Background Autotetraploid rice is a useful germplasm for the breeding of polyploid rice; however, low fertility is a major hindrance for its utilization. Neo-tetraploid rice with high fertility was developed from the crossing of different autotetraploid rice lines. Our previous research showed that the mutant (ny1) of LOC_Os07g32406 (NY1), which was generated by CRISPR/Cas9 knock-out in neo-tetraploid rice, showed low pollen fertility, low seed set, and defective chromosome behavior during meiosis. However, the molecular genetic mechanism underlying the fertility remains largely unknown. Results Here, cytological observations of the NY1 mutant (ny1) indicated that ny1 exhibited abnormal tapetum and middle layer development. RNA-seq analysis displayed a total of 5606 differentially expressed genes (DEGs) in ny1 compared to wild type (H1) during meiosis, of which 2977 were up-regulated and 2629 were down-regulated. Among the down-regulated genes, 16 important genes associated with tapetal development were detected, including EAT1, CYP703A3, CYP704B2, DPW, PTC1, OsABCG26, OsAGO2, SAW1, OsPKS1, OsPKS2, and OsTKPR1. The mutant of EAT1 was generated by CRISPR/Cas9 that showed abnormal tapetum and pollen wall formation, which was similar to ny1. Moreover, 478 meiosis-related genes displayed down-regulation at same stage, including 9 important meiosis-related genes, such as OsREC8, OsSHOC1, SMC1, SMC6a and DCM1, and their expression levels were validated by qRT-PCR. Conclusions Taken together, these results will aid in identifying the key genes associated with pollen fertility, which offered insights into the molecular mechanism underlying pollen development in tetraploid rice.

Oryza alta Swallen is an important germplasm for rice resistance breeding; however, its CCDD genome (2n = 48) resulted in low crossability when the wild rice was crossed with O. sativa and restricted the success of transferring the desirable traits into cultivated rice. Induction of polyploidy is an efficient way for overcoming the low crossability among different species. A new O. alta line, Huaye 5, was developed by our group in 2016, which had high fertility (64.93%) and photoperiod-insensitive. Huaye 5 was used to induce auto-allotetraploidy using tissue culture in the present study. The tissue culture system was established by comparing five basic media (N6, B5, MS, NB and MB), two hormones (2,4-D and 6-BA) for induction and two differentiation media (MS and NB), and then induced auto-allotetraploid in the wild rice line by colchicine. The medium and hormone combinations of NB + 2,4-D (2.5 mg/L) + 6-BA (1.0 mg/L) produced the induction rate of 20%, and MS medium was found to be a suitable medium for callus induction with a differentiation rate of 10.15%, and the treatment of 600 mg/L colchicine for 24 h was the best protocol for inducing auto-allotetraploid. Subsequently, auto-allotetraploid plants (2n = 96) were obtained in the present study and their ploidy levels were detected by using flow cytometry, stomata size and chromosomes count methods. Many inclusions in the parenchyma cells surrounding vascular bundle were observed in auto-allotetraploid rice compared to the parent. We developed a new germplasm from O. alta, and established a protocol of in vitro induction of auto-allotetraploid, which can be used for crossing with autotetraploid rice. Key messageWe have successfully established a protocol for the in vitro induction of auto-allotetraploid in Oryza alta, which can be used to cross with autotetraploid or neo-tetraploid rice.Communicated by Manoj Prasad.

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