L. Rossignol

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.

Interspecific somatic hybridization in potato by protoplast electrofusion

Abstract Somatic hybrid plants between a dihaploid potato, BF15, and one of its wild tuber-bearing relatives, Solanum berthaultii, were produced after protoplast electrofusion with the objective of transferring insect resistance, which is associated with the presence of glandular hairs, into potato lines. Early selection of the putative hybrids was based on differences in the cultural behaviour of the parental and hybrid calli, and in morphological markers. A total of 25 somatic hybrid plants was regenerated from 70 selected calli. The regenerants exhibited strong hybrid vigour and had several morphological traits intermediate to both parents including plant habit, leaf form, and particularly short four-lobed glandular trichomes inherited from the wild parent. DNA analysis of the hybrids using flow cytometry, associated with cytological analysis revealed that 14 were tetraploids, 1 aneuploid, 8 hexaploids and 2 mixoploids. The hybrid nature of the 25 selected plants was confirmed by examining isoenzyme patterns for esterase and peroxidase. Fifteen somatic hybrids were transplanted in soil under a plastic tunnel for a preliminary evaluation of agronomic traits, particularly insect resistance through analysis of browning enzyme activity from four-lobed glandular hairs, and tuber characteristics. The selected somatic hybrids were classified into four groups according to increasing browning enzyme activity. Most somatic hybrids from group 1 were hexaploids, and exhibited a comparable level of browning enzyme activity to that of potato BF15. Their phenotype and tuber characteristics were very close to potato BF15. In groups 3 and 4 all somatic hybrids but one were tetraploids. They showed levels of browning enzyme activity as high as those from the wild species. Some of these hybrids had very high yields, but with tuber characteristics close to those of S. berthaultii. Interestingly, two hexaploid somatic hybrids from group 2 exhibited high activity of browning enzymes and high yields. These traits were associated with the expression of the cultivated phenotype including plant morphology and tuber characteristics. Most somatic hybrid plants tested set fruit with viable seeds.

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.

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.

Production and characterization of intergeneric somatic hybrids through protoplast electrofusion between potato (Solanum tuberosum) and Lycopersicon pennellii

Mesophyll protoplasts of Lycopersicon pennelli Corr., a wild relative of tomato, were electrofused with those from a dihaploid potato clone, cv Nicola, with the objectives of transferring saline tolerance from L. pennellii to cultivated potato. 150 calli were selected from the fusion experiments, finally giving 2 hybrid shoots. Their hybrid nature was verified by examining isoenzyme patterns for esterases (EST), peroxidase (PRX), phosphogluconate dehydrogenase (6-PGD), and glutamate oxaloacetate transaminase (GOT). The hybrid plants had an intermediate morphology, and grew vigorously in vitro. When transplanted to soil, they were less vigorous, due to difficulties in rooting, but were still capable of flowering, and forming short stolons and mishaped tubers, probably resulting from the effects of gene dosage due to the novel association of two genomes from a tuberizing (potato) and a non tuberizing species (L. pennellii). The characteristics of such mishaped tubers provided strong evidence of a hybrid nature for the selected plants. The hybrid plants were highly sterile, producing only 3–7% viable pollen. Tests for salt tolerance showed that the growth of the somatic hybrid plants was reduced by 50% as for L. pennellii, whilst potato did not grow at all under saline conditions.

Transfer of resistance to Verticillium dahliae Kleb. from Solanum torvum S.W. into potato (Solanum tuberosum L.) by protoplast electrofusion

SummaryInterspecific somatic hybrid plants were regenerated after electrofusion of mesophyll protoplasts with the objective of transferring resistance to Verticillium dahliae from Solanum torvum into potato. Early selection of the putative hybrids was based on differences in cultural behaviour of the parental and hybrid calli (particularly the ability of the latter to regenerate early) in combination with morphological markers. Four putative hybrids were recovered from hundreds of calli, probably resulting from complementation of the two parental genomes. The regenerates were tetraploids (2n=4×=48 chromosomes) and exhibited intermediate traits including leaf form, plant morphology and the presence of anthocyanin. The hybrid nature of the four selected plants was confirmed by examining isoenzyme patterns for isocitrate dehydrogenase (Idh), malate dehydrogenase (Mdh), phosphoglucoisomerase (Pgi) and 6-phosphogluconate dehydrogenase (6-Pgd). While the hybrid plants rooted readily and grew vigorously under in vitro conditions, in the greenhouse their development and growth were retarded by difficulties in rooting. When grafted on potato or S. torvum rootstocks, the hybrid plants recovered normal development and growth. Again, they exhibited intermediate morphological traits. Tests for resistance realized in vitro with medium containing 50% Verticillium wilt filtrate showed that all the somatic hybrids were resistant to the fungus filtrate.

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).

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.

High rate of plant regeneration from cultured protoplasts of two medicinal plants: Solanum laciniatum Ait. and Solanum khasianum C.B. Clarke

Summary Solanum laciniatum and S. khasianum plants were propagated by subculturing leafy single node cuttings. The sources of protoplasts were lamina and stems for S. laciniatum , and only lamina for S. khasianum . They were taken from 4-week old plants. Lamina gave the best yield of protoplasts. The first division of protoplasts occurred after 3–5 days of culture in KM8p + 0.2 mg · l −1 2,4-D + 0.5 mg · l −1 zeatin + 1 mg · l −1 NAA. At day 7, the division frequency was estimated at 39.0 % and 26.7 % for lamina and stem protoplasts of S. laciniatum , respectively, and 23.0 % for S. khasianum . After 20–30 days, a dilution of the cultures to 5 times with zeatin or kinetin-containing medium resulted in the formation of meristematic nodules in S. laciniatum with a frequency of 95 %, but only in an increase of callus growth in S. khasianum without any formation of organized structures, especially at a low level of cytokinin (0.1 mg · l −1 ). The nodules were composed of very small meristematic cells. They were partly covered with an epidermis and still connected with the callus. No bipolar structures were observed. The nodules produced only shoots on MS + 0.5 mg · l −1 BAP with a frequency of nearly 90 % and irrespective of protoplast sources. On MS + 2 mg · l −1 zeatin + 0.1 mg · l −1 IAA, more than 80 % of S khasianum calli regenerated buds which further developed into plantlets when transferred onto MS + 0.1 mg · l −1 BAR

Transformation of potato using mannopine and cucumopine strains of Agrobacterium rhizogenes

Mannopine and cucumopine strains of Agrobacterium rhizogenes were used for genetic transformation in two cultivars of potato (Solanum tuberosum L.). An overnight pretreatment of stem fragments with NAA prior to bacterial infection was necessary to induce root formation, otherwise very few roots were produced. Whatever the potato cultivar used, rhizogenesis induced by NAA pretreament depended on the bacterial strain. In fact, when explants from both potato cultivars were pretreated with 26.5 μM NAA, on average 84.4% and 71.9% produced roots after inoculation with the strains 2659 and 2659 GUS respectively. On the contrary, few rhizogenic responses (2.0–17.0%) or no response at all (0.0%) were obtained with the strains 15834 and 8196 GUS whatever NAA concentration used. Tests for confirming stable transformation of plant explants by examining both β-glucuronidase activity and the presence of opines showed that 85% of the selected roots were cotransformed. Most of the transformed roots were highly branched and grew rapidly, compared to non-transformed roots with no branching and poor growth. Transgenic plants were readily regenerated with a frequency reaching 80% of total explants tested for both potato cultivars.