RESUMO
Maize grain protein is deficient in two essential amino acids, lysine and tryptophan, defining it as of low nutritive value. The discovery of opaque2 (o2) gene has led to the development of quality protein maize (QPM) that has enhanced levels of essential amino acids over normal maize. However, the adoption of QPM is still very limited. The present study aims at improving the quality of normal four maize inbred lines (LM11, LM12, LM13 and LM14) of single cross hybrids; Buland (LM11 × LM12) and PMH1 (LM13 × LM14) released in India for different agro-climatic zones by introgressing o2 allele along-with modifiers using marker assisted backcross breeding. Both foreground and background selection coupled with phenotypic selection were employed for selection of o2 specific allele and maximum recovery of the recurrent parent genome (87-90%) with minimum linkage drag across the crosses. The converted QPM lines had < 25% opaqueness which is close to the respective recurrent parents. The QPM versions showed high level of tryptophan content ranging from 0.72 to 1.03 across the four crosses. The newly developed best QPM lines were crossed in original combinations to generate QPM hybrids. The grain yield of improved QPM hybrids was at par and there was significant increase in tryptophan content over the original hybrids.The integrated marker assisted, and phenotypic selection approach holds promise to tackle complex genetics of QPM. The dissemination and adoption of improved QPM versions may help to counteract protein-energy malnutrition in developing countries.
RESUMO
Alfalfa (Medicago sativa L.) is the most widely grown perennial leguminous forage and is an essential component of the livestock industry. Previously, the RNAi-mediated down-regulation of alfalfa SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 8 (MsSPL8) was found to lead to increased branching, regrowth and biomass, as well as enhanced drought tolerance. In this study, we aimed to further characterize the function of MsSPL8 in alfalfa using CRISPR/Cas9-induced mutations in this gene. We successfully generated alfalfa genotypes with small insertions/deletions (indels) at the target site in up to three of four MsSPL8 alleles in the first generation. The efficiency of editing appeared to be tightly linked to the particular gRNA used. The resulting genotypes displayed consistent morphological alterations, even with the presence of up to two wild-type MsSPL8 alleles, including reduced leaf size and early flowering. Other phenotypic effects appeared to be dependent upon mutational dosage, with those plants with the highest number of mutated MsSPL8 alleles also exhibiting significant decreases in internode length, plant height, shoot and root biomass, and root length. Furthermore, MsSPL8 mutants displayed improvements in their ability to withstand water-deficit compared to empty vector control genotypes. Taken together, our findings suggest that allelic mutational dosage can elicit phenotypic gradients in alfalfa, and discrepancies may exist in terms of MsSPL8 function between alfalfa genotypes, growth conditions, or specific alleles. In addition, our results provide the foundation for further research exploring drought tolerance mechanisms in a forage crop.