In vitro regeneration and micro-propagation of enset from Southwestern Ethiopia

Three clones of enset (Ensete ventricosum Welw. Cheesman) from southwestern Ethiopia (Keffa-Shaka zone) were investigated for their potential for micropropagation and regeneration in tissue culture. Corm and leaf tissue of greenhouse-grown plants as well as in vitro germinated zygotic embryos were used as starting material for micro-propagation and regeneration studies. Embryos were excised from disinfected seeds and cultured in vitro. Multiple shoots from both corm- and embryo-explants were obtained using regeneration medium supplemented with 10 μM or 20 μM BAP. Rooting of shoots was achieved using medium with 5 μM IBA, 1 μM BAP and 1 g l⁻¹ activated charcoal. Plantlets obtained by this process were transferred to soil under greenhouse conditions. Optimal conditions, which were determined for clonal propagation of three different genotypes of enset, allowing both in vitro micropropagation and regeneration, are described. This protocol makes for conservation of enset clones, rapid propagation of selected disease-free germplasm and more efficient breeding procedures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Progress in plant protoplast research

During the past years plant regeneration from protoplasts was achieved for a number of important crops (maize, sorghum, rice, wheat, sugar beet). The use of embryogenic tissue for protoplast isolation greatly contributed to this success. There was also some progress in woody plant species and ornamentals. Fusion of protoplasts resulted in may fertile hybrid plants, especially in the Brassicaceae and Solanaceae. These somatic hybridization studies led to introduction of new agronomical traits from sexually incompatible species into the cultivar gene pool and to new nucleus-organelle compositions. The limitations of somatic hybridization, mainly imposed by the taxonomic distance of the parents, and expressed as chromosome loss and reduced fertility, are more clearly recognized now. Asymmetric hybridization with irradiated donor protoplasts resulted in cybrids with new cytoplasmic traits (e. g. intraspecific fusions in Brassica ), as well as in the transfer of only a few donor choromosomes (e. g. intrageneric fusions in Nicotiana ). Most intrageneric fusions, however, resulted only in a limited elimination of donor chromosomes (e. g. in Lycopersicon ), and polyploidization occurred (e. g. in Nicotiana ). Also some success on protoplast transformation was obtained in both monocots and dicots. Fertile transgenic rice plants (Japonica, Indica) were produced after direct gene transfer into protoplasts derived from embryogenic cell suspensions. Particle gun experiments using embryogenic cell suspension of maize resulted in fertile transgenic plants. Transformation of citrus and lettuce by direct gene transfer was also reported.     

Nuclear genomic composition of asymmetric fusion products between irradiated transgenic Solanum brevidens and S. tuberosum: limited elimination of donor chromosomes and polyploidization of the recipient genome

Summary The production of asymmetric somatic hybrid calli after fusion between gamma-irradiated protoplasts from transgenic Solanum brevidens and protoplasts from S. tuberosum are reported. Transgenic (kanamycin-resistant, GUS-positive) S. brevidens plants and hairy root clones were obtained after transformation with Agrobacterium tumefaciens LBA 1060 (pRi1855) (pBI121) and LBA 4404 (pRAL4404) (pBI121), and A. rhizogenes LBA 9402 (pRi1855) (pBI121), respectively. Leaf protoplasts isolated from the transgenic plants or root protoplasts from the hairy root clones were fused with S. tuberosum leaf protoplasts, and several calli were selected on kanamycin-containing medium. The relative nuclear DNA content of the hybrid calli was measured by flow cytometry (FCM), and the percentages of DNA of the S. brevidens and S. tuberosum genomes in the calli were determined by dot blot analysis using species-specific DNA probes. Chromosome-specific restriction fragment length polymorphism (RFLP) markers were used to investigate the elimination of specific S. brevidens chromosomes in the hybrids. The combined data on FCM, dot blot and RFLP analysis revealed that 18–62% of the S. brevidens DNA was eliminated in the hybrid calli and that the RFLP marker for chromosome 7 was absent in seven out of ten calli. The absence of RFLP markers for chromosomes 5 and 11 hardly ever occurred. In most of the hybrids the ploidy level of the S. tuberosum genome had increased considerably.     

Plant regeneration from mesophyll protoplasts of Solanum commersonii dun

Somatic fusion of Solanum commersonii, a frost tolerant wild potato species not crossable with Solanum tuberosum, relies on the possibility to isolate and culture protoplasts. This study was conducted to determine whether protoplasts could be isolated and plants regenerated in three S. commersonii accessions. Shoot cultures for protoplast isolation were maintained on Murashige and Skoog medium. Mesophyll protoplasts were isolated and cultured using a protocol originally described for S. tuberosum with some modifications. Differences were evident among the three accessions for protoplast yield, plating efficiency and regeneration frequency. Protoplast yield ranged from 3.0 to 8.5 × 10⁶ protoplasts per g of fresh tissue. At 1–2 × 10⁴ protoplasts ml⁻¹, which was the optimal plating density, 10–20% of plated protoplasts gave multicellular colonies. Regeneration of shoots was observed in two accessions only, the maximum regeneration frequency being 66%. In one of these accessions the reduction of sucrose concentration in regeneration media improved the regeneration frequency from 14 to 35%. About three hundred plants were rooted in vitro and successfully transferred to soil.     

Electrofusion,a simple and reproducible technique in somatic hybridization of Nicotiana plumbaginifolia mutants

Electrofusion of protoplasts from two complementary nitrate reductase deficient mutants of Nicotiana plumbaginifolia has resulted in somatic hybrid lines. Mesophyll protoplasts isolated from the cofactor mutant CNX 20 and fluorescein diacetate stained protoplasts derived from a cell suspension culture of the NA 36 line, being defective in the apoenzyme, were used in the fusion experiments. In total, 594 lines were recovered which could proliferate on a selective medium with nitrate as the sole nitrogen source. This is including 141 putative hybrid lines which were obtained after transfer of 1048 heterokaryons with a micromanipulator one day after electrofusion. The hybrid character of some of the selected lines was confirmed by nitrate reductase activity measurements. Plants were grown from hybrid calli.Abbreviations NR nitrate reductase - FDA fluorescein diacetate - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP benzylaminopurine - NAA naphthaleneacetic acid - NED N-1-naphtyl-ethylenediamide hydrochloride - PEG polyethylene glycol - AC alternating current - DC direct current     

Production of somatic hybrids between frost-tolerant Solanum commersonii and S. tuberosum: characterization of hybrid plants

Somatic fusion of mesophyll protoplasts was used to produce hybrids between the frost-tolerant species Solanum commersonii (2n=2x=24) and dihaploid S. tuberosum (2n=2x=24). This is a sexually incompatible combination due to the difference in EBN (Endosperm Balance Number, Johnston et al. 1980). Species with different EBNs as a rule are sexually incompatible. Fifty-seven hybrids were analysed for variation in chromosome number, morphological traits, fertility and frost tolerance. About 70% of the hybrids were tetraploid, and 30% hexaploid. Chloroplast counts in stomatal guard cells revealed a low frequency of cytochimeras. The frequency of aneuploids was relatively higher at the hexaploid level (hypohexaploids) than at the tetraploid level (hypotetraploids). The somatic hybrids were much more vigorous than the parents, and showed an intermediate phenotype for several morphological traits and moderate to profuse flowering. Hexaploid hybrid clones were less vigorous and had a lower degree of flowering than the tetraploid hybrid clones. All of the hybrids were female fertile but male sterile except for one, which was fully fertile and self-compatible. Many seeds were produced on the latter clone by selfing and on the male-sterile clones by crossing. The somatic hybrid plants showed an introgression of genes for frost tolerance and an adaptability to cold from S. commersonii. Therefore, the use of these somatic hybrids in breeding for and in genetic esearch on frost tolerance and cold-hardening is suggested.     

Chromosome elimination and mutation in tetraploid somatic hybrids ofSolanum tuberosum andSolanum phureja

The hybridity of eleven somatic hybrids between a diploidS. tuberosum and a diploidS. phureja clone could be verified because the parent karyotypes differed in their C-banding patterns. The hybrids were hypotetraploid and some carried structurally rearranged chromosomes and/or minute centric fragments. The nucleolar chromosomes ofS. phureja were eliminated preferentially. The function of the remaining nucleolar organizer regions was not suppressed. Nuclear DNA content was correlated with the hypotetraploid chromosome numbers.     

Elimination of Solanum phureja nucleolar chromosomes in S. tuberosum + S. phureja somatic hybrids

Summary The karyotype of the dihaploid SVP1 line of S. tuberosum (2n=2x=24) showed two nucleolar chromosomes with differently sized satellites. The diploid SVP5 line (2n=2x=24) and tetraploid regenerants of S. phureja had larger but similar satellites. Somatic hybrids between the diploid lines of these potato species with genome combinations 4 tub + 2 ph (plants 1–3), 2 tub + 4 ph (plants 4–7) and 4 tub + 4 ph (plant 8) had lost 2 phureja nucleolar chromosomes if 4 phureja genomes were present. One phureja nucleolar chromosome of plants 1–3 and both of plants 5 and 7 had rearranged satellites. Elimination of the two nucleolar chromosomes occurred preferentially, was under genetic control, and probably took place during early callus development. NOR activity resulting in rear-rangements between NORs may have caused the elimination.