Development of rice tolerant to heat during flowering

Abstract

High temperature stress on crops results in significant yield reduction. It also affects the grain quality which results in economic loss due to low market price. Rice is also known to be sensitive to high temperature stress particularly at anthesis stage. Temperature exceeding 38°C results in significant decrees in spikelet fertility, anther dehiscense and pollen germination on stigma. An aus type variety Nagina 22 (N22), identified as heat tolerant variety, shows higher rate of spike fertility, anther dehiscense and pollen germination on stigma under high temperature stress (39°C). Whereas heat-sensitive variety Moroberekan shows a sever impairment on those. To identify the molecular mechanism, heat treated anther and spikelet proteome form N22 were analyzed by 2D gel electrophoresis. Deferentially expressed proteins were identified from the spots by sequencing using MALDI-TOF. Using BLAST search these protein sequences were identified which includes heat shock protein, cold shock protein, dirigent like protein, pollen allergens and extensin family protein. Genes coding for the identified proteins were cloned and sequenced from N22. However, no sequence polymorphisms were identified compared to Nipponbare alleles. Gene expression analyses by semi-quantitative and quantitative PCR using RNA from pollinated stigmas confirmed induction of two heat shock protein coding genes upon heat stress. These two genes were selected for validating their role in high temperature stress tolerance by over-expression in heat-sensitive variety background IR64. These genes were cloned into a binary vector pMDC32 under constitutive CaMV 35S promoter and immature rice embryos from IR64 were transformed mediated by agrobacterium. Plants were regenerated through tissue culture. Using southern analysis T0 plants containing single copy of the transgene were selected. Both the T1 lines over-expressing CaMV::17.4kDaHSP and CaMV::16.9kDaHSP shows fairly increased amount of transgene expression compared to the endogenous gene in IR64. Upon heat stress the transgenic plants showed an increased rate of spikelet fertility (maximum 80.98% and 86.52% in 17.4 kDa HSP and 16.9 kDa respectively) than the non-transgenic background IR64 (maximum 57.57%). Whereas the spikelets that were not exposed to heat stress in T1 plants shows the spikelet fertility rate similar to that of non-transgenic IR64. Although the fertility found in the transgenic lines was not as good as N22 (>76%), it can be concluded that the transgene had improved the tolerance to heat stress. Among the two heat shock proteins, the 16.9 kDa sHSP transformed lines show better performance upon heat stress.