Premeiotic ovular culture and candidate gene expression studies for in vitro gamete production in rice

Abstract

Developing genetic variation and recombining the traits of interest are critical factors affecting genetic gain in genetic engineering and crop breeding programmes. The essential steps in a breeding programme, namely, hybridisation and the repeated backcrossing and selection in the field, consume considerable time. Thus, the length of breeding cycles and the steps in breeding programmes delay the timely release of crop varieties. In vitro, gamete production can reduce the time required for this and overcome seasonal impacts and space limitations. The accumulating information on the well-mapped genetic markers and cross-over information, combined with the advanced techniques of in vitro gamete development and in vitro fertilisation, can help crop breeders recombine the desired allelic combinations in a shorter period. Meiosis involves DNA replication and two nuclear division events in germ cells. During meiotic division, DNA double-strand breaks (DSBs) are generated and are repaired via a homology-dependent DNA repair pathway (HDR), resulting in cross-over mediated genetic recombination (Mercier et al., 2015; Osman et al., 2011). During meiotic progression, the somatic cells get committed to the meiotic pathway in the S or G2 phases (Ito and Takegami, 1982). Under appropriate regulation of relevant genes, a somatic cell can behave like a germ cell to undergo meiosis and produce haploid cells, which has been achieved in the animal system. High auxin, temperature, and starvation are suggested to induce meiotic progression like changes in the cells cultured in vitro. Genes responsible for the transition from mitosis to meiosis have been elucidated in several plant species, namely, Arabidopsis, Maize, Rice, etc. OSH1, OsMADS13, OsSPL, OsAM1, OsMEL2, Os Cyclin D2, OsYUC4, OsPAIR1, and OsPAIR2 are some of the genes reported to be involved or regulated during this transition. Six genes among these viz OsMADS13, OsSPL, OsMEL2, OsCyclin D2, OsPAIR1 and OsPAIR2 were selected for analysis in the current study. Except for the OsCyclin D2, a mitosis-related gene, all others are critical to meiosis. When the expression pattern of these genes in the developing panicles of rice is analysed, one can get to know whether the panicle is undergoing premeiotic, meiotic, or postmeiotic development. Itoh and co-workers have reported that the premeiotic, meiotic, and postmeiotic panicles can be identified and isolated by following the length of the panicles (Itoh et al., 2004). In the present study, twenty-two different growth regulator combinations in ½ MS media were tested to support the growth of the post-meiotic ovules of Rice (Oryza sativa L. ssp japonica cv. Nipponbare), where seven among them were found to support the growth and development of the ovules. The seven media were with the following plant hormone combinations - IBA (1 mg/L), IBA + BAP (2+1 mg/L), 2,4-D (2 mg/L), BAP (4 mg/L), 2,4-D + IBA (2+1 mg/L), 2,4-D + BAP (2+1 mg/L) and 2,4-D (4 mg/L). One of the objectives of the current study was to support the premeiotic florets of rice to complete the meiotic progression in vitro and to monitor the expression of the six candidate genes. The candidate gene expression analysis in the premeiotic florets grown in vitro showed an expected gene expression pattern in the seven different media except for the gene OsSPL. Even though the expressions of the other genes varied between the different media conditions, OsMADS13, OsMEL2, OsPAIR1 and OsPAIR2 increased many folds in expression, as expected. The expression of OsCyclin D2 was either downregulated or remained constant under culture conditions. Further, callus culture was established from the scutellum of Oryza sativa L. ssp japonica cv. Nipponbare was subjected to the same culture conditions to test for the changes occurring at the expression levels of the candidate genes. Being cell division/meiosis-related genes, the candidate gene expression changes indicate the influence of culture conditions on the cell division mode. All the candidate genes were expressed in the callus tissues. The expression pattern of meiosis-related genes observed in ½ MS media with IBA + BAP (2+1 mg/L) was similar to the post-meiotic ovule, a sign of probable meiotic induction. However, the OsMADS13 expression level, even though upregulated, was low. All other media have shown an anomalous expression pattern for the OsSPL, OsMADS13, OsMEL2 and OsPAIR1 genes. The study shows that changing the culture conditions could induce meiosis-like gene expression patterns. When a suitable culture condition is given, it may be possible to cause and complete the meiotic transition in the somatic cells of rice. However, more studies are warranted to confirm the induction of meiosis and rule out any anomalous cell behaviour in the culture.

Genes responsible for the transition from mitosis to meiosis have been elucidated in several plant species, namely, Arabidopsis, Maize, Rice, etc. OSH1, OsMADS13, OsSPL, OsAM1, OsMEL2, Os Cyclin D2, OsYUC4, OsPAIR1, and OsPAIR2 are some of the genes reported to be involved or regulated during this transition. Six genes among these viz OsMADS13, OsSPL, OsMEL2, OsCyclin D2, OsPAIR1 and OsPAIR2 were selected for analysis in the current study. Except for the OsCyclin D2, a mitosis-related gene, all others are critical to meiosis. When the expression pattern of these genes in the developing panicles of rice is analysed, one can get to know whether the panicle is undergoing premeiotic, meiotic, or postmeiotic development. Itoh and co-workers have reported that the premeiotic, meiotic, and postmeiotic panicles can be identified and isolated by following the length of the panicles (Itoh et al., 2004). In the present study, twenty-two different growth regulator combinations in ½ MS media were tested to support the growth of the post-meiotic ovules of Rice (Oryza sativa L. ssp japonica cv. Nipponbare), where seven among them were found to support the growth and development of the ovules. The seven media were with the following plant hormone combinations - IBA (1 mg/L), IBA + BAP (2+1 mg/L), 2,4-D (2 mg/L), BAP (4 mg/L), 2,4-D + IBA (2+1 mg/L), 2,4-D + BAP (2+1 mg/L) and 2,4-D (4 mg/L). One of the objectives of the current study was to support the premeiotic florets of rice to complete the meiotic progression in vitro and to monitor the expression of the six candidate genes. The candidate gene expression analysis in the premeiotic florets grown in vitro showed an expected gene expression pattern in the seven different media except for the gene OsSPL. Even though the expressions of the other genes varied between the different media conditions, OsMADS13, OsMEL2, OsPAIR1 and OsPAIR2 increased many folds in expression, as expected. The expression of OsCyclin D2 was either downregulated or remained constant under culture conditions. Further, callus culture was established from the scutellum of Oryza sativa L. ssp japonica cv. Nipponbare was subjected to the same culture conditions to test for the changes occurring at the expression levels of the candidate genes. Being cell division/meiosis-related genes, the candidate gene expression changes indicate the influence of culture conditions on the cell division mode. All the candidate genes were expressed in the callus tissues. The expression pattern of meiosis-related genes observed in ½ MS media with IBA + BAP (2+1 mg/L) was similar to the post-meiotic ovule, a sign of probable meiotic induction. However, the OsMADS13 expression level, even though upregulated, was low. All other media have shown an anomalous expression pattern for the OsSPL, OsMADS13, OsMEL2 and OsPAIR1 genes. The study shows that changing the culture conditions could induce meiosis-like gene expression patterns. When a suitable culture condition is given, it may be possible to cause and complete the meiotic transition in the somatic cells of rice. However, more studies are warranted to confirm the induction of meiosis and rule out any anomalous cell behaviour in the culture.