Intra-callus variability of rice doubled haploids generated through in vitro androgenesis

In vitro androgenesis is among the leading methods in creating source material for crop breeding. Many breeders a priori consider the seed progeny of any doubled haploid a new line, regardless of which callus the line was obtained from. In practice, it often turns out that in field conditions the lines from one callus are outwardly identical, so the breeders discard them, leaving two or three of ones for further work. The validity of such a controversial approach requires experimental confirmation or refutation of polymorphism and genetic variability of doubled haploids of the same callus line. About 100 genes of rice resistance to Pyricularia oryzae Cav. [ Magnaporthe grisea (Hebert Barr.)] are known of which Pi-ta и Pi-ta2 are the most relevant for the Russian Far East. This paper is the first to report intracallus morphological polymorphism and genetic variability for Pi-ta и Pi-ta2 genes due to gametoclonal and somaclonal variability of rice Oryza sativa L. doubled haploids derived from a hybrid plant via in vitro androgenesis. For the first time, a monomorphism in the absence of genetic variability due to callus cell clonal reproduction (mitotic division) was revealed among doubled haploids produced by the same callus line. Our work aimed to study the intracallus morphological and genetic variability of Oryza sativa L. doubled rice haploids derived in vitro androgenetically from a hybrid plant. Experiments were performed in 2017-2018 at the Federal Research Center of Agricultural Biotechnology of the Far East (Russia) and the Crop Resources Institute, Heilongjiang Academy of Agricultural Sciences, (China). A F1 Don 4237 ½ Dolynniy rice hybrid was used. A total of 386 anthers of this hybrid were cultured in vitro with the callus formation rate of 17.1%. Six of eighteen callus lines producing green regenerants were selected for further study. Two seeds of each doubled haploid line were sown in soil and grown in plastic pots (a culture room, 24 °С, 5000 lux, 16 hours/8 hours day/night). One plant of each pair was cut 90 days after sowing to assess the presence/absence of anthocyanin coloration. Plant growth stages (late tillering, stem extension, heading, and flowering) were recorded. Doubled haploids that reached the first two of these stages were assigned to a later group, and those that reached the heading and flowering stages were combined into an early group. The presence or absence of awns, their length and color were estimated after maturation. DNA was extracted from fresh leaves by CTAB method. The DNA quality and quantity were estimated electrophoretically using a 1.0 % agarose gel. Alleles of the Pi-ta and Pi - ta2 genes which determine blast resistance were detected by PCR method. Plants of the Chinese monogenic variety K12 were a positive control for Pi-ta gene, and varieties K27 for Pi - ta2 gene. The doubled haploids of two callus lines, 7.2.2 and 21.2.1, are monomorphic without genetic variability for both resistance genes, Pi-ta and Pi - ta2 . Plant seeds of each callus line (7.2.2 and 21.2.1) will be further combined into two separate breeding lines to intensify breeding due to a larger number of seeds. Doubled haploids of the callus lines 1.2.1, 4.1.2, 8.2.1, 36.2.3 are polymorphic in awn formation, plant growth stage and anthocyanin pigmentation of stem cuts. The callus line 8.2.1 is genetically variable (ten plants carried alleles of both resistance genes, Pi-ta and Pi - ta2 , eight plants possessed none of the alleles, and in 26 plants only Pi - ta2 allele was detected). Thus, upon callus formation via in vitro androgenesis, the two opposite processes occurred are somaclonal variation and cell cloning. Somaclonal variation leads to polymorphism of callus cells and plant-regenerants, while cell cloning determines intra-callus uniformity, as well as the uniformity of some regenerants (and, in many cases, all regeneran