Production of asymmetric somatic hybrid plants between Cichorium intybus L. and Helianthus annuus L

In order to obtain male-sterile asymmetric somatic hybrids between chicory (Cichorium intybus L.) and a sunflower (Helianthus annuus L.) male-sterile cytoplasmic line, mesophyll chicory protoplasts inactivated with iodoacetic acid and hypocotyl sunflower protoplasts irradiated with \u3b3-rays have been fused, using PEG and applying two different procedures. Thirty three plants were regenerated from putative hybrid calli. A cytological analysis of their root-tip cells indicated that most of them had 18 chromosomes, the same number as chicory. Through Southern hybridisation on total DNA using the maize mitochondrial specific gene probes Cox I, Cox II and Cob, three plants were identified as cytoplasmic asymmetric hybrids, as shown by hybridisation bands specific for both chicory and sunflower. One of the regenerated plants produced a novel pattern of hybridisation that was not detected in either parent. When hybridisation of total DNA was carried out with an atpA mitochondrial gene probe the same three cybrids presented both the fertile chicory fragment and the male-sterile sunflower fragment. Finally, Southern hybridisation with an ORF 522 probe, which in sunflower is co-transcribed with the atpA gene, confirmed the hybrid nature of the three plants. The morphology of the cybrids resembled the parental chicory phenotype, and at anthesis their anthers produced fewer pollen grains which could not germinate either \u201din vitro\u201d or \u201din situ.\u201d Cybrid plants grown in the field produced seeds when free-pollination occurre

An efficient and rapid procedure for plantlets regeneration from chicory mesophyll protoplasts

An efficient procedure for plantlet regeneration from chicory mesophyll protoplasts has been developed in order to perform protoplast fusion experiments. Protoplasts were isolated from a genotype of Italian red chicory (CH 363) and purified by centrifugation in a solution containing 13% (w/v) sucrose to collect uniform protoplasts in size. After 2 days culture at a density of 2x104 protoplasts ml-1 of liquid medium, protoplasts were cultured following three different procedures: in liquid medium, stratified in semi-solid medium, and embedded in Ca-alginate droplets. Four different media were used and culture procedures were evaluated recording the protoplast viability, protoplast division frequency and plating efficiency for each experiment. The embedding of protoplasts in Ca-alginate droplets enhanced both division frequency and plating efficiency for chicory mesophyll cells. Furthermore, this procedure shortened the cycle of plant regeneration from protoplasts, which could be completed in eight weeks

Plant regeneration from proroplasts of alfalfa (Medicago sativa) via somatic embryogenesis Regeneração de plantas a partir de protoplastos de alfafa (Medicago sativa) via embriogênese somática

Alfalfa is one of the most frequently studied species from the production of tissue culture-derived embryos point of view. In this study, five alfalfa cultivars were analyzed with reference to their ability to regenerate plants from protoplast cultures via somatic embryogenesis. Plant regeneration from leaf-derived protoplasts isolated from the cultivar Rangelander was achieved using a protocol defined for alfalfa cell suspension-derived embryogenesis. Because of its high efficiency, this procedure is recommended for protoplast electroporation-mediated genetic transformation of alfalfa.<br>A alfafa é uma das espécies mais freqüentemente estudadas do ponto de vista da produção de embriões somáticos derivados da cultura de tecidos. Neste trabalho, cinco cultivares de alfafa foram analisados com referência à capacidade de regenerar plantas a partir de culturas de protoplastos via embriogênese somática. Regeneração de plantas a partir de protoplastos isolados de folhas da cultivar Rangelander foi obtida usando-se um protocolo definido para embriogênese somática derivada de suspensões celulares de alfafa. Em função da sua alta eficiência, recomenda-se o uso deste procedimento para transformação genética de alfafa mediada por eletroporação de protoplastos