Rice anther culture: callus initiation and androclonal variation in progenies of regenerated plants.

Anthers from rice (Oryza sativa L.) subspecies japonica initiated more callus than their indica or indica x japonica counterparts. A mild stress, either by slow desiccation or heat shock, prior to the plating of anthers enhanced the ability to initiate callus. Slow dessication of anthers enhanced the ability of the japonica anthers to initiate callus even in medium that was supplemented with NaCl. The ability to initiate callus by the anthers plated on NaCl-supplemented medium decreased as the NaCl level in the medium increased. Among the regenerated plants 2.5% were albino and another 2% were haploid. Androclonal variation for tiller numbers, shoot height, plant dry matter and flowering were noticed in the progenies of the regenerated plants.

Plant regeneration from cell suspension cultures of Vigna aconitifolia.

Plant regeneration has been achieved routinely from established cell suspension culture lines of Vigna aconitifolia (moth bean), a highly drought tolerant grain legume. The cultures originated from three-week-old leaf callus. Several media including MS, B5, AA, SL, PCM, SH and L-6 were tested for their effects on cell growth. Maximum growth was observed in L-6 medium containing 44.5 µM 2,4-D. After 6 to 8 weeks the suspensions were filtered through 500, 250, 125 and 60 µm sieves, respectively, for four to five subcultures. An embryogenic cell line (VA-686) was obtained from the cell fraction collected below 250 µm. The VA-686 cell line is being maintained on L-6 medium with 4.5 µM 2,4-D and 2.3 µM Zeatin. Somatic embryogenesis was induced by transferring the cells to L-6 medium with 4.6 µM zeatin in which green cell clusters were produced. The somatic embryos developed from most of the cell clusters when plated on L-6 agar medium with 2.3 µM BA.Plantlets were obtained from the embryos on L-6 medium with 10.0 µM IBA. The regenerated plants were grown to maturity in the greenhouse.

Regeneration from long-term cell suspension cultures of tepary bean (Phaseolus acutifolius).

Plant regeneration has been achieved from long-term cell suspension cultures established from leaf derived callus of tepary bean (Phaseolus acutifolius). The proportion of densely cytoplasmic cells in suspension culture increased when cultured in the L-6 medium with 54 µM NAA and 2 µM KN. Filtration of the cells at each of five consecutive subcultures resulted in the isolation of a plant regenerating cell line (TB 686), which is being maintained in L-6 medium with 4.5 µM 2,4-D and 2.3 µM zeatin. Differentiated green cell aggregates were obtained when cells from maintenance medium were transferred to the same medium with 10 µM BA. Embryo-like structures developed from these aggregates on L-6 medium with 2.3 µM zeatin, 0.69 µM GA3 and 1.5 µM NAA. Plantlets regenerated from these structures when they were cultured on L-6 medium with 7.0 µM NAA and 1.0 µM KN. Plant regeneration from the cell line remained relatively constant for 270 days. Regenerated plants were grown to maturity in the greenhouse.

Somatic embryogenesis in cell cultures of Glycine species.

This report describes the development of procedures for the production of somatic embryos in cell cultures of Glycine species including soybean. The conditions for callus induction and initiation of rapidly growing cell suspension cultures were defined. Methods for inducing embryogenesis were tested on 16 lines of several Glycine species and cultivars of soybean. The SB-26 Culture of a G. soja gave the best results and was used in the experiments. Embryogenesis required the presence of picloram or 2,4-D. AMO 1618, CCC, PP-333 and Ancymidol enhanced the embryogenesis frequency. Plants of the G. soja (SB-26) were grown to maturity from seed-derived shoot tips. Characteristics of the plants are discussed.

Cell division and differentiation in protoplasts from cell cultures of Glycine species and leaf tissue of soybean.

Protoplasts were isolated from cell cultures of G. soja and G. tabacina, respectively. The isolation procedure employed Percoll for the separation and concentration of protoplasts. The cultured protoplasts formed cells which developed into embryo-like structures. Protoplasts also were isolated from leaf tissue of soybean cv. Williams 82. Upon culture, the protoplasts regenerated cell walls and divided to form cell cultures.

Chromosome stability of cell suspension cultures of Nicotiana spp.

Cell suspension cultures of Nicotiana were initiated using conditions designed to selectively favor stable chromosome number. These conditions included use of leaf explants to initiate cultures, growth of cells in culture medium containing 2,4-D, and transfer of cells with short subculture intervals. Four cell lines derived from Nicotiana tissue with 2n=24, 48, or 72 were established and retain stable chromosome number. Each line could be regenerated to recover plants that retained the somatic chromosome number during culture. Establishment of haploid and diploid cell lines with stable chromosome number is important for mutant isolation and protoplast fusion.

Fine structure of fusion products from soybean cell culture and pea leaf protoplasts.

Protoplasts from pea (Pisum sativum L.) leaves and cultured soybean (Glycine max L.) cells were fused by means of polyethylene glycol and subsequently cultured for one week. Both agglutinated protoplasts and cultured fusion products were examined by electron microscopy. Agglutination occurred over large areas of the plasma membranes. The membrane contanct was discontinuous and irregularly spaced. Many cultured fusion products regenerated cell walls and divided to form cell clusters. Fusion of pea and soybean interphase nuclei occurred in some cells. The detection of heterochromatin typical of pea in the synkaryon, even after division, suggests the cells were hybrids. The cytoplasm of the cells from the fusion products contained both soybean leucoplasts and pea chloroplasts. The chloroplasts had apparently ceased dividing and some showed signs of degenerating. Large multinucleate fusion products developed cell walls but failed to divide.

The fusion of erythrocytes by treatment with proteolytic enzymes and polyethylene glycol.

Polyethylene glycol (PEG) has been utilized to induce homokaryocyte formation in avian and mammalian erythrocytes previously treated with proteolytic enzymes. PEG of molecular weight 6,000-7,5000 was found superior to 1,500 and 20,000 MW PEG. Cells exposed to protease alone, prior to PEG treatment, fused to a high degree (60-95% multinucleated cells), whereas trypsin or pepsin treatment alone allowed very little fusion (2.5%). Trypsin lowered the effectiveness of protease when used in combination. Cells which were not treated with proteolytic enzymes agglutinated in the presence of PEG but did not fuse to a significant extent (0.01%). Fusion was also markedly dependent upon the rate at which PEG was eluted during the fusion process. Electron microscopy indicated that fusion began during the elution of PEG from the agglutinated cells.

Electron-microscope observations of mitosis and cytokinesis in multinucleate protoplasts of soybean.

Multinucleate soybean protoplasts produced by spontaneous fusion during enzyme digestion of the cell wall initiated cell division after approximately 40 h in culture. The structure of these protoplasts during mitosis and cytokinesis was studied with both light and electron microscopes. Most nuclei did not fuse but divided synchronously. Interphase nuclei was commonly connected by short narrow nuclear bridges. At prophase and metaphase the nuclei appeared typical of those in most higher plants; technical difficulties prevented an adequate examination of protoplasts at anaphase. Telophase was characterized by cytokinesis involving phragmoplast and cell plate formation; however, complete partitioning of the cytoplasm by cell plants was not observed. Numerous coated vesicles were present near to or continuous with the cell plate and plasmalemma. The presence of a few dividing protoplasts with at least double the normal chromosome number suggests that some nuclear fusion occurred prior to mitosis. Very little cell wall material was detected at the margin of the dividing protoplasts.

Plant protoplast fusion and growth of intergeneric hybrid cells.

Interspecific and intergeneric fusions of plant protoplasts were induced by polyethylene glycol (PEG) 1540 or 4000. The frequency of heterokaryocyte formation (or rate of fusion) was much higher when PEG was eluted with a high pH-high Ca(2+) solution or a salt solution than when it was eluted with a protoplast culture medium. The frequency of heterokaryocyte formation was also affected by the types of enzymes used for wall degradation, duration of enzyme incubation and molality of the PEG solutions.The maximum frequency of heterokaryocyte formation was 23% for V. hajastana Grossh.-soybean (Glycine max L.) and barley (Hordeum vulgare L.)-soybean, 35% for pea (Pisum sativum L.)-soybean, 20% for pea-V. hajastana, 14% for corn (Zea mays L.)-soybean and 10% for V. villosa Roth-V. hajastana.40% of the barley-soybean, corn-soybean and pea-soybean heterokaryocytes divided at least once. Some divided many times and formed clusters of up to 100 cells in 2 weeks. The heterokaryocytes of soybean-V. hajastana, V. villosa-V. hajastana also divided. Of the PEG-treated protoplasts of N. langsdorffii and N. glauca 13.5% developed into tumor-like calli. The morphology of these calli was very much like that of the tumors produced on amphidiploid plants of N. langsdorffii x glauca.Nuclear staining indicated that heterokaryocytes of V. hajastana-soybean, pea-soybean, corn-soybean and barley-soybean could undergo mitosis. Nuclear divisions in a heterokaryocyte were usually synchronized or almost synchronized. Nuclear fusion and true hybrid formation usually occurred during the first mitotic division after protoplast fusion. A hybrid of barley-soybean in third cell division was observed. The frequency of heterokaryocytes which underwent nuclear fusion has not been determined. Multipole formation and chimeral cell colonies were also observed.

Immunological methods for the agglutination of protoplasts from cell suspension cultures of different genera.

Protoplasts from cell suspension cultures of Vicia hajastana Grossh., soybean (Glycine max L.) and brome grass (Bromus inermis Leyss.) were tightly agglutinated by immune sera prepared against them in rabbits. After incubation, the aggregated protoplasts became adpressed over a considerable area of their surface. Antibody prepared against Vicia protoplasts agglutinated both Vicia and soybean protoplasts alone, as well as a mixture of the two. Soybean and bromegrass antibody likewise cross-reacted with and agglutinated Vicia protoplasts. The heterologous reactions were nearly as strong as, and in some cases stronger than, the homologous. When sheep anti-rabbit globulin was reacted with a mixture of the protoplasts previously coated with homologous antibody, agglutination occurred much more quickly and the aggregates could not be dispersed without physical damage. Carbol-fuchsin staining of nuclei showed that Vicia and soybean protoplasts were randomly mixed in the aggregate. The protoplasts were viable and underwent division after the antibody treatment. The immune serum, which presumably contained complement, lysed the protoplasts unless it was heat-treated prior to use.