Darasinh Sihachakr

The origin and evolution of sweet potato (Ipomoea batatas Lam.) and its wild relatives through the cytogenetic approaches

The distribution and organization of 5S and 18S-5.8S-26S (18S) rDNA were studied in 10 varieties of hexaploid Ipomoea batatas, five accessions of tetraploid Ipomeoa trifida, and six related species (five diploids, I. trifida, I. triloba, I. tiliacea, I. leucantha and I. setosa and one tetraploid, I. tabascana), by using fluorescence in situ hybridization (FISH). The FISH data obtained indicated that polyploidization was followed by decrease in the number of 18S rDNA loci in higher ploidy level and provided evidence for major genomic rearrangements and/or diploidization in polyploid I. batatas. Among the five diploid species examined, I. trifida appeared to be the most closely related to I. batatas. By contrast, I. leucantha was closed to I. tiliacea, but both species were distant from sweet potato. I. triloba and I. setosa were distantly related to the rest of Ipomoea batatas complex. The close relationship between I. trifida and I. batatas was also demonstrated by the presence of one 18S and CMA marker in these two chromosome complements only. Based on chromosome morphology, tetraploid I. trifida appeared to be more closely related to sweet potato than I. tabascana. Taking all data obtained in this study, I. trifida might be the progenitor of I. batatas, and I. tabascana, interspecific hybrid between these two species.

Applications of biotechnology in eggplant

Eggplant (Solanum melongena L.), an economically important vegetable crop in many countries in Asia and Africa, often has insufficient levels of resistance to biotic and abiotic stresses. Genetic resources of eggplant have been assessed for resistance against its most serious diseases and pests (bacterial and fungal wilts, nematodes and shoot and fruit borer). Attempts at crossing eggplant with its wild relatives resulted in limited success due to sexual incompatibilities. However, the ability of eggplant to respond well in tissue culture, notably plant regeneration, has allowed the application of biotechnology, particularly the exploitation of somaclonal variation, haploidisation, somatic hybridisation and genetic transformation for gene transfer. Somaclonal variation has been used to obtain lines with increased resistance to salt and little leaf disease. Traits of resistance against bacterial and fungal wilts have successfully been introduced into the cultivated eggplant through somatic hybridisation. However, most somatic hybrids were sterile when the parental lines were distantly related. In contrast, the use of close relatives as fusion partners or highly asymmetric fusion resulted in the production of fertile hybrids with resistance traits and a morphology close to the cultivated eggplant, thus avoiding the series of backcrosses necessary for introgression of desired traits into eggplant. As far as molecular markers and genetic engineering are concerned, the information available for eggplant is very scanty. Two genetic linkage maps have been established by using RAPD and RFLP markers. In order to analyse the genetic relationships between eggplant and its relatives, some studies based on AFLP and ctDNA analyses have also been conducted. So far only resistance against insects, and parthenocarpic fruit development have successfully been developed in eggplant using Agrobacterium tumefasciens transformation. However, some work on genetic engineering of eggplant for other biotic and abiotic stresses has recently been initiated.

Biotechnology of eggplant

Eggplant (Solanum melongena L.) is an important vegetable crop grown in various tropical and temperate parts of the world. There is a wide genetic diversity in the cultivated as well as the wild species of eggplant. Cultivated varieties of eggplant are susceptible to a wide array of pests and pathogens as well as to various abiotic stress conditions. In contrast, the majority of wild species are resistant to nearly all known pests and pathogens of eggplant and thereby are a source of desirable traits for crop improvement. Tissue culture protocols for organogenesis, somatic embryogenesis, anther culture and protoplast culture have been well established for the eggplant. Somatic hybridisation has also been attempted for transferring useful genes from wild species to the cultivated plants through protoplast fusion. However, the information on genetic engineering and molecular biology of eggplant is very limited. Transgenic eggplants for insect resistance, for the production of parthenocarpic fruits and abiotic stress tolerance have been accomplished. However, transgenics of eggplant are yet to be developed for improvement of other agronomic traits, including disease and pest resistance, and quality and shelf life of fruits. Molecular markers to complement traditional breeding programs are being developed for genome mapping of agronomic traits. The present review summarises efforts to improve eggplant genetics with an emphasis on the use of biotechnology to introgress genes from wild species into cultivated eggplant.

Thaumatin gene confers resistance to fungal pathogens as well as tolerance to abiotic stresses in transgenic tobacco plants

We report here the development of transgenic tobacco plants with thaumatin gene of Thaumatococcus daniellii under the control of a strong constitutive promoter-CaMV 35S. Both polymerase chain reaction and genomic Southern analysis confirmed the integration of transgene. Transgenic plants exhibited enhanced resistance with delayed disease symptoms against fungal diseases caused by Pythium aphanidermatum and Rhizoctonia solani. The leaf extract from transgenic plants effectively inhibited the mycelial growth of these pathogenic fungi in vitro. The transgenic seeds exhibited higher germination percentage and seedling survival under salinity and PEG-mediated drought stress as compared to the untransformed controls. These observations suggest that thaumatin gene can confer tolerance to both fungal pathogens and abiotic stresses.

Androgenic dihaploids from somatic hybrids between Solanum melongena and S. aethiopicum group gilo as a source of resistance to Fusarium oxysporum f. sp. melongenae

Abstract. Dihaploid plants were obtained through anther culture of somatic hybrids between eggplant and Solanum aethiopicumgilo. The androgenic origin of the dihaploids was demonstrated by ploidy determination (flow cytometry and chloroplast counting) and isozyme and molecular (I-SSR and RAPDs) analyses. The androgenic plants showed significant morphological variability in the traits analysed. Pollen viability in the diploid androgenic plants was drastically reduced with respect to the somatic hybrids; however most of the dihaploids produced parthenocarpic fruits. S. aethiopicum and the somatic hybrids showed complete resistance to fungal wilt caused by Fusarium oxysporum f. sp. melongenae. Out of the 41 dihaploids inoculated, 34 were symptomless. The population of androgenic plants developed may represent a useful source for introgression of the Fusarium resistance trait into eggplant.

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.

Resistance to bacterial wilt in somatic hybrids between Solanum tuberosum and Solanum phureja

Somatic hybrid plants were produced after protoplast electrofusion between a dihaploid potato, cv. BF15, and a wild tuber-bearing relative, Solanum phureja, with a view to transferring bacterial wilt resistance into potato lines. A total of ten putative hybrids were selected. DNA analysis using flow cytometry revealed that six were tetraploids, two mixoploids, one amphiploid and one octoploid. In the greenhouse, the putative hybrids exhibited strong vigor and were morphologically intermediate, including leaf form, flowers and tuber characteristics. The hybrid nature of the ten selected plants was confirmed by examining isoenzyme patterns for esterases and peroxidases, and analysis of RAPD and SSR markers. Analysis of chloroplast genome revealed that eight hybrids possessed chloroplast (ct) DNA of the wild species, S. phureja, and only two contained Solanum tuberosum ct type. Six hybrid clones, including five tetraploids and one amphiploid, were evaluated for resistance to bacterial wilt by using race 1 and race 3 strains of Ralstonia solanacearum, originating from Reunion Island. Inoculations were performed by an in vitro root dipping method. The cultivated potato was susceptible to both bacterial strains tested. All somatic hybrids except two were tolerant to race 1 strain, and susceptible to race 3 strain. Interestingly, the amphiploid hybrid clone showed a good tolerance to both strains.

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.

Source of resistance against Ralstonia solanacearum in fertile somatic hybrids of eggplant (Solanum melongena L.) with Solanum aethiopicum L

Solanum aethiopicum is reported to carry resistance to bacterial wilt disease caused by Ralstonia solanacearum, which is one of the most important diseases of eggplant (Solanum melongena). These two species can sexually be crossed but the fertility of their progeny is very low. In order to transfer the resistance and improve the fertility, somatic hybrids between S. melongena cv. Dourga and two groups of S. aethiopicum were produced by electrical fusion of mesophyll protoplasts. Thirty hybrid plants were regenerated. When transferred to the greenhouse and transplanted in the field, they were vigorous and showed intermediate morphological traits. Their ploidy level was determined by DNA analysis through flow cytometry, and their hybrid nature was confirmed by examining isozymes and RAPDs patterns. Chloroplast DNA microsatellite analysis revealed that 18 hybrids had the chloroplasts of the eggplant and 12 those of the wild species. The parents and 16 hybrids were evaluated in the field for their fertility and resistance to bacterial wilt using a race 1, biovar 3 strain of R. solanacearum. All hybrids were fertile and set fruit with viable seeds. Their yield was either intermediate or as high as that of the cultivated eggplant. Both groups of S. aethiopicum were found tolerant to R. solanacearum, as about 50% of plants wilted after 8 weeks. The cultivated eggplant was susceptible with 100% of wilted plants 2 weeks after inoculation. All somatic hybrids tested were as tolerant as the wild species, except six hybrids showing a better level of resistance.

Somatic hybrid plants produced by electrofusion between dihaploid potatoes: BF15 (H1), Aminca (H6) and Cardinal (H3).

In order to regenerate somatic hybrids, mesophyll protoplasts from a dihaploid potato, BF15 (H1), were electrofused with those from two other dihaploid clones, Aminca (H6) and Cardinal (H3). Determination of the ploidy level by flow cytometry showed that 10% of plants regenerated from the fusion experiment with “BF15 + Aminca” were diploids, 14% triploids, 63% tetraploids and very few were mixoploids or had a higher ploidy level. Using morphological markers and vigour in plant growth, we were able to recover a total of 24 somatic hybrid plants, respectively 20 and 4 hybrids (accounting for 12% and 13% of regenerants) from the fusions “BF15 + Aminca” and “BF15 + Cardinal”. Most of the somatic hybrids were at the expected tetraploid level (2n=4x=48). The hybrid nature was confirmed by examining isoenzyme patterns for malate dehydrogenase (MDH) and isocitrate dehydrogenase (ICD).