Biotechnology : To improve the breeding process

Marker Assisted Selection

Genetic Modification, particularly transgenes, is a very unpredictable and dangerous process. There is strong opposition because ethical and health  issues are involved. Scientists themselves admit it is not very accurate. That is why a new system is being advocated, called Marker Assisted Selection (MAS). This technique does not involve insertion of genetic material but it aims to identify the gene responsible for a particular trait.

However, this method too can be viewed with suspicion. In an attempt to clear these doubts we interviewed eminent breeder scientist Dr. Debal Deb. He is a freelance scientist with post-doctoral research in human ecology (Indian Institute of Science, Bangalore) and ecological economics (University of California, Berkeley). A series of questions about existing apprehensions was put to him. Here we have documented his response.

Jagannath:    There are two varieties that are being introduced – one is a flood-resistant variety which is called “Swarna Sub-1’ which has already been given to the farmers and the other is a drought-resistant variety called ‘Sahabhagi’ which is being tested in Chhattisgarh. They say that this is the result of marker assisted selection. Sir, we would like to know what is this marker assisted selection?

Debal:    Marker assisted selection or MAS is a technology which transfers genes or quantitative trait loci (QTL) from a population of a crop to another population of a crop where that particular gene is missing from the latter’s genome. The technique deals with transferring QTL or genes from one population to another target population of a species. In other words, a population of a species may have a sequence of DNA which is agronomically more important than its equivalent in the corresponding genetic locus on the same chromosome in the target population of the same species. In the jargon of population biology, both the populations belong to the metapopulation of the species.So it could be that in this particular case, i.e. in the case of this drought tolerant variety being introduced, the gene which is responsible to confer that particular property to the rice variety in question might be missing in some populations of the metapopulation. So, what the scientists essentially do is select that particular gene or the desired form of the gene (technically called allele of the gene) that is located in a specific site on the chromosome, and then isolate some  “marker” gene which is closely linked with that particular gene in question and then transfer both the chosen trait gene and the marker gene to the target population where this particular marker and the chosen gene or the allele is missing. Now this is the principle which has been used over thousands of years by farmers in their traditional selective breedings. All kinds of hybridization programs and crop breeding programs have made use of this selection procedure. But this kind of traditional selection takes a long time because the farmer has to wait until germination of the seeds and their maturation. And then the crop has to be tested whether that particular chosen trait has been transferred into the new population. Then there would be a proportion of this population that may not inherit this desired gene, so they have to be rejected, and the proportion that will inherit the marker and the gene will be interbred. In contrast, modern MAS is rapid, precise, and can be achieved within a single crop generation. 

Jagannath:    Debalda, is there any specific need for MAS when traditional stress-resistant varieties already exist and more are being discovered? For instance, in the post-tsunami period it was observed that many of the rice varieties have tolerated the stress.

Debal:    MAS is much faster and precise than ordinary varietals breeding procedure. But you are right, we already have a number of indigenous varieties all over India, almost in every State of India, which are known to be salinity tolerant, drought tolerant, or flood tolerant, etc. And … you’re right … there have been many varieties which have been serendipitously discovered in the sense that they were not known to be tolerant of that particular stress until that had occurred. So it is a kind of result of natural experiments using that particular genome. So after that natural experiment, we’ve seen that out of maybe 200 varieties, only 2 varieties have survived in which case you can expect that those varieties may have the stress-tolerant genes. This is serendipitous discovery, made by farmers. In many of these cases the environmental stress could be  too much rain, too late rain, too early rain and so on. So essentially we have in our provision, that is, farmers’ provision, a large number of varieties which are stress tolerant.

The problem is the distribution of those varieties, or getting those varieties to the reach of the farmers who need them. Unfortunately, the agriculture department of any State has never thought it a priority (a) to identify and conserve those stress-tolerant and (b) once they’re identified or even known that they exist, to make them available or accessible to the farmers.

In fact, none of these gene banks, whether it is an international gene bank or a national gene bank, has ever made any of the seeds directly accessible to any farmer on record although in principle it is possible … that if the farmer could write that we need this particular variety which is lost from the farmers’ field and are in store of some gene bank they should be made available, but unfortunately this has never been made possible or it has never happened. My own experience is that in spite of my repeated requests to IRRI, I’ve not received a single Bengal variety or Indian variety that have gone extinct from the farmers’ fields.

Now, MAS could be used as a machinery to make the farmer dependable and more reliable on this kind of crop seeds. The risk is that with introduction of these new MAS varieties, especially when it involves commercialization by private corporations, MAS might replace all the existing varieties - even when the scientists’ intention is good. Good in the sense that the farmers would be able to grow his crop that would be able to withstand stressful conditions, at the expense of all the other existing varieties which are already in the hands of the farmer. They might find an extra incentive to lose them. This is exactly what has happened with the Green Revolution. And I think this is another extension of the GR to focus on MAS as the ‘only solution’ or ‘the best solution’ to marginal environmental conditions. Underlying the policy distribution of these ‘better’ varieties, is  the assumption is that whatever varieties are now existing are less good or worse or inferior, and therefore, should be chucked out.

However, the policy should be geared to make MAS varieties available only where none of the local varieties are available which have those properties. Wherever the local landraces with the desired characteristics are available, the government should be promoting these varieties at a larger scale so that they don’t get lost. Until that is taken care of, only a handful of modern varieties will be dominating the entire scenario and the hundreds of varieties which are still remaining will become extinct. 

Jagannath:    Could there be any problem with regard to bio-safety issues like genetic contamination associated with MAS?

Debal:    Because MAS does not involve any alien gene or any marker DNAs which are transferred across the species or genus or taxonomic kingdom barriers, there is no such bio-safety issues involved. However there are certain aspects of population genetics of concern, in that there might be some markers composed of DNA fragments of some unknown properties. In these cases, the stretch of the marker DNA may, at least hypothetically, include some non-coding DNA segment as well, which are associated with that particular locus to be trasnferred. In some cases it is likely that some of the non-coding DNA faction might have certain other functions which are yet unknown. And as the research in RNA is expanding the horizon of our understanding of gene regulation mechanism, we are increasingly becoming aware of the roles of the non-coding factions of DNA which are more important than we ever thought of. Like certain portion of the non-coding DNA regulates the expression of certain genes, regulates the transcription process and even translation of mRNA into proteins. Certain proteins which are translated from the DNA might also be involved in enhancing or thwarting the processes of DNA transcription, translation, or the replication of certain genes. So in case those marker DNAs, which are in use, are of those categories of great significance in the transcriptional or translational processes then they might have certain kind of an unprecedented effects which was not intended in the marker assisted breeding itself. In some cases it is likely that certain weedy characteristics, or some other undesired properties of a particular variety might be enhanced. And through cross pollination, whether between neighbouring cultivated varieties or even uncultivated wild rice like Oryza rufipogon – which naturally grow in the farm fields, those undesired traits may become widespread. And the frequency of those undesired traits might be the price of the desired traits which we are trying to propagate through MAS….. So this uncertainty is dormant there.

However, if it is done in the laboratory and if sufficient care is taken in order to test its transcriptional or translational or gene regulation function, then the uncertainty could be eliminated.  And that is also possible as long as it is in the control of public research, in government research institutions where we can expect certain marginal responsibility and accountability; - not when it is conducted entirely for profit motive in the hands of private enterprises.

Jagannath: In relation to MAS, the farmer would basically like to know whether the yield will be stable and whether the disease and pest incidences will be less. And the consumer would like to know whether there is any possibility of unexpected toxicity in food or health effects.

Debal: For the farmer there is no way of knowing what exactly would be the effect. They have to believe in it because the seeds are being given by an authoritative institution, whether it is a govt. institution or some company, who is supposed to know better because they made use of better technology, costlier technology and so on. Secondly, the farmer has no way of testing beforehand, prior to planting the seeds on their field, whether it is really performing in accordance with the prediction. From the consumer point of view , she has no way of knowing whether it will have any adverse health effect and she has no concern either whether the rice that is being sold on the market was drought-tolerant or flood tolerant, because it has nothing to do with the nutrient content or the taste of it. It is a completely different characteristic which is not essentially regulated or related to the flood-tolerant or stress-tolerant gene. So forget about the consumer effect. The consumer might be aware of the bio-safety issues, but … after all it is the duty of the govt. institution to ascertain whether there is any adverse health effect from transgenic or even chemically grown conventional varieties. But it is very important for the farmers to know whether this is really reliable variety.

For example what happened with the Bt Cotton? We already know that the farmers had full faith in the companies promoting the Bt Cotton and when it did not perform or when the Bt gene did not express sufficiently in some key locations or key tissues of the cotton crop, it had a devastating effect and the crop was attacked by the pests which was the target of the Bt Cotton had been designed to control. And then by the time that testing was done, it was either misrepresented, misreported, underreported or simply ignored. The result was that the whole burden was borne by the farmer himself and not by the government, nor by the company. The same situation is with the MAS seeds. We have to take on faith what is given as MAS rice, even if it is not so. If it is really MAS rice then I don’t think there would be any appreciable health effect but if it is not, that is, if that is a transgenic variety in disguise, and promoted in the name of MAS, then of course the whole series of uncertainties and accountabilities follow. As long as it is true that it is MAS then I don’t think there would be any adverse effect, because the desired gene as well as the marker DNA that is transferred along with the gene, are not alien DNA from distantly related or unrelated species.

Jagannath: For the hybrid seeds and also the GM varieties, they are fertilizer intensive and also water intensive, but do you foresee it has problems with the MAS varieties?

Debal: It depends on the genes being transferred. If the genetic material is transferred from a rice variety or a wild relative of rice to another rice  variety, then the gene is a part of the species’ genome. I don’t think the resultant variety is necessarily going to be dependent on agro-chemicals or water. If it is drought-tolerant for example, then by definition it does not need extra water. And if it is flood tolerant and if the farmer needs to grow it on a water-scarce farm, then of course he has to depend on water supply, otherwise it cannot survive. It depends on the type of the gene transferred and the agronomic characteristic of the resultant varriety.

Jagannath: So, one thing MAS definitely will do is further reduce our diversity in seeds and affect traditional seeds. 

Debal: If the promotion of MAS or any other variety is promoted by the govt. as the ‘best’ or ‘only solution’ to the food production problem, at the expense of all other existing varieties, then certainly farmers will be made more and more dependent on the supply of this seed. Because today in the modern market system, seed is always supplied by the market – the seeds are not exchanged normally among the farmers, even when there is a scope, then of course the farmers will have to look forward to heading more and more towards the supply of the same seed at the expense of all other seeds that they used to cultivate. And as I said before, there are dozens of traditional varieties which are stress tolerant but the farmers may not be growing them, because they might be inculcated  to believe that those are worthless varieties, inferior varieties. And once abandoned for two or three years, it’s lost forever, because the seeds will have lost its germinating ability. This is the same process that has been operating over the past 45 years. And that is how we have lost thousands and thousands of varieties. And if this MAS variety is promoted in the same way the same thing will happen.

Jagannath: MAS will also take crop breeding practices out of the control of farmers and breeders and it will make them more dependent on market forces.

Debal: Yes. To a large extent, farmers have already lost much of their traditional knowledge system and skills because they are made dependent on the market supply of all the seeds that they use for cultivation. The traditional varieties that some of the farmers are still cultivating are not improved by any of these farmers except in very rare cases. By and large, the farmers have completely forgotten the techniques of selecting pure lines or improving the varieties with desired characteristics. MAS is a highly sophisticated modern technology which cannot be accessible to the farmers at all, whether it is in the hands of the govt. or private sector. And therefore once the farmers are made dependent on the supply of those seeds for their particular agronomic characteristics whether it is flood tolerance or drought tolerance, they will have no way of improving those varieties or getting a parallel system of breeding those kind of seeds from their own stock of traditional varieties. So it is not that MAS by itself will be responsible for erosion of farmer’s control over production and crop breeding, but the socio-cultural learning process that disempowers the farmer, dissociates the farmer from the crop breeding science at the grassroots level. The reciprocal process of erosion of traditional agricultural knowledge and crop genetic diversity is already afoot. And this can be enhanced by creating further dependence on the external supply of seeds, in which case the erosion of cultural systems (like seed exchange), erosion of crop genetic diversity and the loss of local control over the seed resources will be enhanced. And in the process, self-reliance, self-sufficiency of farmers, and agricultural sustainability, will be lost. I call that an instance of hijacking of sustainability. 

Jagannath: Can this technology be patented?

Debal: Yes. The technique of MAS has already been patented. But a couple of scientists have declared that they have donated this kind of patents and the technology will be accessible to any researcher. The patentability or patenting itself may not be a crucial factor for the farmer’s access to the varieties. So it is not as problematic as the transgenic crop. But the varieties with certain  traits may be coming under the intellectual property rights of certain companies. In some cases they might say that in one particular commercialized variety – whether it is transgenic or not – might contain some gene which should enable that particular variety to tolerate say, 40 ppt of salinity and above. So in that case, it is an unique variety which has specific predicted properties and that is very likely to be patented, in which case that will entail a legal implication or commercial implication. But we hope that MAS, as long as it is in the hands of the public institution, that will not be of much concern to the farmers. 

Jagannath: There is apprehension that as the govt. is not denying that MAS is a form of genetic engineering they may also introduce such varieties as the Golden rice and so on, saying that well, we are already using genetically engineered varieties.

Debal: That genetic engineering is a broad term. And bio-technology is another very wide term. Bio-technology involves tissue culture, monoclonal antibody, MAS and transgenics. But genetic engineering specifically involves recombinant DNA, and transfer of DNA fragments from one organism to the other without recourse to the normal natural course of inheritance of genes.

So, in this case the MAS is one form of genetic engineering in the sense that it involves transfer of genes. We can only differentiate it from what we call GM crop, which is essentially a transgenic crop in this way – transgenics involves transfer of genes across species, genus or even kingdom barriers, so that genes from bacteria or insect is possible to transfer into a crop. In the case of MAS, it also involves certain genetic engineering tools to transfer the genes, but it is confined to the genome of a particular species, or at most the periphery of its wild relatives. So the activists or the people who are concerned should be aware that transgenics and MAS are of qualitatively different categories.

Whenever there is an attempt at confusing these two methods, that pernicious attempt should be foibled, so that there is no scope of smuggling transgenic varieties in the guise of MAS or even to justify transgenics. So, let us be very technically specific: what exactly do you mean by this genetic engineering? If it is MAS, then call it MAS. Is it transgenic? Then call it transgenic, and then we are abandoning all transgenic varieties since we are against its bad science in the hands of commerce. 

Jagannath: The IRRI scientists are of the opinion that traditional varieties with levels of abiotic stress tolerance exist, but with poor agronomic and quality characteristics. Do you agree?

Debal: It remains to be defined what they consider as poor agronomic characteristics. There is not a single modern high yielding variety and modern transgenics in rice that can incorporate aroma. There is not a single rice variety born of modern crop breeding research which has been successful in transferring full aromatic properties .  If you consider just the seed output – then of course, the yield of  basmati or gobindobhog or lilabati and you name any of our aromatic varieties, is low - yield in terms of absolute grain yield per unit area of production. But if we consider that grain yield is one criterion by which we can consider that it is of “poor characteristic”, then we have to admit that aroma is another unique characteristic, which is a very rich characteristic for which basmati is exported.

Nobody in Europe buys basmati for its productive potential. It is for its aroma. And that gives India its large export earning from crops. The secondary importance of the yield itself is quite problematic from the agronomic point of view. Yield by any scientific definition is the output per unit of input. Again, by this criterion, any kind of indigenous crop is much more high-yielding than any so-called high-yielding modern varieties, simply because for the latter, the input is enormous, both in terms of economic value and in terms of quantitative estimation, whether it is urea or water – whatever. So if we consider just water for example, the output of IR-36 or IET series of varieties, it is quite high-yielding in terms of absolute grain output, but if we divide this output by that millions of litres of water, and compare this with the relatively smaller output but with negligible input of water for the drought-tolerant traditional varieties, then we find the fallacy of the definition of yield. Unfortunately official  agronomists take special care to confuse this. And this misuse of the term ‘yield’ to replace ‘absolute grain output’ is always in practice to confuse and misguide the actual estimation of yield itself. Secondly, none of these characteristics that the genetic engineers and the MAS scientists are using, whether it is flood tolerance or drought tolerance or salinity tolerance were invented by modern crop science. All these traits are already there in the crop genome, because were it not for their presence they would not have been able to make any kind of MAS variety at all in the first place. Because it is there, scientists are now able to manipulate those genes.

So all the crop properties which are so called ‘poor’ as well as ‘rich’ characteristics, have already been known over thousands of years. It is already on record by IRRI and Bioversity International that more than 110,000 rice varieties existed in India. Most of these we have lost. And of the lost varieties there is no way of telling which unique traits characterized those lost varieties, and what are the characteristics or genes still available to be tapped in future. So, until we have this definite information, scientists have no legitimacy to say that all the traditional varieties have ‘poor’ characteristics.

Jagannath: My last question Dr. Deb – how would MAS affect your own rice breeding activities?

Debal: First of all I won’t get access to this MAS – and (laughs) I don’t need to. It’s not just me. I think all the indigenous farmers or the farmer-breeders or any scientist who knows the art of plant breeding know that Indian rice varieties have all kind of characteristics which are desirable from the agronomic point of view, from the farmers’ point of view. It only requires the scientific understanding to identify them and make use of them. Even from the purely scientific interest, we have in our accession two unique varieties – I’m not talking of the agronomic properties but its unique genetic characteristics – like each spikelet containing double rice grains. And each of these grains is capable of developing a viable embryo – so the variety has the capability of doubling the output. But I’m not considering that. I’m just telling of this unique trait, indicating the existence of genes - most likely a single gene that induces ovary duplication, which has not yet been identified. We have another variety which has three grains in a single spikelet -- sateen. I collected another variety from Orissa that has a pair of wide ‘wings’ on the rice hull.

Now once such unique genetic traits are identified by morphological examination, then our genetic engineers might jump upon those genes. But we don't yet know all the genes in rice genome, and their products. The rice genome was published some decades ago. But nobody has ever identified which part of the genome is responsible for causing duplication and triplication of the rice ovaries. In some cases we have also found 5 or 6 ovaries in the same spikelet of sateen rice. That indicates that the gene continues to function, in the spikelet to multiply the ovary. To know the function of specific genes is the work of functional genomics, which is still in its infacny. Just listing the arranged nucleotides and the DNA strings and printing the genome is not enough to know the organism. 

To be continue....