Effect of Cytokinins on Shoot Regeneration and Gene Expression during Shoot Apical Meristem Transformation in Cotton

Abstract

Upland Cotton (Gossypium hirsutum L.) is the most economically important fiber and oil crop in the world, a model polyploid crop for transgenic improvement, and a high-quality source of protein around the world. Cytokinins (CKs) are one of the main regulators of the shoot apical meristem (SAM) and play an important role in the maintenance and growth of this pivotal stem cell niche. In the SAM, cytokinins and auxins interact in a negative feedback loop controlled by WUSCHEL and CLV to determine whether the cells grow laterally, vertically, proliferate, or differentiate. Cotton is a highly recalcitrant crop, which has greatly slowed down efforts to introduce beneficial genes for abiotic and biotic stress resistance, fiber length and quality, and oil production. Shoot apical meristem transformation (SAMT) is a promising in vitro method that has been used to bypass the genotype-dependence of conventional transformation methods and increase transformation efficiency in recalcitrant crops like cotton. An important step in SAMT is the shoot proliferation phase which causes the plant to produce multiple shoots that have been shown to increase the chances of obtaining a transgenic plant. Shoot proliferation is induced using different cytokinin concentrations, depending on concentration and hormone selection, the results can be drastically different. In this research thirty-one different concentrations of CK were used to determine which one is most efficient at regenerating multiple shoots in TM-1 cotton during this critical transformation phase. After six weeks on shoot multiplication medium (SMM), it was found that 3mg/L BAP was most effective for inducing shoot multiplication with an average of 5  0.4 shoots per explant. It was then tested alongside two other concentrations that performed well (2.5 & 3.5mg/L BAP) against an established SMM concentration used in SAMT. qRT-PCR was performed to evaluate what effect the elevated CK concentrations have on gene expression of key developmental regulators during SAMT and it was found that WOX11, WOX13, KNAT2, CUC2, WUS, and CLV3 were downregulated compared to the control SAMT concentration in Coker-312, while BBM was found to be elevated at 3.5mg/L BAP for all three tested genotypes.