Application Of Molecular Genetics To Crops

“The science of biotechnology is likely to be to the first half of the 21st century what the computer was to the second half of the 20th century. Its implications are profound, its potential benefits massive. Britain is well placed to keep our lead in Europe. I want to make it clear: we don’t intend to let our leadership fall behind and are prepared to back that commitment with investment.” are the words of Tony Blair at the European Bioscience Conference (November 2000) (cited in Francesca Tencalla, 2005), which is the best way to present my positive answer towards the question. The molecular genetics positively impacts the plant breeding paradigm.

Introduction:

Centuries ago, the science was unknown to the farmers but the present agricultural scenario is changing its traditional frame of conventional breeding to the most recent and modernized frame of molecular plant breeding. Today, the world agriculture is facing great challenges on four fronts visually, Production, Population, Pollution, and Politics. Also, the change in climate (global warming) is the next major challenge for the crops, as unlike animals they can not move from one place to another. The prospects for feeding humanity as we are in 21st century often are portrayed in a daunting light to keep pace with the population growth. As we are told that world’s population has been growing faster than crop production, since the early 1980’s and is expected to be 8 billion in next or two decades. At this point, we need to think in-depth about the opportunities to meet these challenges and to improve the crop production at a subsequent rate. Are the agronomic practices merely going to play the key role or the refined crop protection measures will be the major part of increase in crop production, at this junction of scientific development? No other than, molecular genetics and the application of molecular techniques to enhance the efficiency of plant breeding, will play the crucial role in crop improvement, is a promise to the next generations (Ben Miflin, 2000).

At the turn of the 20th century, the discovery of principles of natural selection and hybridization by Darwin and the rediscovery of Mendelian genetics served as the basis for plant breeding and genetics. In the same way, tremendous increase in crop yields in the 21st century has been powered by the development of plant biotechnology, -omics era of molecular biology, and molecular genetics. The recent techniques of molecular genetics are emerging continuously to overcome the demand of growing population (Moose et al., 2008).

The chief objective of molecular genetics is to enhance crop production and crop produce usefulness by genetic modification of crop plants; this is precisely what plant breeding has been doing from the day prehistoric man began to domesticate the orphan (wild) species. The molecular genetic techniques aim to give traditional breeding a technological boost, says Jorge Dubcovsky, a wheat molecular geneticist from University of California, whose group is presently working on Marker Assisted selection in wheat for 23 separate traits, conferring resistance to insect-pests, fungi and viruses. “His enthusiastic claim is that this research could offer the wings for crop improvement to plant breeding what jet engine has brought to air travel.”

Basic Terms:

“Plant Breeding is an art as well as science of improving genetic makeup of plants in relation to their economic use (Paul et al., 2006; Singh B.D., 2003).”

“Molecular genetics refers to the study of molecules, our genes, their structure and functions at molecular level. Also studies the transfer of genes from one generation to the next. It employs the principles of genetics and methods of molecular biology (NCBI, Wikipedia).”

“Recently, Crop improvement is the science of value addition to the existing crop species by using the technique, which marries conventional plant breeding with molecular biology, to get theatrical gains in yield (Austin R.B., 1986).”

Hundred years of Genetics:

(From domestication to transformation)

Plant breeding deals with the aspect of crop production. In early days, plant breeding was mainly based on skills and the abilities of the breeder involved in the programme. But as the genetic engineering and the tools of molecular genetics were elucidated, breeding methods and programmes were designed in their light. Plant breeding began with the domestication, when primitive men cultivated the first crop for the benefit of mankind. Thereafter, the process of exploiting the crops for improvement of their commercial values has a long history in itself. As early as 700 B.C., Babylonians and Assyrians performed artificial pollination in date palm. Then, the first artificial hybrid named Fairchilds’ mule produced by Thomas Fairchild by crossing carnation with sweet William in 1717. These provide the clear evidence of plant breeding exist as an art before the discovery of Mendelian genetics.

In 1900, the rediscovery of Gregor John Mendel’s paper provided the foundation for the vast knowledge of genetics for crop improvement. A noteworthy development resulted from the discoveries of G.H. Shull on inbreeding in maize (Zea mays), led to the production of hybrid varieties in maize, sorghum, cotton, rice and several other crops. Green Revolution, one of the greatest achievements in the modern plant breeding has been the introduction of dwarf gene in cereals, particularly in wheat (Triticum aestivum) and rice (Oryza sativa), (Borlaug, 2000).

In 1960, Allozymes the first biochemical genetic markers were on hand. The next decade provided new tools to geneticists of restriction fragment length polymorphism (RFLP) and Southern blotting. Taq polymerase was found in 1980 and shortly Polymerase chain reaction was developed, which is now routinely used in plant breeding research. The recently developed technology is single nucleotide polymorphic markers based on high density DNA arrays, a technique known as ‘Gene Chips’ (Rodomiro Ortiz, 1998). In 1980, the era of biotechnology began with the successful production of the first transgenic plant using Agrobacterium (Moose and Mumm, 2008).

The methods of crop improvement have changed dramatically through last 25 years. The continued exploitation of biotechnology and the integration of genomic tools in crop improvement widen the plant breeding research. The novel genetic approaches like next generation sequencing (NGS), high-throughput marker genotyping, advanced-backcross QTL analysis, introgression libraries (ILs), multi-parent advanced generation intercross (MAGIC) population, can be harnessed to recognize the genetic variations within the crop species and between cultivated and wild species (Varshney et al., 2009). The seeds of molecular genetics are immensely sprouting and sooner will grow into a self-sufficient fruitful plant to alleviate the world hunger.

Application and Impact of Molecular Genetics on Crop Improvement:

The Father of Green Revolution, Dr. Norman Borlaug said in an interview: “Biotechnology helps farmers produce higher yields on less land. This is a very environmentally favorable benefit.” He justified his statement by giving the fact that the world’s grain output in 1950 was 692 million tonnes. After forty years or so later, the world’s farmers sown the crops in about the same acreage of land but they harvested 1.9 billion tonnes that counts the 170% increase in global production. To get this increased production in 1999 using the same conventional breeding, farmers would have needed an additional land of 1.8 billion hectares, instead of using 600 million as used in 1950 (www.actionbioscience.org).

Opportunely, plant breeding research is at an arena where there are remarkable advances being made at molecular levels and these endow with opportunities to augment the molecular technologies available for crop improvement as outlined below.

Distant hybridization:

With the advancement of molecular genetics, it is now possible to transfer genes between distantly related plants. Now genes can be transferred interspecific and intergeneric. Recombinant DNA technology can be amplified to transfer the desirable genes from lower level organisms. For example, Progenies derived from rice (Oryza sativa ssp. japonica) plants pollinated by Oenothera biennis exhibited numerous morphological and developmental traits. Results from amplified fragment length polymorphism (AFLP) analysis showed that several rice lines contained extensive genetic variations, which included disappearance of rice parental bands and/or appearance of novel bands (Chu Xiu Chang et al., 2007).

Recombinant DNA technology:

There are two methods for crop improvement using r-DNA technology namely, direct and indirect methods. The direct method involves the introduction of novel gene(s) to crops by transformation outside the constraints of sexual crossing. Whereas, the indirect method involves the improving crops by the development of molecular markers (Miflin, CIHEAM). These methods have their specific significance in plant breeding so, are discussed as under.

Molecular Markers for crop improvement:

Molecular marker refers to the easily detectable marker linked to a desirable trait. The plant breeding analyses like, early generation selection, enrichment of complex F1 generation, choice of donor parent in backcrossing, recovery of recurrent parent genotype in backcrossing, linkage block analysis and selection, exploit molecular markers as a valuable tool. Application of molecular markers in plant breeding includes Germplasm characterization/fingerprinting, determining seed purity, systematic sampling of Germplasm, and phylogenetic analysis. Molecular markers played a key role in replacing bioassays (Varshney et al., 2008).

Crop plants exhibit 20-50,000 genes, out of which only few are of our interest in crop improvement. Conventional linkage maps are based on these genes of interest which have distinct morphological effect. But there are limitations in such maps as they cannot map out the genes governing quantitative traits, are time consuming and tedious. Therefore, emphases were made to focus on molecular markers for linkage mapping. There are many types of molecular markers visually isozymes, restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPD), cleaved amplified polymorphic sites (CAPS), simple sequence repeats (SSR), amplified length fragment polymorphism (AFLP), and the latest includes single nucleotide polymorphism (SNP) and single feature polymorphism (SFP), (Bertrand C. Y Collard and David J Mackill, 2008; Varshney et al., 2006).

Out of these, SSR or microsatellite is the most widely used marker in major cereal crops (Bertrand C. Y Collard and David J Mackill, 2008). The SNP and DArT (diversity array technology) markers are other high-throughput markers, which can be used to prepare the whole genome map even without the availability of sequence data for the crop (Varshney et al., 2006). In recent years, noteworthy and stimulating progress has been made in marker assisted technology and the development of markers linked to the gene of interest. Some important achievements are given in table 1.

Molecular Assisted Selection (MAS):

MAS (figure 1) speed ups and makes conventional breeding easier as herein, linked molecular markers are used for indirect selection of desirable traits that were difficult to select earlier in seedling stage (Anushri Varshney et al., 2004). Jonathan et al., 1998, while performing their research on Musa found that due to its triploid nature, the highly relevant generation and the precise linkage maps is not usually attainable. So, to generate linkage map, they first generated traditional linkage map based on diploid relatives and extrapolation to the polyploidy crop and then did polyploidy mapping based on single dose markers in population derived from crosses between tetraploid and diploid genotypes. Thus, it became possible to generate the linkage map of current Musa, which is an allotetraploid.

Bioinformatics:

Bioinformatics is often overlooked side of molecular marker strategies. As we know that molecular technologies produces a large number of data, with great chances of error introduction during handling and interpretation. The correct accreditation of data is necessary for getting the precise results after a long run of experiments, so for maintaining that huge amount of data bioinformatics enabled such software that provide accurate results and are available through World Wide Web (Simmonds et al., 1999).

Quantitative trait Loci (QTL):

The most interesting use of molecular markers is the efficient selection of traits. They also made it possible to select polygenic traits controlled by QTLs, by the use of molecular markers like RFLPs and isozymes. Interesting work has been done on QTL by Tanksley et al., on tomatoes, Stuber et al., on maize, McCough and Doerge on rice and Bradshaw et al. on potatoes. The future research can be made in improving the disease resistance in cereal crops (Simmonds et al., 1999).

Development of nodules in cereals:

Leguminous plants have root nodules that contain nitrogen fixing bacteria known as Rhizobium. These bacteria fix atmospheric nitrogen in root nodules in the form of nitrates. If this gene can be transferred or active root nodules can be developed in cereal crops like wheat, rice, maize, sorghum, pearl millet, barley etc. thus, the reliability of crops on chemical fertilizers would be reduced leading to a significant reduction in cost of production as well as environmental pollution. The introduction of symbiotic biological nitrogen fixation into the major non-legume crops of the world would be one of the most significant contributions that biotechnology could make to agricultural sustainability.

With this objective, Edward C. Cocking et al., studied the interaction of rhizobia with the root systems of cereals. They have already confirmed that some naturally occurring rhizobia, such as Parasponia species those isolated from root nodules of non-legume and Aeschynomene from stem nodules of tropical legume species, are able to enter the root systems of maize, rice and wheat by ‘crack entry’ means the part of plant where lateral roots emerge through the root cortex, so it results to the both inter and intra cellular penetration of rhizobiain, particularly in the cortex of emerging lateral roots.

“In the recent study, they have interacted oxygen tolerant Azorhizobium caulinodans ORS571 (kindly donated by Dr J K Ladha, IRRI) isolated from stem nodules of the tropical legume Sesbania rostrata with the root systems of rice and wheat. We have found that intracellular invasion of cells of the cortex of roots of both rice (IR42 and Lemont) and wheat (Wembley) results in plants that are active in nitrogen fixation as determined using acetylene reduction assays” (Edward C. Cocking et al.).

The Plants for future:

(Development of transgenic plants)

Transgenic plant is the product of genetic engineering, with artificially inserted foreign gene (Adina Breiman and Esra Galun, 1997). The most important tool in plant biotechnology is the transgenic plant production. This technique requires the study of chromosome at molecular levels, genome sequencing and the identification, selection and then insertion of gene of interest form same species in modified form, or related wild species, unrelated species, genus, across kingdom or from microbes (bacteria, virus, fungi). The successful commercialization of first transgenic crop in 1996 is the remarkable achievement in plant breeding and biotechnology. In 2008, globally both the number of countries (25 countries are GM growers) as well as the number of farmers (13.3 million farmers) growing transgenic crops in 125 million hectares of land, has increased significantly (Table-2), (ISAAA web pages).

This technology boosts yields, reduce pesticide usage, increase fertilizer efficiency, enhance disease, drought, pest, resistances to various crops. Recently, Sugarbeet RR herbicide resistant variety is grown in USA, gives significant positive results. Another milestone of transgenic breeding is GOLDEN RICE: genetically modified rice that contain bets carotene which can produce vitamin-A, once consumed (Potrykus, 2001). The production of golden rice presents a second green revolution, where certain countries will become the sole producers of major money making crop.

It is believed that this would be the most dominating science in future that will play the most crucial role in the crop improvement and can satisfy the growing world hunger.

Future prospects:

We already have genetically modified crops for various interesting traits, which are developed so far generally involve only the addition of a single gene. Looking to the future, it’s unclear whether complex traits, which are thought to involve multiple genes, will be amenable to manipulation through genetic engineering (Jonathan Knight, 2003).

All these techniques are costly and the research in public sectors is less due to lack of funds. There is the thrust of evolving the more precise, accelerating and cheaper molecular methods that can revolutionized the world agriculture. DNA chip technology and user friendly marker system are the evolving trends in future.

For sustainable and self sufficient agriculture, we need carry out continuous efforts in positive direction. There should be the willingness to accept the research and research products globally, politically and socially as well. Then only we can expect second green revolution.

Conclusion:

We are at the stage of broad and rapid era of science development in all the fields. Time is ripe for the second green revolution which would not only concentrate on increased productivity but also on value added traits to reduce cost of production, pollution dangers and improved quality. Rapid development of plant biotechnology, particularly molecular genetics will serve as the basis for the second green revolution. To speed up the production process economically, to fulfill the aspirations of huge populace, to achieve diversification and adding value to the primary produce so as to make agriculture enterprise farmers as well as environmental friendly. Advanced technologies are expected to materialize many of our expectations in the 21st century. On the other hand, if we arrogantly enjoy the past but ignore the new challenges, or underestimates our capabilities and feel afraid of innovations like GM crops, it is possible that we miss the good opportunities, as said in this Chinese proverb, “Ninety miles is only the half way of a hundred-mile journey.”

Confucius once said: “The passage of time is just like the flow of the river, which goes on day and night, forever.” The past glories are the momentum for our new journey; the journey of science, journey of development, journey towards the state of self reliance, while the lessons from the past may teach us to be smarter.

We need molecular genetics to make historic contributions to the rejuvenation of the plant breeding and thus the agriculture.