A definition of biotechnology from the U.S office of technology assessment defines, “Any technique that uses living organism to make or modify products, to improve plants or animals, or to develop micro-organisms for specific purpose”. Its use in recent times have dramatically increase and open the door of possibilities especially in case of plant breeding. In traditional approach of plant breeding there was little control over how or where a gene is expressed but because of new method precise control of gene expression is achieved. Such new approaches have really changed the face of plant breeding in very short period of time.
Some of the approaches of biotechnologies and its application in plant breeding is discussed below:
a. Tissue culture:
It refers to culturing of explants (meristems) in laboratory on specific nutrient media until they can be moved into the soil. The tiny growing tip of plant is used in culture media to produce entire plant (totipotency). Micro-propagation, is production of multiple copies of single plant using tissue culture technique. Micro-propagation of potato-bud and garlic plants in the laboratory can eliminate virus diseases which ensures new crop is planted with virus free materials. Micro-propagation also helps to multiply individual superior plants to create parents plants used in production of hybrid seed (e.g. orchid and gerbera). This technique basically shortened the breeding cycles. Similarly, embryo culture helps to rescue hybrid plant from wide crosses, which often fail to produce viable seeds enabling breeder to produce distant hybrid plant. The immature embryo or ovule is rescued and cultured in-vitro for formation into whole plant. This technique is for overcoming post fertilization barrier. It is mainly used for transfer of disease resistance genes from wild relatives to domesticated crops.
b. Protoplast fusion:
It is another method for producing new genetic variations. A protoplast is plant cell whose outer cell wall has been removed, allowing their DNA to be combined under certain condition. The fused cell then induced to regenerate into whole plants that have traits of both the parents. e. g. Interspecific somatic fusion of amphidiploid hybrid of rapeseed, resynthesized from Brassica Oleracea and B. rapa. Protoplast fusion also helps in transfer of male sterility from radish to Brassica species which is used in production of F1- hybrids.
c. Double haploids:
Double haploids have considerable importance in plant breeding if completely homozygous lines are needed quickly, if traits are complex and in recessive manner under different environmental factor. Double haploids technique is nowadays frequently used in maize breeding. The method is Intraspecific in-vivo haploid induction and lines that produce high number of haploids are called “Inducers”. However, haploid induction rate is very low i.e about 5%. Anther culture works on the concept of Double haploids. Anther culture is used in the production of F1 hybrid varieties. The first step is to develop inbred parent lines by repeated self-pollination. The haploid plant that contain one copy of each chromosome and by the application of Chemical (colchicine) identical set of chromosome can be formed which restores its fertility. So, inbred line is develop in short time. Haploid plant can be obtained from sugar beet, barley, wheat and maize.
d. Markers assisted breeding and genomics:
Markers based breeding tends to shape the future of breeding program in predictable manner. Markers are used to transfer and test for inheritance of gene. Both protein and DNA based markers are used for this purpose. Markers are being useful for genetic mapping of genes for quantitative traits, markers assisted selection and analysis of genomic structure. In some of the crops, the problem of linkage drag can be overcome through the use of use of markers based breeding for e.g. in Potato. Hence, markers can be used as useful method only to transfer the desired part of DNA and not the unnecessary segments to produce desired trait. For example, an isozyme marker speeded the development of commercial tomato
varieties containing the Mi gene (which confers resistance to the root knot nematode, a serious worldwide pest). Markers are also used to identify the haploids plants (anthocyanin pigment in endosperm in maize kernel). RFLPs, SNPs, SSRs are some of the example of genomic markers used.
e. Recombinant DNA technique:
This technique isolate the specific segment of DNA molecules, insert it into other DNA molecules and amplify them to produce large copies of DNA molecules. This is done by the researchers to produce organisms of specific interest, clone them and study it. These technique rely on the use of restriction enzymes (e.g. EcoRI). In short, a desired gene is cut using restriction enzymes, inserted into vector and then finally introduce into bacterial cell to amplify in large amounts. Once gene have been cloned, they are ready for transfer in one or more than one individual.
This precise transfer of gene of interest into new cell to develop novel plant is major achievement in plant breeding. The mostly used technique is agrobacterium mediated gene transfer (Indirect method). This indirect method is mostly prevalent because of its capability of stable transformation ability. However, many direct technique have been developed such as;
Particle bombardment (particle gun mediated): applied to all species
Silicon carbide fibre: used for maize and tobacco.
Electroporation of intact cell, micro-injection etc. But these direct method only provides the transient expression of gene. These techniques helps to produce transgenic crops. These technique plants are also referred as genetically modified organisms (GMOs). E.g. Insect resistance and herbicide tolerant gene in soyabean, maize, cotton and canola (major global biotech crops). So this technique enable breeder to exploit any specific gene for crop-improvement purpose. These transgenic crops also reduce the use of agrochemicals like herbicide and pesticide and increase the economy of farmers and helping the consumer as well.
f. Genome editing:
Genome editing works in the principle of restriction endonuclease. It is used for the purpose of site directed mutagenesis. It generally works for the TILLING purpose. (Target Induced Local Lesions In Genomes) also known as reverse genetics concept. Some of the site –directed mutagenesis are:
Zinc-finger nuclease (ZFN) technology
The major purpose is cut specific segment of DNA sequence and fill with the desired segment of DNA into that gap to create new sequence (mutation). CRISPR/Cas9 is recent breakthrough and mostly used technique these days. It helps in alteration of genome sequence and prevents the problem of linkage drag. This technique has high specificity and efficiency.
Biotechnology has and will amaze us in coming days as well. All the improvement and advancement in plant breeding has been due to biotechnological advances. Not only the breeders but also the environmental problems are minimized due to the use of transgenic crops. The success and adoption rate of genetically modified crops is increasing rate worldwide. Intervention of new methods and approaches have reaches new limits and created many new opportunities to look forward. So, the future of precise and high accuracy of plant breeding lies in invention and discovery of different biotechnological application for crop improvement.