Transgenics: Transgenic plants, genetically modified organisms (GMOs), living modified organisms (LMOs) and Genetically Engineered Organisms (GEOs) are synonyms and represent products of the process of transgenesis.
In literary terms, transgenics (trans + genie), as the word denotes, means transfer of genetic material (DNA fragment carrying known genes) from across the biological systems, i.e. from viruses to man (donors) into plants cells (recipient), through in-vitro techniques.
The plants derived from these cells are termed as genetically transformed plants or transgenics plants. These plants transmit and express introduced traits in successive generations.
Importance of transgenics in agriculture
Biotechnology, based on recombinant DNA technology, is being advocated as an important adjunct to conventional plant breeding for sustainable development in agriculture. Tools and techniques of recombinant DNA technology have widened the definition of ‘gene pool’ in plant breeding because it is now possnble to mobilize candidate genes of interest into plants from hitherto inaccessmle bioresources.
Encouraged by these developments, a wider role of biotechnology in ecotechnologybased precision farming of future, especially for developing countries like India has been envisaged. There should be a judicious integration of biotechnological approaches in each of the following major components of conventional crop improvement programmes, viz.
(1.) integrated gene management,
(2.) integrated nutrient management,
(3.) integrated pest management,
(4.) water management,
(5.) Soil health care, and
(6.) post-harvest management.
The development of genetlcally tailored plants of tomorrow endowed with attributes enumerated above is a distinct possibility through recombinant DNA technologies.
In recent years, the practical utility of alien genes through transgenesis has been extensively demonstrated and transgenics plants harbouring genes for insect pest and herbicide tolerance, improved post-harvest shelf life, and for quality have been developed in a number of crop plants and are being grown commercially in both developed and developing countries.
Landmark discoveries leading to development of transgenics
The 20th century began with the rediscovery of Mendel’s Laws of Inheritance and ended with full genome sequence of a number of organisms, including Arabidopsis among plants and human being among animals. These hundred years are dotted with revolutionizing scientific discoveries, which led to the development of recombinant DNA technology and in turn to transgenics.
These discoveries established that all the characters in living beings are governed by genes; each gene with a well defined nucleotide sequence in DNA occupies a pre-determined location in the chromosome; there is site specific cleavage of DNA by restriction enzymes; restricted DNA fragments can be cloned into appropriate vectors; these vectors mediate the mobilization of cloned genes into the recipient; the expression of the introduced gene in the recipient plant cell can be manipulated; and transformed plant cell can be regenerated into a transgenic plant.
The recombinant DNA technology developed so far permits only random insertion of foreign DNA in the chromosomes and, therefore, the level of expression of the inserted gene and its stability is greatly influenced by the neighbouring DNA sequences in the chromosome.
Methods of gene transfer in plants
The spectacular demonstrations that the plant cells are totipotent, i.e. have the potential to develop into a full plant and that they can be transformed with foreign DNA has paved way for genetic engineering of plants for desired traits.A large number of methods of gene transfer in plant cells have been tried over years, and are discussed here.
Direct gene transfer
(i) Pollen mediated,
(iv) particle bombardment or biolistics,
(vi) ultrasound-induced DNA uptake,
(vii) silicon carbide fibre-mediated DNA uptake,
(viii) laser-mediated DNA uptake,
(ix) liposomemediated DNA uptake,
(x) PEG-mediated DNA uptake, and
Vector mediated gene transfer
(i) Agrobacterium mediated,
(ii) agro-infection, and
(iii) viral vector.
Of the above listed methods, two methods, i.e. Agrobacterium-mediated and biolistics, have been predominantly used in transformation work; While both of these methods are quite effective, each has distinct advantages and shortcomings, and their deployment has been largely influenced by the characteristics of the plant tissues to be transformed.
Agrobacterium-mediated gene transfer: Agrobacterium tumeaciens is a gramnegative soil bacterium, which causes tumor formation (called crown gall) on a large number of dicotyledons as well as some monocots and gymnosperms.
Crown gall induction is due to the transfer of a specific DNA fragment, the T-DNA (transferred DNA), from a large tumor-inducing (Ti) plasmid within the bacterium, to the plant cell. Three bacterial genetic elements are required for T-DNA transfer to plants:
(i) T-DNA border sequences that consist of 25 bp direct repeats, flanking and defining the T-DNA. Usually, all DNA sequences between the borders are transferred to the plant.
(ii) The Virulence (Vir) gene-encoded by the Ti-plasmid in a region outside of the TDNA. Vir A and Vir G form a 2 component regulatory system responsible for transcriptional activation of the vir operons. The other vir genes are involved in the processing of T-DNA targeting to the nucleus and are probably involved in precise TDNA integration into plant genome.
(iii) The third bacterial element necessary for TDNA transfer consists of a number of chromosomal gene, chvB, pscA, exocC and att-with functions like response to plant wounds and attachment of the bacterium to the plant cell.
Particle bombardment or biolistics: The invention of the direct gene transfer technique of particle bombardment was a major development in plant genetic manipulation, as it had enabled transformation of many plants not amenable to Agrobacterium based gene transfer techniques.
The method involved particle gun accelerated microprojectiles, typically tungsten or gold particles (1.2 mm in diameter) coated with plasmid DNA of interest, to velocities at which they can penetrate plant cell walls. The most widely used particle gun is PDS-1000/He marketed by BioRad, which uses helium gas for propulsion of the DNA coated gold/tungsten particles. Gold is preferred over tungsten because gold is biologically inert whereas tungsten degrades DNA over time and can be toxic to some cell types.
“Transgenics” have been produced from bombardment of various explants like shoot apices, immature zygotic embryos, inflorescences etc. in maize, wheat, tobacoo, barley and some other plants.
Development of transgenics crops
In recent years, in addition to the development of biotic stress tolerant GM crops, conscious efforts are underway for engineering plants endowed with improved or altered nutritional quality.
Transgenics for nutritional quality
Transgenic rice for higher β-carotene (pro vitamin A): B-carotene, a precursor of vitamin A, is naturally present in a number of fruits and vegetables. However, due to food habits and lack of affordability, about 9.2 million pre-school children suffer from vitaminA deficiency and at least 60,000 children go blind each year.
Hence, an adequate supply of B-carotene through staple food is almost a necessity for overcoming vitamin A deficiency. The transgenic “Golden rice” developed, over-expressing B-carotene, in rice seems to be an ideal approach for providing, at least in part, the vitamin A requirement to the target population.
The work on the transfer of the candidate genes in the local elite cultivars of rice by backcrossing as well as by direct transformation has been initiated. This is an example which will go a long way in –
(i) demonstrating how a technology of this kind can be exploited for the benefit of a vast and vulnerable segment of the population and
(ii) kindling an international debate on the propriety of patenting genes meant for common good as also for sharing of IPR for general welfare of mankind.
Transgenics for enhanced vitamin E: Vitamin E (tocopherol) is present in plants in two predominant isomeric forms, α-tocopherol and γ-tocopherol. Alpha tocopherol is considered more desirable for human and animal consumption because it has higher biological activity than γ-tocopherol.
Incidently γ-tocopherol is present in plants in higher amount as compared to α-tocopherol. It is now known that gene for γ-tocopherol methyl-transferase (γ-TMT) is involved in the conversion of y-tocopherol to α-tocopherol. γ-TMT was over expressed in Arabidopsis ‘transgenics‘ plants.
Transgenics for enhanced mineral content: Plants take up different minerals relevant to human nutrition and health from soil and accumulate in specific tissues. Transgenic strategies for improvement of mineral content in plants are being designed in a number of laboratories.
Genes encoding proteins (divalent metal transporter) involved in membrane transport of different mineral nutrients, viz. Fe2+, Zn2+ Mn2+, Cu2+, Ni2+,Ca2+/H+ and Mg2+/H+ have been identified. Fuxther molecular and functional analyses are in progress for deciding transgenic route to be adopted.
Iron-rich transgenic rice for fighting iron deficiency (anaemia) has already been developed. This has been achieved by introducing and expressing ferritin gene from Phaseolus vulgaris in rice grains.
Also, to increase bioavailability of iron, a gene for thermo-tolerant phytase from Aspergillus fumigatus was mobilized and overexpression of endogenous cysteine-rich metallothionein like protein was achieved. The “transgenics” rice with higher iron content, rich in phytase, and cystein peptide is expected to address iron deficiency so widely spread in human population.
Transgenics for enhanced plant productivity
Genetic engineering of plants for productivity traits is still in its infancy internationally, in spite of the availability of voluminous information on carbon and nitrogen metabolism; the two most important and key processes contributing to growth and productivity in plants.
Unfortunately, plant yield is a complex trait, contributed collectively by a large number of biochemical and morphological attributes where each one of these is governed by polygenes. Thus, as of today, a simple trans-genetic route mobilizing one or two qualitative genes has not provided the required breakthroughs in increasing plant productivity.
Transgenics for tolerance to biotic stresses
Modern farming practices which led to the emergence of ‘Green Revolution’ have aggravated the problem of pest damage in almost all the agro-ecosystems but more so in the high-input agriculture in India. Extensive and, at times injudicious, use of pesticides has also forced insects to evolve pesticide tolerant biotypes, thus further compounding already worst situation.
It is estimated that biotic stresses, including insects, fungal, bacterial, Viral and weed related ones put together reduced is crop yields by 10-40% depending upon the crop, region and severity of pest infestation. In monetary terms, the loss estimated to exceed Rs 50,000 crore every year (1 crore = 10 million). Though the consumption of pesticides in India is only about 280 g/ha as compared to 2 kg/ha in Europe and 10 kg/ha in Japan, yet there is widespread contamination of food and food products and environment with pesticide residues, primarily because of uneven and injudicious application of pesticides.
Transgenics for abiotic stress tolerance
More than 7 million ha of land in India suffers from salinity and alkalinity problems. With the expansion of irrigation systems in the past in areas lacking proper drainage system, the areas under salinity and alkalinity have further increased. In addmon, about 40% of the cropped area is still rainfed and the crops periodically suffer from moderate to severe drought.
Coastal and other low-lying areas experience seasonal water submergence. It is thus evident that abiotic stresses with their severity and recurrence cause heavy losses in agricultural production. The breeding for abiotic stress tolerance, therefore, assumes special significance in Indian agriculture.
Fortunately abiotic stress tolerant genotypes in relative terms are available in the germplasm of a number of crop plants but could not be made use of effectively in the absence of an efficient selection procedure. Marker aided selection approach, therefore, deserves high priority for breeding abiotic stress genotypes.