Related or distant species of cultivated cs are a large pool of many desirable genes. Gene transfer from these species through conventional breeding is difficult owing to post- and pre-zygotic sexual incompatibilities. Somatic hybridization via protoplast fusion is a possible alternative for gene transfer from these species to cultivated crops. Since the early days of somatic hybridization many intergeneric somatic hybrids have been developed through symmetric fusion, asymmetric fusion and microfusion. Somatic hybrids are mainly selected by using markers such as specific media or fusion parents with special features, biochemical mutants, antibiotic resistance and complementation strategy. The hybridity of the regenerants is determined based on morphological, cytological and molecular analysis. The inheritance patterns of nuclear and cytoplasmic genomes in the somatic hybrids are diverse. Nuclear DNA from both fusion parents co-exists congruously in some hybrids with translocation and rearrangement of chromosomes, but spontaneous elimination of chromosomes from either or both fusion parents has been observed very often. In asymmetric fusion, chromosome elimination is an important issue that is a complicated process influenced by many factors, such as irradiation dose, phylogenetic relatedness, ploidy level of fusion parent and regenerants. As for chloroplast genome, uniparental segregation is mainly detected, though co-existence is also reported in some cases. The mitochondrial genome, in contrast to chloroplast, undergoes recombination and very frequent rearrangements. Somatic cell fusion has potential applications for crop genetic improvement by overcoming sexual incompatibility or reproductive barriers, and by realizing novel combinations of nuclear and/or cytoplasmic genomes.

Intergeneric somatic hybridization and its application to crop genetic improvement

The subjects of this article are protoplast isolations and protoplast fusions, in particular their history, a review of factors influencing the protoplasts isolation and fusion, selection of hybrid plants and utilization of somatic hybrids in plant breeding. Somatic hybridization through protoplast fusion can overcome sexual incompatibility among plant species or genera; transfer genes of resistance to diseases (viral, bacterial, fungal), pests, herbicides and others stress factors; obtain cybrid plants; transfer cytoplasmic male sterility or incease content of secondary metabolites in hybrid plants. The article is focussed mainly on the family Brassicaceae because among representatives are significant crops for the human population. Various successful combination of intraspecific, interspecific and intergeneric protoplast fusion were reported between representatives of the family Brassicaceae with the genus Brassica which belonged to the first agricultural crops used for the isolation of protoplast.

/pdf/protoplast-cultures-and-protoplast-fusion-focused-on-3oxben7ny7.pdf

Protoplast cultures and protoplast fusion focused on Brassicaceae: A review

Somatic Hybridization and Applications in Plant Breeding

An improved protocol for Brassica protoplast culture and plant regeneration was developed. Isolated protoplasts from four-weeks-old in vitro shoot tip culture of Brassica oleracea var. botrytis cv. Siria F1 and Brassica napus doubled haploid of breeding line OP-1 were cultured at a density of 9.8-11.2 × 10 4 protoplasts/ml in darkness at 25°C in a modified medium containing 2% glucose, 0.25 mg/l 2,4-D, 1 mg/l BAP and 1 mg/l NAA. The first divisions of protoplasts were observed on the third day of culture in B. oleracea and on the fourth day in B. napus. The protoplast cultures were diluted with low osmotic medium on 7 th and 11 th day. The frequency of dividing cells was about 80% in B. oleracea and 50% in B. napus. After one month, the microcalli of approximately 0.5-1 mm in size were transferred into an induction medium with various combinations of growth regulators. Minimum duration of enzyme treatment time and extended dark period in the initial phase of culture increased the survival rate of protoplasts. Organogenesis started when the calli enlarged in size on an induction medium (1 mg/l NAA, 0.02 mg/l GA 3 , 1 mg/l 2iP) with 2% sucrose and 0.8% agar. Regeneration frequency of calli was found to be 69-75% in B. oleracea and 2-3% in B. napus. Well-developed shoots were transferred for rooting to a half-strength MS medium without growth regulators. More than 100 B. oleracea regenerants were transferred into soil, and they produced normal heads and set seeds. This very simple procedure is efficient and suitable mainly for B. oleracea var. botrytis and represents a background for fusion experiments.

/pdf/a-simple-procedure-for-mesophyll-protoplast-culture-and-v68lwl1zj5.pdf

A Simple Procedure for Mesophyll Protoplast Culture and Plant Regeneration in Brassica oleracea L. and Brassica napus L.

American ginseng (Panax quinquefolius L.) is one of the most valuable herbs in the world. Its major active components are ginsenosides. In order to produce ginsenoside heterogeneously, somatic hybridization, a novel approach for genetic introgression, was employed in this study. Protoplasts derived from respective calli of carrot (Daucus carota var. sativus Hoffm.) and American ginseng (P. quinquefolius L.) were used as the fusion partners. Hybrid calli derived from single cell lines containing chromatin of American ginseng were confirmed by the analyses of isozyme, Random amplified polymorphic DNA (RAPD) and genomic in situ hybridization (GISH). High performance liquid chromatography (HPLC) results showed that the ginseng monomer Rb1 was synthesized in seven of the hybrid calli identified as well as in the parent American ginseng calli but not in the parent carrot calli. Results indicated that hybrid introgression lines could produce ginsenoside Rb1 and the ginsenoside Rb1 biosynthesis pathway has been introgressed into carrot cells via somatic hybridization. From the point of biosafety view concerning the consumer acceptance, the potential predominance to produce ginsenosides with somatic hybridization other than with genetic transformation is discussed.

Ginsenoside Rb1 in asymmetric somatic hybrid calli of Daucus carota with Panax quinquefolius

Somatic hybridization of Brassica oleracea (cauliflower, cabbage, kohlrabi) and Armoracia rusticana (horseradish) protoplasts was performed to test the possibility of the formation of intergeneric hybrids. Out of three B. oleracea crops tested only cauliflower (B. oleracea var. botrytis) yielded viable hybrids when fused with horseradish protoplasts. Regenerated plants were asymmetric hybrids as revealed by morphological, karyological, and DNA analysis. In these plants, extensive elimination of A. rusticana genome occurred during subsequent cultivation. Using a chloroplast DNA probe no A. rusticana-specific hybridization signals were detected thus indicating a rapid elimination of horseradish cytoplasmic (chloroplast) components.

Construction of intergeneric somatic hybrids between Brassica oleracea and Armoracia rusticana

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Construction of intergeneric somatic hybrids between Brassica oleracea and Armoracia rusticana