By Niranjan A. Kambi

In order to illustrate how genetically modified organisms (GMOs) are synthesised in the lab using modern genetic engineering techniques, let’s take a concrete example. What could make for a better case than GM mustard—the latest to be approved by the Genetic Engineering Appraisal Committee (GEAC) and is also at the centre of a major controversy involving GEAC, civil societies and farmers.

Let’s begin with a quick background about Indian mustard. Mustard, also referred to as Rapeseed (Mustard seed), has become one of the most vital sources of vegetable oil in the world over the last two decades. It is cultivated in over 60 countries across the world including Australia, Canada, China, France, India, and the USA.

In India, more than 90 per cent of mustard is cultivated in seven states—Assam, Gujarat, Haryana, Madhya Pradesh, Rajasthan, Uttar Pradesh and West Bengal. The predominant species is Brassica juncea which  accounts for over 90 per cent of the area under mustard cultivation.

B junco is either cultivated individually or mixed with other crops such as chickpea, wheat, lentils and sugarcane among others. It is used as a vegetable (the famous sarson ka saag is prepared using mustard leaves), condiment, fodder for cattle and as a source of oil (extracted from seeds). It is also used in Ayurvedic treatment of muscle pain, rheumatism and arthritis.

The primary justification for developing genetically modified (GM) mustard has been the development of hybrid varieties — which may show higher productivity traits as compared to self-breeding plants — to eventually wean us off foreign mustard or canola oil imports. To increase the probability of Indian mustard’s cross-breeding, researchers from the Centre for Genetic Manipulation of Crop Plants (CGMCP) and University of Delhi’s Department of Genetics have adopted a technique developed by Belgian researchers to produce male-sterile (MS) plants which could then be cross-pollinated by wind or insects.

Two genes — barnase and barstar — were isolated from a soil bacteria called Bacillus amyloliquefaciens. While barnase carries codes for a ribonuclease enzyme, a protein that destroys RNAs inside a cell, barstar is a protein that inhibits barnase.

Scientists from Belgium have figured a way to express barnase gene in a specific tissue called tapetum, inside the male reproductive organs of one of the two parental lines of tobacco and oilseed rape. The tapetum plays a vital role in nourishing the male gametophyte and the expression of barnase leads to RNA degradation and degeneration of tapetum resulting in MS plants.

They also introduced another gene called barstar which is an inhibitor of barnase and used to restore fertility in barnase-containing lines of oilseed rape thus called restorer of fertility (RF).

Scientists at CGMCP extended this approach and introduced barnase and barstar in two Indian varieties of mustard — Varuna (barnase) and EH-2 (barstar).  Therefore, the hybrid seeds of the GM mustard (DMH-11) were produced by wind or bee pollination delivering pollens from RF to MS lines.

The resulting hybrid seeds were fully fertile.

According to these scientists, the process of generating a viable barnase-barstar system which shows restored fertility in first generation progeny of B juncea was highly inefficient. In their experiments, only one of 30 cross-combinations involving three barnase lines and 14 barstar lines (consisting of 12 wild type and 2 modified barstar lines) was able to restore fertility in the next generation.

It is interesting to find out how barnase and barstar’s expression was restricted to tapetum. As we learnt earlier, a promoter is a specific DNA sequence at the start of a gene which helps in initiation of its expression. The sequence of DNA found only at the beginning of the genes expressed in tapetum of tobacco plant (Nicotiana tobacum) also referred to as tapetum-specific TA29 promoter was isolated and inserted at the start of the barnase and barstar gene or the protein coding sequence. Therefore, the TA29 promoter made sure that barnase and barstar expression was tapetum-specific.

In order to ensure that barnase and barstar genes were indeed inserted into their respective genomes in host plants, scientists added yet another gene unit — referred to as GM gene cassette — to the entire sequence. This new unit functioned as a marker giving scientists the ability to test whether the barnase or barstar was successfully incorporated into the mustard’s genome by a process called transformation (see below for details).

This gene codes for an enzyme called Phosphoinothricin acetyltransferase (PAT) derived from the fungi, Streptomyces hygroscopicus. It provides resistance to Basta (glufosinate ammonium), a herbicide which is manufactured and sold by agrochemical company, Bayer. The promoter for expressing the bar gene called CaMV 35S promoter, was taken from a virus called Cauliflower Mosaic Virus. This has been selected for its strong promoter activity or in lay terms a promoter on steroids as it robustly drives the expression of any gene that is put under its control. This feature of the promoter makes it a popular choice for driving genes in almost 80 per cent of GM plants.

In fact, when researchers from CGMCP introduced the CaMV 35S promoter to drive the bar gene, it inadvertently also expressed barnase gene (as barnase gene in their original GM cassette was also downstream of the CaMV 35S promoter and adjacent to bar gene). This “leaky” expression of barnase in multiple tissues of the mustard led to extremely low yield of MS plants. Also, the plant expressing barnase gene in all their tissues showed abnormalities in the structure of the plant tissues, poor fertility in female plants, low frequencies of seed germination along with improper segregation of transgene in the plant progenies.

Scientists ensured that barnase was not expressed indiscriminately in all plant tissues by introducing an extra sequence of DNA called the spacer sequence between CaMV 35S promoter and the TA29 promoter. The spacer sequence contained fragments of DNA representing truncated versions of two more foreign genes – topoisomerase gene from pea plant and acetolactate synthase gene from Arabidopsis, another plant. This sequence was inserted (see fig) in order prevent leaky expression of barnase from CaMV 35S promoter.

 Map of barnase gene construct.

Map of barstar gene construct.

We also need to understand the process by which this complex sequence of DNA was inserted into a mustard plant’s genome. Traditionally, the GM gene cassette is introduced into the genome of the host plant by means of two methods.

One is simply bombarding a tissue culture of plant cells with gold or tungsten nanoparticles coated with GM DNA, sort of “genetic bullets” fired into millions of plant cells in a culture. A second method involves using a bacterium that naturally infects the plant cells as carrier, which in this case is A tumafaciens.

In the case of GM mustard, the latter was used. After insertion, the transformed plant cells were selected by glufosinate application as the cells that do not take up the DNA do not survive the treatment due to absence of a bar gene. The transformed cells were then treated with plant hormones, which coax the cells to proliferate and differentiate into small GM plantlets and were then transferred to soil and allowed to grow.

Once the plants started growing, scientists weeded out all the specimens in which the desirable trait is not seen and only select those with GM phenotype or trait. This is yet another step which illustrates the inefficient and uncertain nature of this technology. The process of inserting the cassette is highly non-specific, not to mention crude and there is no control over where the insertion happens in the plant’s genome. The insertion can occur at a place of another intact gene, thus, disrupting the function of that gene like shot in the dark.

A lack of precision coupled with all the unknown and unintended effects or risks makes this method unnatural. Hence, plants with such modifications should be subject to much higher safety standards than the natural non-GM food.

How many different organisms contributed the DNA to GM Mustard?

Source: Biosafety Assessment Report for GM mustard issued by GEAC under the MoE&F

Therefore, the DNA sequences are assembled from eight unrelated organisms – three species of soil bacteria, four promoters from two plant viruses and tobacco, another flowering plant, spacer sequences from two more different plants, Pea and Arabidopsis, and finally two terminator sequences from a soil bacteria and a virus.

This is a clear demonstration of the artificiality of a GMO approach, as it is next to impossible for all these genetic elements from disparate organisms to come together into a single organism by natural mechanisms.

In this regard, there is another argument that agri-biotech companies selling GMOs and GMO-proponents advance. They allude to the natural phenomena of movement of genetic material across unrelated organisms also known as horizontal gene transfer or HGT as opposed to the vertical gene transfer from parents to offspring that is characteristic of normal breeding.

HGT has been observed mostly in between distinct species of bacteria. GMO-proponents conflate this natural HGT and gene transfer through genetic engineering and therefore, make the claim that genetic modification is just a faster method of HGT that occurs in nature and is therefore as safe as the natural varieties. However, HGT between higher organisms like plants or animals is rare and occurs only under specific conditions.

A textbook example of HGT is the presence of viral DNA fragments in the human genome called human endogenous retroviral elements (HERVs). These elements are the inherited remnants of retroviral infections that afflicted our ancestors in the past. It is suggested that these viral DNAs were inserted into the human genome in the germline DNA or the DNA of our reproductive cells. HERVs are estimated to be almost 8 per cent of the entire human genome, which almost three times the part of genome that codes for proteins.

However, some facts are in order. Firstly, virtually all HERVs are not expressed and therefore cells have active mechanisms for silencing these elements. Secondly, the extended periods of evolution and natural selection is what gives HERVs their natural and ‘safe’ status. In other words, nature had all the time to make sure that HERVs during the evolution of human genome were rendered safe. Therefore, the safety due to HERVs and other natural HGTs cannot be construed as justification for insertion of foreign pathogenic DNA in GM technology as the latter entails lack of complete knowledge on the part of geneticists, the short-term scale of its introduction and the effects of the artificial insertions of foreign DNA. And this coupled with an inadequate assessment of biosafety that are in place in most countries makes GM technology highly risky and nothing like what occurs in nature.

To summarise, genetic engineering is fundamentally different from natural breeding of plants and poses special risks which needs to be taken seriously and addressed accordingly. This is evident from the fact that a moratorium is placed on using GMOs in India and in most countries in Europe.

The author would like to thank members of Science Desk, Swetha Godavarthi for reviewing and IndSciComm for discussions on the topic.