Application of cell and tissue culture and in vitro selection for disease resistance breeding
Centre for Plant Breeding and Reproduction Research (CPRO-DLO)Euphytica (Impact Factor: 1.39). 07/1991; 56(3):269-285. DOI: 10.1007/BF00042373
Somaclonal variation, i.e. the variation induced by cell and tissue culture, offers an opportunity to broaden the genetic variation of crops. As a result of somaclonal variation a wide range of plant characteristics can be altered. However, the selection of agronomically important traits, e.g. disease resistance, has many limitations. The efficiency of selection can be increased by the application of in vitro selection procedures. Selection strategies that may be applied to obtain disease resistant somaclonal variants are described. Their merits and limitations, in relation to the efficiency of the procedures, the frequency of disease resistant variants and the genetics of the resistance obtained, are discussed.
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- "In vitro culture systems are known to be a potential source of variant plants from which those with desired traits may be selected. This approach has been widely applied in the improvement of a number of agricultural species (Van den Bulk 1991; Jain 2001; Lakshmanan et al. 2005; Snyman et al. 2011). In many such studies, physical (Ali et al. 2007; Sharma et al. 2010) or chemical (Imelda et al. 2000; Shah et al. 2009) mutagens, together with in vitro culture, have been used to increase mutation frequency to obtain disease-tolerant regenerated plants, e.g. "
ABSTRACT: Eldana saccharina is a destructive pest of the sugarcane crop in South Africa. Fusarium sacchari PNG40 (a fungal strain harmful to E. saccharina) has the potential to be an endophytic biological control agent of this stalk borer. However, the fungus causes Fusarium stalk rot in sugarcane, thus limiting its usefulness. Sugarcane plants tolerant to F. sacchari PNG40 were obtained by exposing embryogenic calli (cultivar N41) to the chemical mutagen ethyl methanesulfonate (EMS), followed by in vitro selection during somatic embryogenesis and plantlet regeneration on media containing F. sacchari culture filtrates (CF). The incorporation of 100 ppm CF in the culture media at the embryo maturation stage, at germination, or at both, resulted in callus necrosis and consequent plantlet yield reduction. Subsequent trimming of the roots of regenerated plants and their exposure to 1500 ppm CF served as a further selection treatment. More plants produced from EMS-treated calli (10.2 %) displayed improved root re-growth in the presence of CF pressure than those from non-treated calli (6 %). Greenhouse tests performed by inoculating putative-tolerant plants with F. sacchari PNG40, re-isolation of Fusarium spp. from asymptomatic plants and establishment of the identity of fungal isolates as PNG40 using molecular analysis, confirmed the tolerance of the plants. Random Amplified Polymorphic DNA (RAPD) analysis of the plants revealed genetic differences between some mutants and the parent cultivar. Currently, insect bioassay trials to test the effect of PNG40 on E. saccharina using the tolerant genotypes which permit endophytic colonisation by the fungus are ongoing and results on stalk damage, survival and weights of artificially inoculated larvae will be presented. The in vitro mutagenesis and selection approach employed in the present study has potential application in generating tolerance to other diseases of sugarcane.
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- "Numerous authors pointed at advantages of using in vitro cultures over resistance testing in natural conditions: unfavorable weather and climate conditions are avoided, a small space is exploited for testing a large number of individuals, and mass screening of mutants for resistance is facilitated. It has been also suggested that in experiments involving resistance selection an increase in percentage of resistant regenerants can be expected when a preselection in in vitro conditions is applied [van den Bulk 1991, Švabova and Lebeda 2005, Sowik et al. 2008]. "
ABSTRACT: In this study in vitro root culture protocol was elaborated in order to diversify screening methods and develop quick and reliable assay to test the level of chosen Daphne genotype resistance to fungal pathogen Thielaviopsis basicola. Plantlets of Daphne caucasica, D. cneorum, D. jasminea, and D. pontica were propagated aseptically on medium composed of WPM mineral salts and MS vitamins, supplemented with 1.0 mg dm-3 2iP, 0.1 mg dm-3 NAA, 0.5 g dm-3 PVP, 0.5 g dm-3 MES, 0.65 g dm-3 calcium gluconate, and 20 g dm-3 sucrose. Root cultures were initiated from adventitious roots regenerated on micropropagated shoots. Proliferative root cultures of examined Daphne species were obtained both on solidified and liquid medium, supplemented with various doses of NAA. Cultures of detached roots proved to be a convenient system of testing to T. basicola resistance. Daphne roots cultured in vitro were distinctly contaminated under laboratory conditions. Susceptibility of cultured organs differed between species. This simple method is appropriate to be put into practice for quick selection of resistant/ tolerant genotypes to the soil-borne fungal pathogens affecting plants via root system.
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- "Other types of chromosomal mutations, e.g. chromosome breakage and chromosome loss have been documented in plants derived from in vitro cultures (Larkin and Scowcroft 1981; Van den Bulk 1991; Bairu et al. 2011). We determined the chromosome number to identify aneuploids within R0 plants. "
ABSTRACT: Protoplasts isolated from three accessions of cultivated carrot and 5-day-old protoplast-derived aggregates were subjected to selection to identify somaclonal variants with enhanced tolerance to the fungal disease black rot incited by Alternaria radicina. Different concentrations [1, 2, 3.5, 5, 10, 20, 35 and 50 % (v/v)] of a fungal culture filtrate (FCF) from 2-week-old liquid cultures of A. radicina were used. Protoplasts and aggregates were subjected to short-term selection for a period of 10 days. All FCF concentrations added to the cultures on the day of isolation decreased protoplast survival frequency and plating efficiency, while FCF applied 5 days later inhibited cell divisions in 5–50 % concentrations. The responses of protoplasts to the treatment were genotype dependent. Most R0 plants were regenerated in all accessions from cell lines grown with 1 % FCF, while only a few plants were produced from 2 to 3.5 % FCF-treated cultures of ‘Dolanka’ and the breeding line ‘9304B’, respectively. Nineteen-percent of putative stress-tolerant regenerants were tetraploids, while only 5 % tetraploids were observed in the control. The incidence of unique random amplified polymorphic DNA fragments indicating possible chromosomal rearrangements was low and did not differ among regenerants after selection and those derived from the control. Mobilization of miniature inverted repeat transposable elements was not observed. Some R0 individuals regenerated both from FCF-treated and untreated cultures showed lower susceptibility to A. radicina in a laboratory assay in comparison to control plants grown from seed. Regenerants from FCF-treated cultures showed lower frequency of flowering plants and a higher rate of male sterility. Pollen viability of the putative stress-tolerant regenerants varied over a wide range (6–98 %), independently of in vitro selection conditions. Our data suggest that A. radicina FCF may be feasible for the in vitro selection to generate plants with superior phenotypic performance against A. radicina.
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