Page 318 - AWSAR 2.0

294 || AWSAR Awarded Popular Science Stories - 2019
Can this male-sterility be transferred to any popular variety, so that we can improve its vigour and yield? Yes, we can. But it is usually done through traditional crossing and selection (“conventional breeding”) to transfer this character, which took and can take a minimum of 6-7 generations. One potential solution is: directly modifying the DNA of the male-sterility gene.
Scientists found that male sterility in rice is controlled by different regions of DNA including Os02g0214300 known as tms5. By studying the DNA regions of tms5 in both male-sterile wild relatives and normal plants, it was found that a simple deletion (loss of some nucleobases) in the tms5 region was responsible for this sterile male part. With the help of CRISPR/Cas9, we can artificially cut and remove few bases in tms5, so that, they would also be sterile when grown at a higher temperature and produce the seeds at lower temperatures.
We took Improved White
Ponni (shortly IWP), a fine-
grained rice variety popular in
South India. It is a high yielder
and has other desirable qualities
such as moderate resistance to
pests and diseases. However,
IWP is tall (more than 150 cm)
and takes around 140 days to
mature. This is not preferable
in the case of hybrids. So, by
mutating the IWP with gamma-
rays (energetic rays released
by radioactive elements), we
developed some shorter and
early maturing plants (~90 cm
plant height and 120 days to maturity) without any change in other characters of IWP. We named one such mutant as IWP22-2. We confirmed the mutations by reading the entire sequence of the DNA of IWP and IWP22-2 using modern sequencing technologies (Next
Generation Sequencing). Now this IWP22-2 can be modified as a “male-sterile” line.
We precisely located the region of tms5 in IWP22-2. We designed targets (called “primers”) for this region so that the Cas9 could identify and attack it. Since the rice plant does not have the Cas9 enzyme, these primers were stuck in a plasmid DNA called pRGEB32 possessing Cas9, with the help of the DNA sticking enzymes (DNA ligase). We multiplied this plasmid inside the Escherichia coli bacterium and transferred it to Agrobacterium tumifaciens. The A. tumifaciens helped us transfer this plasmid into the rice tissue. The Cas9 in the plasmid specifically had cut the targeted DNA region of the IWP22- 2 rice variety. The outcome was significant. The plants showed mutations in the region where the Cas9 attacked the DNA. This would further be evaluated by raising them inside a protected greenhouse to confirm male-sterility.
The male-sterile lines developed in such a manner can be useful in many ways. Although India has released many hybrid rice varieties, certain issues like difficulty in developing “male-sterile” lines, the inability of the male parents to fertilise the female plants and poor performance of the “male- sterile” lines are making it difficult to exploit commercially.
We believe the male- sterile lines developed with CRISPR/Cas9 could reduce these problems since it takes only 2-3 generations to develop
a complete thermosensitive male-sterile plant and can be crossed with any superior male parent to develop hybrids. And the developed varieties will lose the foreign plasmid DNA in a single generation, the plants remain ““transgene-clean” hereafter. Apart from
The entire length of DNA is chains of these four bases arranged in a specific order (the number of such ATGCs in rice is whopping ~420 Mbp, that is, 420,000,000 base pairs, which are comparatively smaller than many other organisms!) hidden deep inside the nucleus of every cell.