Doubled haploid (DH) production accelerates the development of homozygous lines in a single generation. In maize, haploids are widely produced by the use of haploid inducer Stock 6, earlier reported in 1959. Three independent studies reported haploid induction in maize which is triggered due to a 4 bp frame-shift mutation in matrilineal ( ZmPLA1 ) gene. The present study was focused on the generation of mutants for ZmPLA1 gene in maize inbred line LM13 through site-directed mutagenesis via CRISPR/Cas9-mediated ribonucleoprotein (RNP) complex method to increase the haploid induction rate. Three single guide RNAs (sgRNAs) for the ZmPLA1 gene locus were used for transforming the 14 days old immature embryos via bombardment. 373 regenerated plants were subjected to mutation detection followed by Sanger’s sequencing. Out of three putative mutants identified, one mutant depicted one base pair substitution and one base pair deletion at the target site.
Background Doubled haploid technology offers the fastest route of inbred line development by rapidly fixing the desirable combinations in a single year. However, the differential response of haploid induction to genetic background of maternal lines accompanied with low induction rate and high mortality rate due to artificial chromosomal doubling of haploid seedlings creates hindrance in doubled haploid production on a commercial scale under tropical conditions. To speed up the hybrid breeding programme in sub-tropical maize, efforts are reported here to optimize the protocol for efficient production of fixed lines using haploid inducers. The second-generation haploid inducers i.e. CIM2GTAILs obtained from CIMMYT, Mexico were used for haploid induction in 13 F1s of diverse backgrounds. For standardization of chromosomal doubling protocol, various concentrations of colchicine and two seedling growth stages were used to determine the extent of chromosomal doubling and survival rate of doubled haploid plants. Results A high mean haploid induction rate is obtained from CIM2GTAIL P2 (10%) as compared to CIM2GTAIL P1 (7.46%). Out of four treatments, CIMMYT reported protocol of chromosome doubling in tropical maize comprising combination of 0.07% colchicine and 0.1% DMSO at V2 stage is highly effective for acquiring doubled haploid plants in sub-tropical adapted maize with high survival rate of 52.7%. However, increasing the colchicine concentration from 0.07 to 0.1% led to high mortality rate. Conclusion According to the findings, the haploid induction rate, survival rate and overall success rate varied depending upon the genotype of the inducer and the source population along with the concentrations of chemical used. The optimized protocol developed using CIMMYT haploid inducer CIM2GTAIL P2 for efficient doubled haploid production will not only fasten the breeding programme but will also reduce the production cost of doubled haploid with great efficiency in sub-tropical maize.
The genetic studies of ear height and its related traits were carried out by making crosses between two contrasting parents for ear height i.e, AKON40A (high ear placement) and LM23 (low ear placement) to generate F1 hybrids, which were selfed to produce F2 population and backcrossed with both parents to generate BC1.1 and BC1.2 generations. Generation mean analysis was performed on all six generations for a total of 12 morphological and phenological traits. The genetic components revealed the presence of additive gene action, additive × additive and additive × dominance gene interaction for ear height and plant height. Out of 284 SSR primer pairs, 146 primer pairs confirmed parental polymorphism, while nine primers were exhibited polymorphic in bulked segregant analysis (BSA). These nine primers were used for constructing linkage map in F2 population. Three linkage groups were formed in which LG1 spanned 26.9 cM on chromosome 3 while, LG2 and LG3 spanned 42.1 and 6.7 cM on chromosome 6 and 7, respectively. Gene governing the trait ear height was mapped on chromosome 6 between the SSR primers umc1979 and umc2317 having LOD value of ~ 3 and the phenotypic variance (R2) of 8.5. The prevalence of additive gene action for ear height suggested that recurrent selection can be utilized to improve source population for reducing ear placement by accumulating favourable alleles. Improved source population then can be utilized for inbred line development leading to maize hybrids having medium to low ear placement.
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