roxb.) and V. unguiculata (L.) Walp (syn. V. sinensis (L.) Saviex Hassk) using an enzyme solution comprising Cellulase 2.5%, Macerozyme, Hemicellulase and Driselase each at a 0.5% level in 0.5 M sorbitol. Isolated protoplasts were cultured in Murashige and Skoogs (1962) basal liquid medium supplemented with BA, NAA, 2,4-D (1 mg/l each) and sucrose (14%). After four weeks, protoplast colonies were transferred to the same medium with a reduced level of sucrose (7%). Colonies proliferated into actively growing calli. Further attempts to regenerate plants from such calli were not successful. However, protoclones of V. unguiculata differentiated roots on auxin/cytokinin supplemented media. Alternative methods for shoot differentiation from protoplastderived cultures were tried by the use of Agrobacterium tumefaciens “shooter” strains pGV 2215 or pGV 2298 or wild type strain B6S3.

In Vitro Manipulation of Barley and Other Cereals

Already, it can be seen and more so in the future, it is expected, that plant cell biology and molecular biology will have a major impact on agriculture by supplementing the present activities of plant breeders in expanding and diversifying the gene pool of crop species and in speeding up the breeding process. Different strategies are used which apply in vitro methods to generate diversity within existing populations, to identify rare, but desired individual plants, and to broaden the genetic pool of breeding material. Of major interest are the production of homozygous lines by anther- and micropsore-culture, the in vitro selection of cultured cells to create stress or disease resistant plants, protoplast fusion and somatic hybridization to overcome the natural barriers of incompatibility or to establish new combinations of organellar and nuclear genomes in somatic hybrid or cybrid plants, and finally the transfer of isolated genes to achieve a directed, highly defined genetic modification of a specific crop plant. Progress with cereals and grasses in the past has been rather slow when compared to species such as Nicotiana tabacum, Solanum tuberosum or Brassica napus. The field has been reviewed recently in numerous articles (Gobel and Lorz, 1988; Lorz et al., 1988; Ozias-Akins and Vasil, 1988), thus mostly recent experiments from our laboratory and new developments will be discussed.

Regeneration and Differentiation of Protoplasts in Grain Legumes

Somatic hybridization following protoplast fusion is a reliable tool to combine genotypes which are difficult to cross sexually Since the first report of sustained division and regeneration of plants from protoplasts in Nicotiana tabacum, success has been achieved in many plant families. Unfortunately, unlike other plants, regeneration of protoplasts of chief food crops have proved difficult though sporadic reports are available (Hammatt et al. 1986). Regeneration of plants from protoplasts of a few forage legumes have been achieved, but grain legumes are relatively retractable to available culture stategies. We have investigated regeneration potential of different species of the genus Vigna in order to use them for our future somatic hybridization programme.

Transformation of the grain legumeVigna aconitifolia Jacq Marechal byAgrobacterium tumefaciens, regeneration of shoots

Hypocotyl, stem, leaf and shoot-apex of one-week-old seedlings ofVigna aconitifolia were inoculated with the nopaline wild type (T37) ofAgrobacterium tumefaciens to induce tumours. Hypocotyl segments of 1 cm length fromin vitro grown plants were also co-cultivated with a bacterial suspension to obtain transformed tissue. Axenic cultures obtained from both the experiments when cultured on basal medium proliferated into callus which differentiated into shoot buds on the same medium after, repeated subcultures. The transformed nature of the shoots was confirmed by their failure to produce roots, growth on hormone-free medium and the presence of nopaline and nopaline dehydrogenase.

Tissue culture studies in mothbean — Factors influencing plant regneration from seedling explants of different cultivars

Hypocotyl and stem explants of 12 cultivars of mothbean callused and differentiated embryoids/shoot buds on basal medium devoid of growth regulators. Cotyledon and leaf explants failed to differentiate. Factors that influence differentiation of embryoids/shoot buds such as pre-conditioning of the germinating seeds with cytokinins and antiauxins, size and orientation of the explant on the medium and use of thin-layer peelings as explant were investigated. The shoot buds/embryoids developed into full fledged plantlets with roots on the same medium and were reared to maturity in field.

Callus formation and plant regeneration from protonemal protoplasts of the mossAnoectangium thomsonii Mitt.

The protoplasts of the mossAnoectangium thomsonii, isolated from protonemal cells underwent divisions in 48–72 hours on modified MS medium enriched with growth regulators, 2,4-D and kinetin, 10 % sucrose and coconut water (5 %). Subculture of protoplast derived cells in a medium of relatively low osmotic potential (5 % sucrose) produced dark green calli which could be maintained completely undifferentiated.