A relationship between ploidy level and the number of chloroplasts in stomatal guard cells in diploid and amphidiploid Brassica species

Doubled haploid lines production through isolated anthers and isolated microspores cultures has been used widely for genetic studies and plant breeding of Brassica crops. The ploidy level of microspore derived plants varies, and normally haploid, diploid and mixoploid plants could be obtained in vitro. The determination of ploidy level is essential in doubled haploid pure line production. The determination of ploidy level by counting the number of chloroplast in stomatal guard cells (NCSGC) is less time consuming, laborious and expensive comparing to chromosome counting in root tip cells or mother pollen cells and flow cytometry methods. Several studies have been reported concerning relationship between ploidy level and number of chloroplast in stomatal guard cells of Brassica rapa, B. napus and B. oleracea species, however a small number of genotypes had been analyzed. In our study, the NCSGCs of haploid ( n ) and diploid (2 n ) Chinese cabbage ( B. rapa ssp. pekinensis ), winter oilseed rape ( B. napus var. napus ) and haploid, di- and tetraploid white cabbage ( B. oleracea var. capitata ) microspore derived plants were estimated, and also the influence of plant growth temperature (6±2 °Ñ è 24±2 °Ñ) and development stage (vegetative or generative) was investigated. High correlation between the ploidy level of microspore-derived plants and NCSGC is found for white cabbage ( Brassica oleracea, r = 0.94), Chinese cabbage ( B. rapa, r = 0.90) and oilseed rape ( B. napus, r = 0.94). The chloroplast average number in stomatal guard cells was very similar among the same ploidy genotypes of Chinese cabbage as well as rapeseed, while the variation of chloroplast number in diploid and tetraploid white cabbage plants was significant. In a range of early, middle and late maturing diploid white cabbage inbred lines there was established the tendency to form more chloroplasts in the early lines (Pl, Sus and others) and less in the late lines (AM2, Sa1, Ges2 and others), with the difference up to 1.7 times, that is comparable to the difference between haploid and diploid plants of Chinese cabbage or rapeseed. The chloroplast number in stomatal guard cells is 4.2-7.8 and 7.9-13.6 for Chinese cabbage ( B. rapa ) haploids and diploids, respectively, 7.5-12.4 and 14.1-20.3 for rapeseed ( B. napus ) amphihaploids and amphidiploid, respectively, and 7.7-9.9, 11.7-17.9 and 18.0-26.5 for white cabbage ( B. oleracea ) haploids, diploids and tetraploids, respectively. No significant influence of vegetative or generative stage of plant development or growth temperature on NCSGC.