Robert Haïcour

Electrofusion for the production of somatic hybrid plants of Solanum melongena L. and Solanum khasianum C.B. Clark

Abstract Electrofusion has successfully been used for the production of somatic hybrid plants of Solanum melongena (eggplant) and S. khasianum. This fusion was carried out in a movable multi-electrode (2 mm apart) fusion chamber (500–700 μl capacity) containing a mixture (1:1) of mesophyll protoplasts of both species. Following an alignment of protoplasts induced by an A.C. fields of 125 V/cm and 1 Mhz, fusion was initiated by an exposure of the protoplast samples to a train of 3–4 D.C. pulses of 1.2 kV/cm, each 20 μs. The fusion rate was estimated at 30–40%, at least 30% of which were binary fusions. The mixture of fused protoplasts cultured in KM8p medium containing 0.2 mg/l 2,4-D, 0.5 mg/l zeatin, 1 mg/l NAA and 6.5% (w/v) glucose produced abundant calli, some of which gave rise to shoots on regeneration medium. Although no selection methods have been used, a total of 83 somatic hybrid plants were recovered from 83 individual calli in 3 fusion experiments. They accounted for 40–50% of all the regenerated plants. Several traits of the hybrids were intermediate to those of the parents. All the hybrid plants flowered preciously. The pollen viability averaged 12%, but none of them had set fruits. A random sample of the hybrids gave chromosome numbers ranging from 46 to 48. These numbers approximated to the expected tetraploid level ( 2n = 4x = 48 chromosomes ) The hybridity was confirmed by the banding patterns ofperoxidase activities whcih were composed of the bands of both parents.

Somatic hybrid plants produced by electrofusion between Solanum melongena L. and Solanum torvum Sw

SummarySomatic hybrid plants between eggplant (Solanum melongena) and Solanum torvum have been produced by the electrofusion of mesophyll protoplasts in a movable multi-electrode fusion chamber. Using hair structure as a selection criteria, we identified a total of 19 somatic hybrids, which represented an overall average of 15.3% of the 124 regenerated plants obtained in the two fusion experiments. Several morphological traits were intermediate to those of the parents, including trichome density and structure, height, leaf form and inflorescence. Cytological analyses revealed that the chromosome numbers of the somatic hybrids approximated the expected tetraploid level (2n=4x=48). Fifteen hybrid plants were homogeneous and had relatively stable chromosome numbers (46–48), while four other hybrids had variable chromosome numbers (35–48) and exhibited greater morphological variation. The hybridity of these 19 somatic hybrid plants was confirmed by analyses of phosphoglucomutase (Pgm) and esterase zymograms.

Foreign gene delivery into monocotyledonous species

Monocotyledonous plants are generally more recalcitrant to genetic transformation than dicotyledonous species. The absence of reliable Agrobacterium-mediated transformation methods and the difficulties associated with the culture of monocotyledonous tissues in vitro are mainly responsible for this situation. Until recently, the genetic transformation of monocotyledons was essentially performed by direct transfer of DNA into regenerable protoplasts or intact cells cultured in vitro, via polyethylene glycol treatment, electroporation or particle bombardment. Since 1990, the use of particle gun technology has revolutionized the genetic engineering of monocotyledonous species, allowing transformation to be more independent of the in vitro culture requirements. Today, at least one genotype of each major monocotyledonous crop species, including cereals, can be genetically transformed.

Plant regeneration from cultured protoplasts of the cooking banana cv. Bluggoe (Musa spp., ABB group)

SummarySuspensions of embryogenic cells of a triploid banana (Musa spp., cv. Bluggoe) were initiated from the uppermost part of meristematic buds, and used as protoplast source. After 20 weeks in culture, the suspension contained a mixture of globular structures or globules and embryogenic cell clusters, as well as single cells. Two types of protoplasts were obtained from embryogenic suspension culture: small (20–30 μm) and larger (30–50 μm) protoplasts with a dense cytoplasm and large starch grains respectively. The small protoplasts probably originated from embryogenic cell clusters, and also from pseudocambial cells of globules, while larger protoplasts were probably released from oval starchy cells and those of the globule peripheral area. In co-culture with a suitable feeder, consisting of suspensions of diploid banana cells, the protoplasts of triploid banana reformed the cell wall within 24 h and underwent sustained divisions leading to the formation of small clusters of 2–3 cells within 7 days. The latter developed directly into embryos without passing through an apparent callus phase. 10% of such embryos gave rise to plantlets when subcultured in 2.2 μM 6-benzylaminopurine and 2 μM 4 amino-3,5,6-trichloropicolinic acid for 1 week, before transfer to MS medium containing 10 μM 6-benzylaminopurine. The rest of the embryos underwent intensive direct secondary embryogenesis which could lead to the formation of plantlets with a frequency of up to 50% upon further transfer to hormone-free medium.

Somatic Hybridization of Eggplant (Solanum melongena L.) with Its Close and Wild Relatives

The eggplant (Solanum melongena L.) is an economically important vegetable crop in tropical and warm temperate regions. It is grown on about 432 × 103 ha worldwide, yielding 5.7 million t in 1989, with China (2.28 million t), Turkey (0.70 million t), Japan (0.58 million t), and Egypt (0.45 million t) being the main producers (FAO 1989).

Effect of genotype, gelling agent, and auxin on the induction of somatic embryogenesis in sweet potato (Ipomoea batatas Lam.).

Lateral buds of six cultivars of sweet potato were induced to form embryogenic callus in a culture medium solidified with two types of gelling agents, Agar or Gelrite, and supplemented with various concentrations of auxins, 2,4-D, 2,4,5-T and Picloram. Of the six cultivars screened, only three gave an embryogenic response. Best results with an average of 3.53% embryogenic response were obtained with the medium solidified with Agar, while in Gelrite only 0.45% of lateral buds gave rise to embryogenic callus. The interaction between the genotype and auxins was highly significant; particularly the optimal response was obtained with cv. Zho and 865 yielding 10.7 and 14.7% somatic embryogenesis, respectively, in the medium containing 2,4,5-T or Picloram. The plant conversion was dramatically improved by subculture of the embryogenic callus on the medium with the combination of 1 microM 2,4-D and 1 microM Kinetin or 5 microM ABA alone before transfer of mature embryos onto hormone-free medium. The embryogenic callus of sweet potato and its sustained ability to further regenerate plants have regularly been maintained for several years by frequent subculture in 5 microM 2,4,5-T or the combination of 10 microM 2,4-D and 1 microM BAP or kinetin. The embryo-derived plants seemed apparently genetically stable and similar to the hexaploid parental plants, based on morphological analysis and their ploidy level determined by using flow cytometry.

Callus formation from cultured protoplasts of banana (Musa sp.)

Abstract Protoplasts were isolated from cell suspension initiated from calli of immature seeds of Musa acuminata ssp. burmannica cv Long Tavoy (AA). When co-cultured at high density with a reliable feeder culture, the protoplasts underwent sustained divisions and formed callus. Cytological studies showed that the cells of protoplast-derived calli had embryogenic characteristics as the cell suspensions used as protoplast source. Analysis of isoenzymes, in particular phosphoglucomutase (PGM) and alcohol dehydrogenase (ADH), distinguished the Lolium feeder cells from the protoplast-derived calli of banana and confirmed the Musa nature of these calli. The response of protoplasts depended upon the specificity of the feeder-physical barrier interaction. As a matter of fact, Lolium feeder induced a high rate of growth of protoplast-derived calli when combined with the use of Millipore membrane, used as a physical barrier keeping separate the protoplast culture from the feeder. Banana feeder gave similar results if nylon mesh was used as a physical barrier. The addition of dimethylsulfoxide (DMSO) to the feeder culture neither increased the proliferation of nurse cells nor affected the growth of protoplast-derived calli of banana.

Direct regeneration of transformed plants from stem fragments of potato inoculated with Agrobacterium rhizogenes

Mannopine and cucumopine strains of Agrobacterium rhizogenes were used for genetic transformation in two cultivars of potato (Solanum tuberisum L.). An overnight pretreatment of internodes with α-naphthaleneacetic acid prior to bacterial infection was found to strongly inhibit shoot formation. On the contrary, infection with bacterial strains enhanced the frequency of shoot formation, compared with the controls, except for the strain 15834 which completely inhibited shoot formation in both potato cultivars. Shoots developed directly from the upper part of both inoculated and control explants, at a frequency ranging from 1 to 5 shoots per fragment. Among 93 shoots regenerated, 9 were found to be opine positive, and exhibited an altered phenotype with shortened internodes. Histological study revealed that the transformed shoots developed directly from cells of the internode sections, and not from induced roots. When grown in an insect-proof tunnel, the transformed plants had both altered and normal phenotypes and were able to produce tubers.

In Vitro Production of Verticillium dahliae-Resistant Potato Plants

The cultivated potato (Solanum tuberosum L.) is a tetraploid (2n = 4x = 48) and is thought to have derived from a primitive diploid species, Solanum stenotomum (2n = 2x = 24). Since tetraploids are more vigorous and more productive, the other degrees of ploidy have been progressively eliminated. The outcome of crosses between tetraploid parental lines is often unpredictable. Since the parents are highly heterozygous, the Fx usually shows a large genetic variation for a number of characteristics, diminishing the chance of finding a new variety. In addition, breeding efforts may be hampered by the fact that sexual incompatibility and male sterility can also occur between and within potato parental lines.