Response of carrot protoplasts and protoplast-derived aggregates to selection using a fungal culture filtrate of Alternaria radicina

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.

Application of the salt stress to the protoplast cultures of the carrot (Daucus carota L.) and evaluation of the response of regenerants to soil salinity

“…Our study showed that 50 mM NaCl stress applied in vitro induced polyploidy of protoplast-derived regenerants in two out of three tested accessions. The increase in ploidy has been reported for regenerants subjected to biotic stress (Hartman et al 1984;Rutkowska-Krause et al 2003;Grzebelus et al 2013); however to the best of our knowledge this is the first example of generating polyploidy under salt stress. Research on Arabidopsis showed, that plants with higher ploidy seem to be more adaptive to the adverse conditions compare to diploids what might be a result of enrichment in genes related with response to different stress and hormonal responses observed under stress Ramirez-Parra 2014, 2015).…”

Comparison of Four Systems to Test the Tolerance of ‘Fortune’ Mandarin Tissue Cultured Plants to Alternaria alternata

“…This last result would suggest that the filtrate could contain many other unknown compounds, beyond ACT, that might be also playing positive or negative roles in the in vitro culture of ‘Fortune’, separately and/or synergistically with ACT. The fungal culture filtrate has been used by few authors for inducing resistance to some species of the Alternaria genus through tissue culture techniques [ 9 , 14 , 16 , 17 ]. The components of this filtrate were not analyzed in any of these studies; hence, the exact content of the culture media, and even the presence of mycotoxin and its concentration, were actually unknown.…”

Somaclonal Variation in Chrysanthemum × morifolium Protoplast Regenerants

“…This could result in more applications than purely the modification of ornamental properties. Grzebelus et al (2013) identified protoclonal variants with increased Alternaria tolerance after exposing carrot protoplasts to the fungus filtrate. Likewise, biotic stress exposure of Chrysanthemum protoplasts may provide a tool to enhance tolerance to particular diseases in regenerants via protoclonal variation.…”

“…Rice protoplast regenerants were aberrant in plant height, flag leaf length and width, panicle length, number of primary branches, number of spikelets per panicle and number of seeds per panicle (Lee et al, 1999). Carrot protoplasts selected with Alternaria radicina fungal culture filtrate yielded individuals with decreased susceptibility toward the pathogen (Grzebelus et al, 2013). As for ornamentals, regenerants from mesophyll and cell suspension derived Dianthus protoplasts exhibited variations like an abnormal morphology with a decreased chromosome number, precocious flowering and vigorous growth with tetraploidy (Shiba and Mii, 2005).…”