Efficient isolated microspore culture protocol for callus induction and plantlet regeneration in japonica rice (Oryza sativa L.)

Abstract

Background Isolated microspore culture is a useful biotechnological technique applied in modern plant breeding programs as it can produce doubled haploid (DH) plants and accelerate the development of new varieties. Furthermore, as a single-cell culture technique, the isolated microspore culture provides an excellent platform for studying microspore embryogenesis. However, the reports on isolated microspore culture are rather limited in rice due to the low callus induction rate, poor regeneration capability, and high genotypic dependency. The present study developed an effective isolated microspore culture protocol for high-frequency androgenesis in four japonica rice genotypes. Several factors affecting the isolated microspore culture were studied to evaluate their effects on callus induction and plantlet regeneration. Results Low-temperature pre-treatment at 4 ℃ for 10–15 days could effectively promote microspore embryogenesis in japonica rice. A simple and efficient method was proposed for identifying the microspore developmental stage. The anthers in yellow-green florets located on the second type of primary branch on the rice panicle were found to be the optimal stage for isolated microspore culture. The most effective induction media for callus induction were IM2 and IM3, depending on the genotype. The optimal concentration of 2, 4-D in the medium for callus induction was 1 mg/L. Callus induction was negatively affected by a high concentration of KT over 1.5 mg/L. The differentiation medium suitable for japonica rice microspore callus comprised 1/2 MS, 2 mg/L 6-BA, 0.5 mg/L NAA, 30 g/L sucrose, and 6 g/L agar. The regeneration frequency of the four genotypes ranged from 61–211 green plantlets per 100 mg calli, with Chongxiangjing showing the highest regeneration frequency. Conclusions This study presented an efficient protocol for improved callus induction and green plantlet regeneration in japonica rice via isolated microspore culture, which could provide valuable support for rice breeding and genetic research.

Effects of the duration of a low-temperature pretreatment and hormone concentrations on anther cultures and the regeneration of awnless triticale

“…In terms of plant height, the plant height of 19 plants was between 51.77-120.30 cm, and most of the plants were higher than 90 cm, and 6, 7, 18, and 19 plants were between 110-120.30 cm, which was basically close to the plant height of mature triticale varieties. Except the number of branches of 3,8,12,14,17,30,33,34,35 and 36 was 3-9, the rest of the plants were more than 10 branches, and the traits were good. The spike length of plants 6, 7, 11, 18, 19, 30 and 34 was higher than 10 cm, while the other plants was less than 10 cm.…”

“…A pretreatment at high or low temperatures can increase the callus or embryoid body induction rate [12]. This pretreatment can signi cantly increase the rice, wheat, and pepper anther callus induction rate [13,14]. A low-temperature pretreatment can cause the developing microspores to deviate from the gametophyte developmental pathway to the sporophyte developmental pathway, leading to the production of haploid callus or embryoid bodies, which subsequently develop into plants [15].…”