Delhi Scientists Genome Edit the Mustard Plant for Bland Oil But Retain Insect-Repelling Pungent Compounds in Leaves

Genome-edited mustard plants in Dr. Naveen Bisht's greenhouse. Photo courtesy Dr. Bisht.

What worth is salt if it loses its salty taste, a revered religious figure is said to have remarked two millennia ago. Traditional consumers of mustard in north and eastern India could pose the same question about the oil seed. What use is it if it loses its pungency?

Naveen Bisht

For breeders of mustard and rapeseed, which belong to the same Brassica family, the answer is not so stark. They have seen a vast market for the oilseeds when stripped of their pungency. Such oils with less than 30 micromoles (mcmol) of a compound called glucosinolates per gram of seed weight qualify as Canola quality and are preferred by those who haven’t acquired a taste for the pungent oil. High amounts of glucosinolates in rapeseed-mustard deoiled meal also repel cattle, poultry and swine. When scrubbed off the compounds, mustard oil cake can be a protein-rich substitute for soybean-based animal feed.

But glucosinolates have evolved in Brassica plants for a reason. They are their defence against predatory pests and pathogens. In their absence, insecticides must be applied, which increases the cost of cultivation. The problem is particularly acute in Europe, which in 2018 banned the use of insecticides similar to nicotine called neonicotinoids in flowering crops like rapeseed that attract bees and other pollinators.

This is where a new research development by Naveen C. Bisht and his team from the Delhi-based National Institute of Plant Genome Research (NIPGR) of the Department of Biotechnology (DBT), fits in. Using genome editing technology, they have developed mustard that has low glucosinolates in seeds but not in the rest of the plant. They homed in on two genes – GTR1 and GTR2 -that transport glucosinolates from their source in leaves and pod walls to the seeds. These genes have six similar variants each. The team has precisely edited 10 of them using an agrobacterium to deliver enzymes that act as scissors to the genetic sites where the transporter genes are located.

In the first-generation self-pollinated plants, after genome editing, glucosinolates in seeds ranged from 6.21 to 145.88 mcmol per gram of seed weight compared to an average of 146.09 mcmol in the non-edited plants, the scientists reported in an article in the Plant Biotechnology Journal. In the second generation, the compounds were as low as 3.40 mcmol in some of the mutated plants. In the third generation, the levels ranged from 17.72 mcmol to 30.46 mcmol. In 14 of the second-generation mutants, the glucosinolate level was 79.12 to 92.34 mcmol in leaves after 30 days, similar to or higher than in the non-edited plants. The glucosinolate levels in roots were also similar to those in the non-edited plants. The mutation was done on a popular mustard variety called Varuna.

Other scientists elsewhere have also achieved low glucosinolate levels in mustard seed. One group mutated four of six variants of the glucosinolate transporter gene (GTR2) using chemicals and reported a 60 percent reduction. However, this reduction was not at par with Canola quality. It also did not provide information about the concentration of the compounds in the rest of the plant, or about seed size and yield. Another group mutated three variants of the transporter gene GTR2 in rapeseed and achieved a significant reduction in seed glucosinolates. But the mutants were not agronomically viable as there was also a significant reduction of the compounds in the leaf. Seed weight and size and the composition of their fatty acids were also affected.

“Our study is very advanced,” says Bisht, whose team includes colleagues Avni Mann, Juhi Kumari, Roshan Kumar and Pawan Kumar as well as his collaborators from the Centre for Genetic Manipulation of Crop Plants (CGMCP) of Delhi University’s South Campus –  Akshay Pradhan and  Deepak Pental.

The team began work in 2017 – before the environment ministry and the Department of Biotechnology eased the regulations for genome editing last year. The relaxed guidelines allow an institute’s biosafety committee (IBSC) to move the trials of genome-edited crops from confined conditions to open fields, provided they are scrubbed clean of foreign genes that are used to insert the genetic scissors. Plants with traits from foreign species, whether genome edited or genetically modified (GM) are still subject to a rigorous process before they are approved for commercial cultivation by the Genetic Engineering Appraisal Committee (GEAC).

Bisht says the genome-edited low glucosinolates-in-seed mutants are stable and fertile, that is, they can be multiplied through self-pollination and the mutated traits will be inherited by progeny plants. The next stage is to produce enough seeds during the coming winter season. About 2.5 kg of seed are needed for all India coordinated research trials that are conducted under the aegis of the Indian Council of Agricultural Research. The seed multiplication will be done this winter. The all-India trials will be done in the winter of 2024-25. But commercial cultivation will require license from the holders of patents to genome editing CRISPR-Cas9 technology. These include a clutch of institutes like the Broad Institute of the Massachusetts Institute of Technology (MIT), Harvard University and Rockefeller University. The appropriate central government R&D agencies will have to negotiate with the patent holders for license to commercialize the genome edited products within the country.

Work is parallelly going on in reducing erucic acid levels in the genome-edited low glucosinolate mustard, Bisht says. Erucic acid is poorly ingested in the human gut, says Pental, former vice chancellor of Delhi University and founder of CGMCP. Pental’s team has developed “double low” mustard, that is mustard which has low levels of glucosinolates and erucic acid using conventional breeding. The double low mustard oil has high oleic fractions, just like olive oil. Pental’s team has also developed genetically modified mustard. Last year, the government approved the mustard hybrid, DMH-11, for commercial cultivation, India’s first GM food crop to get permission for cultivation by farmers. But commercialisation has been held back by litigation in the Supreme Court filed anti-GM activists.

India depends hugely on imported edible oil for its cooking oil requirements. Last year, it imported 14 million tonnes costing nearly $20 billion. Palm oil comprised 56 percent of the imports. Low glucosinolate mustard will not replace palm oil, because it cannot compete on price. But bland mustard oil can reduce the imports of sunflower and soybean oil, which comprised 44 percent of the imports last year.

(This report by Vivian Fernandes was first published in BQPrime)

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