Somatic embryogenesis and fertile green plant regeneration from suspension cell-derived protoplasts of rye ( Secale cereale L.)

Abstract

A method for somatic embryogenesis and fertile green plant regeneration from suspension cell-derived protoplasts of rye (Secale cereale L. cv. Auvinen) was developed. Fast-growing and friable embryogenic calli with a high regeneration capacity were induced from immature rye inflorescences using modified MS medium. These friable embryogenic calli were used for suspension culture initiation in liquid AA medium. A high yield of protoplasts was obtained from suspension cell clumps after 3-5 days of subculture. Isolated protoplasts were cultured in KM8p medium. The frequency of protoplast cell divisions and colony formations in liquid culture medium were similar to those on agarose-solidified medium. Compact embryogenic calli were developed from protoplast-derived microcalli in growth medium mMS. Approximately 7% of the transferred embryogenic calli produced green shoots on N6 regeneration medium. Of 33 green plants, 28 were fertile with normal flowering and seed set. The ratio of green and albino plantlets was 1:4. Rye protoplast-derived green plants showed normal diploid characters as determined by flow cytometer analysis and chromosome counting.

Expression analysis of somatic embryogenesis-related SERK, LEC1, VP1 and NiR ortologues in rye (Secale cereale L.)

“…Rye is known to be one of the most recalcitrant cereals with regard to its regeneration ability (Ma et al 2003), which is highly dependent on genotype (Linacero and Vasquez 1990; Rakoczy-Trojanowska and Malepszy 1993, 1995). Immature embryos are the most widely used explants and have been used successfully to regenerate rye (Ward and Jordan 2001).…”

Identification of QTLs associated with albino plant formation and some new facts concerning green versus albino ratio determinants in triticale (×Triticosecale Wittm.) anther culture

“…The biggest number of OTLs connected with AR/100A were localized in subgenome R and the positive effect originated from 'Modus' allele. It could be connected with general recalcitrance in regard to the in vitro culture response in rye (Ma et al 2003;Ma and Pulli 2004;Targońska et al 2013). Generally problems associated with rye anther and microspore culture are poor embryogenic callus induction, low green plant regeneration and high proportion of albinos (Ma and Pulli 2004).…”

The specificity and genetic background of the rye (Secale cereale L.) tissue culture response

“…Ma et al (2003) developed a method for embryogenic callus induction and fertile plant regeneration from suspension cell-derived rye protoplasts, but only 7 % of the embryogenic calli transferred to solid MS medium produced green shoots.…”

“…Biotechnological methods, e.g., double haploid production, genetic transformation or selection of plants with beneficial somaclonal changes could effectively improve the breeding of rye. However, their application is limited by a lack of an efficient procedure of plant regeneration in vitro, as rye is one of the most recalcitrant cereals in regard to in vitro plant regeneration ability (Ma et al 2003) that is, additionally, highly dependent on genotype (Linacero and Vazquez 1990; Rakoczy-Trojanowska and Malepszy 1993, 1995; Popelka and Altpeter 2001). The regeneration efficiencies from immature embryos of the best responding rye genotypes such as lines: L318, L20 or L4 are around 60.2, 59.6 and 52.9 %, respectively (Popelka and Altpeter 2001; Rakoczy-Trojanowska and Malepszy 1995), but in most of the genotypes it is not higher than a few percent.…”