Improved protocol for efficacious in vitro androgenesis and development of doubled haploids in temperate japonica rice

Abstract

DH (Doubled haploid) is the immortal mapping population and an outcome of single meiotic cycle, contributed from male partner. An improved procedure was developed for high frequency androgenesis in japonica genotypes, K-332 and GS-88 and their F 1 s. A total of 207 fertile, green, di-haploid plants were generated from K-332 × GS-88 hybrids using the improved anther culture protocol. The investigation was carried out to evaluate callus induction potential and regeneration response for the genotypes and the derived F 1 s on N6 media and modified N6 media (N6 M ). Whereas, N6 failed to induce callusing, agarose solidified N6 M media supplemented with 4% maltose, growth regulators; NAA (2 mg/l), 2, 4-D (0.5 mg/l), Kinetin (0.5 mg/l), and silver nitrate induced high calli percentage of 27.6% in F 1 s, 9.5% and 6.7% in GS-88 and K-332 respectively. Murashige and Skoog (MS) media supplemented with 3% sucrose, and the hormonal combination BAP (2 mg/l), Kinetin (1 mg/l) and NAA (1 mg/l) induced high green shoot regeneration rates (0–60.0%). The effect of cold pre-treatment at 4°C and the stage of anther collection and their interaction was studied. The effect of cold pre-treatment (CP) of collected boots at 4°C (for CP 2 : 2, CP 4 : 4, CP 6 : 6 and CP 8 : 8 days) at different stages of panicle emergence (BES 4-6 : 4–6, BES 7-10 : 7–10, BES 11-13 : 11–13, BES >13 : more than 13 inches was worked out in relation to the effect on response of calli induction, albino regeneration, green plant regeneration and number of shoots/green calli. CP referred to the number of days for which the collected boots were incubated before they were inoculated. BES was the length (inches) between flag leaf and penultimate leaf at the time of boot collection. We concluded that CP 6 and BES 7-10 showed better response to callus proliferation and regeneration of plantlets across genotypes. The appropriate pre-treatment, stage of anther collection and favourable media composition resulted in high calli induction and green plant regeneration rates in recalcitrant japonica genotypes. The modified N6 media resulted into efficient callus induction and is expected to be useful for studies which aim at rapid generation of mapping populations for genetic studies.

Genetic variation of doubled haploids derived from anther culture of M1 red rice plants

“…The 11 DH plants have red pericarp as their parental line. The successful rate of acclimatization was 80 % which is lower than that of the other studies such as 91.7% in F1 hybrid of Indica rice (Dewi et al 2017), 89% in F2 generation of Mediterranean Japonica rice (López-Cristofanini et al 2018), and 100% in F1 generation of temperate Japonica rice (Sakina et al 2020). The spontaneous DH plants were identified by their normal morphological appearance with more than 90% grain fertility whereas haploid plant had smaller appearance with sterile panicle (Figure 1i-1j).…”

Improvement of Anther Culture to integrate Doubled Haploid Technology in Temperate Rice (Oryza sativa L.) Breeding

“…A large quantity of calli on the N 6 NDK induction medium was produced, independent of genotype, in AC of the five tested japonica genotypes, while there have been some reports of an effect of genotype on the number of produced calli [14,25,33]. Hence, the improvement of culture conditions and media composition can mitigate genotype dependency in AC.…”

“…In indica rice, a combination of 2,4-D and 6-benzylaminopurin (BAP) was used to increase the efficiency of androgenesis induction in AC [11,12], while Mayakaduwa and Silva [13] applied a combination of 2,4-D, NAA and kinetin. A combination of NAA, BAP and kinetin was also applied for induction of androgenesis in japonica and japonica × indica crossing combinations [28,33].…”

Haploid Induction in Indica Rice: Exploring New Opportunities

“…Surface disinfected panicle boots are subjected to cold stress (at 10 • C for 7-10 days) to convert microspores from the gametophytic to the sporophytic pathway of development. Callus induction and regeneration are regulated by specific plant growth regulators and incubation conditions [10,[18][19][20][21]. The regenerated plants may be haploid, in which case they are chromosome-doubled, or they may have undergone spontaneous doubling of chromosomes in vitro to yield doubled haploids that are fully homozygous [7,22,23].…”

“…Given that cytological observations are time-consuming, easily observable morphological markers that correlate well with microspore development are used in rice anther culture to correctly identify pollen maturity prior to culture [48]. The external morphological trait that is frequently used with rice anther culture is the internode distance between the flag and the penultimate leaf of the panicle boot [18,21,[48][49][50][51]. Panicle length at the time of harvest, as well as the length ratio between anther and spikelet, have also been used as guides to determine microspore maturity [17,52,53].…”