Change in nuclear DNA content and pollen size with polyploidisation in the sweet potato (Ipomoea batatas, Convolvulaceae) complex.

Genome size evolution and its relationship with pollen grain size has been investigated in sweet potato (Ipomoea batatas), an economically important crop which is closely related to diploid and tetraploid species, assessing the nuclear DNA content of 22 accessions from five Ipomoea species, ten sweet potato varieties and two outgroup taxa. Nuclear DNA amounts were determined using flow cytometry. Pollen grains were studied using scanning and transmission electron microscopy. 2C DNA content of hexaploid I. batatas ranged between 3.12-3.29 pg; the mean monoploid genome size being 0.539 pg (527 Mbp), similar to the related diploid accessions. In tetraploid species I. trifida and I. tabascana, 2C DNA content was, respectively, 2.07 and 2.03 pg. In the diploid species closely related to sweet potato e.g. I. ×leucantha, I. tiliacea, I. trifida and I. triloba, 2C DNA content was 1.01-1.12 pg. However, two diploid outgroup species, I. setosa and I. purpurea, were clearly different from the other diploid species, with 2C of 1.47-1.49 pg; they also have larger chromosomes. The I. batatas genome presents 60.0% AT bases. DNA content and ploidy level were positively correlated within this complex. In I. batatas and the more closely related species I. trifida, the genome size and ploidy levels were correlated with pollen size. Our results allow us to propose alternative or complementary hypotheses to that currently proposed for the formation of hexaploid Ipomoea batatas.

Maternal inheritance of a chloroplast microsatellite marker in controlled hybrids between Fraxinus excelsior and Fraxinus angustifolia.

Restriction fragment length polymorphism, polymerase chain reaction-restriction fragment length polymorphism and simple sequence repeat (SSR or microsatellites) analyses were performed to detect chloroplast DNA polymorphisms between two ash species, Fraxinus excelsior and F. angustifolia. Only one SSR locus was found to be polymorphic, confirming the very close relatedness of these species. Inheritance of this marker was studied in hybrids obtained from controlled crosses between the two tree species. Results indicated, for the first time in Oleaceae, that chloroplasts are maternally inherited. This chloroplast SSR marker is now used concomitantly with nuclear markers to analyse ash populations in sympatric areas.

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.

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.

Isozyme modifications and plant regeneration through somatic embryogenesis in sweet potato (Ipomoea batatas (L.) Lam.).

The potential of somatic embryogenesis was evaluated for 10 cultivars of sweet potato through extensive embryogenic response and isozyme analysis. Embryogenic callus was induced by incubating lateral buds on Murashige and Skoog medium containing 10 µM 2,4-dichlorophenoxyacetic acid for 6-8 weeks. The frequency of embryogenic response was low, and varied with genotypes, ranging from 0 to 17%. Embryo to plantlet formation could be enhanced by the use of the combination of 2,4-dichlorophenoxyacetic acid with kinetin, both used at 0.01 µM. Embryogenic callus with its potential of plantlet formation has constantly been maintained for over two years. However, after several subcultures, 0.5 to 12% of embryogenic callus reverted irreversibly into friable fast-growing non-embryogenic callus whose ability to regenerate shoots was then definitively lost. The isozymes of esterase, peroxidase, glutamate oxaloacetate transaminase and acid phosphatase investigated in this study were found appropriate to distinguish compact embryogenic from friable non-embryogenic callus in sweet potato. In fact, the callus reversion was associated with a loss of bands or a decline in isozyme activity. On the contrary, very small changes in isozyme activity or no specific changes at all were observed during the differentiation of embryogenic callus into globular embryos.

Production and characterization of fertile somatic hybrids of eggplant (Solanum melongena L.) with Solanum aethiopicum L.

In order to produce fertile somatic hybrids, mesophyll protoplasts from eggplant were electrofused with those from one of its close related species, Solanum aethiopicum L. Aculeatum group. On the basis of differences in the cultural behavior of the parental and hybrid protoplasts, 35 somatic hybrid plants were recovered from 85 selected calli. When taken to maturity either in the greenhouse or in the field, the hybrid plants were vigorous, all rapidly overtopping parental individuals. The putative hybrids were intermediate with respect to morphological traits, and all of their organs were larger, particularly the leaves and stems. DNA analysis of the hybrids using flow cytometry in combination with cytological analysis showed that 32 were tetraploids, 1 hexaploid and 2 mixoploids. The hybrid nature of the 35 selected plants was confirmed by a comparison of the isoenzyme patterns of isocitrate dehydrogenase (Idh), 6-phosphogluconate dehydrogenase (6-Pgd) and phosphoglucomutase (Pgm). Chloroplast DNA (ctDNA) restriction analysis using Bam HI revealed that among the 27 hybrid plants analyzed, 10 had S. aethiopicum patterns and the 17 remaining hybrids exhibited bands identical with those of eggplant without any changes. All of the somatic hybrid plants flowered. Both parental plants had 94% stainable pollen, while the hybrids varied widely in pollen viability ranging from 30% to 85%. The somatic hybrids showed high significant variation in fruit production. Nevertheless, there was a tendency for low fertility to be associated often with S. aethiopicum chloroplast type and/or with an abnormal ploidy level, while good fertility was mostly associated with the tetraploid level and eggplant chloroplasts. Interestingly, 2 tetraploid somatic hybrid clones were among the most productive, yielding up to 9 kg/plant. As far as the fertility of the F1 sexual counterpart was concerned, only 2 fruits of 50 g were obtained. Hybrid fertility in relation to phylogenetic affinities of the fusion partners is discussed.

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

Suspensions 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 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).

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

Somatic 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 protoplast culture of sweet potato (Ipomoea batatas Lam.).

This is the first report on successful plant regeneration from protoplasts of sweet potato. Two cultivars (Guyana and Duclos XI) of sweet potato plants propagated under in vitro conditions were used as the source of protoplasts. Green compact calli with meristematic areas were induced in the medium supplemented with 2mg1(-1) zeatin, and plant regeneration occurred when these calli were transferred onto the medium with zeatin level reduced to 0.25mg1(-1). Plant regeneration was found to be genotype-dependent, since it was only obtained for cultivar Duclos XI.