In this article we will discuss about the biotechnology in plant breeding.
In the conventional plant breeding programme, the development of a new variety or hybrid takes about five to twelve years, starting from inbred production and then hybridization and selection of F1 hybrids. To overcome the sexual barrier (pre-fertilisation and post-fertilisation), there is the need of modern non-conventional breeding methods.
One of the approaches is the use of ‘Biotechnology’ through different cell and tissue culture techniques and genetic engineering methods. Somatic hybrid production by protoplast culture-fusion technique, use of different molecular biological techniques and alien gene incorporation into the genetic background of cultivated species thus become obvious.
Tissue Culture Techniques and their Applications in Plant Breeding:
The culturing of plant cells or tissues in synthetic medium and their development into mature plants has immense potential for plant improvement.
There are few major avenues which were opened by plant tissue culture can be listed below:
Micro-Propagation:
The technique of micro-propagation or regeneration of plantlets from any tissue has been successfully achieved in case of wheat, rice, sugarcane, maize, barley and many other crop plants. But this technique is specially useful for propagation of medicinal plants which grow slowly and cannot be bred in the conventional methods.
Moreover the vegetative propagating plants, such as banana which multiply by rhizome and one plant can yield about 10 plants per year, through micro-propagation as many as 2,00,000 plant- lets can be obtained. Equally this technique is applicable to tree plants like teak, eucalyptus, etc.
The meristem culture helps to get the disease free plant and also the vegetatively propagated crop plants can be maintained in disease free condition for long time. Clonal propagation method used for some heterozygous plants, especially the ornamentals, helps a lot in breeding programme.
Maintenance and multiplication of self-incompatible inbred line (male sterile line) is possible by tissue culture methods very easily. Mutagens can be applied to single cell and the effect can be detected easily, isolated and utilised fully for new variety production through tissue culture.
Embryo Culture:
Distant hybridization programme sometimes yields non-viable embryo, then the embryo culture method and embryo rescue help to obtain the viable hybrids. Embryo rescue or embryo culture is with the objective to rescue the embryo which aborts at an early stage of development, i.e., no mature seed can be obtained.
The hybrid embryos are excised and put on a synthetic medium so that they can develop seedlings. Most extensive-use of this technique has been observed in raising the interspecific and inter-generic crosses within the tribe Triticeae of Poaceae.
Distant hybridization programme sometimes eliminates the chromosomes of one parent, thus the culture of hybrid embryo allows to develop the haploid plant. For example, the inter-generic cross between maize (Zea mays) and wheat (Triticum aestivum) has resulted in production of monoploid wheat plant.
Protoplast Culture and Fusion:
Protoplast culture technique itself has an immense potential for crop improvement programme, as the alien gene introduction or incorporation is more easier in this way and transgenic or genetically modified crops can be regenerated.
Protoplast fusion, i.e., somatic hybrid production shows a new path to overcome the sexual barrier between distantly related wild and crop plants. This will help to transfer some useful characters like disease resistance, salt tolerance, drought tolerance, etc.
Somatic hybrids between rice (Oryza sativa) and barnyard grass (Echinochloa oryzicola) has been obtained. The most extensive programme has been in progress in the family Brassicaceae where the different traits like drought tolerance from Eruca, pathogen resistance from Sinapis, cytoplasmic male sterility or CMS from Diplo-taxis have been transferred in cultivated Brassica.
Cybrids or cytoplasmic hybrids are obtained through following methods:
(i) Fusion of normal protoplast with enucleated protoplast from other parent,
(ii) Fusion of normal protoplast from one parent and protoplast with non-viable nuclei from other parent. In Brassica, the success has been achieved using this technique, i.e., CMS line with ‘Ogura’ cytoplasm, herbicide (atrazine) resistant trait has been transferred to cultivated variety.
Haploid Culture:
Haploid plants from anther and pollen culture and diploidisation of these haploids help a lot to get the homozygous inbred lines which are to be used in breeding programme.
The haploid plantlet production is aimed through another culture or pollen culture, where the embryoids developed from this culture (haploid) may be treated with colchicine to get diploid homozygous plants which may be used in breeding programme. In China more than 100 rice varieties developed using the technique to give an increased yield.
This type of haploid production technique has been successfully used for breeding of barley, maize, sugarcane, oilseed rape and some other crops.
The greatest usefulness of another culture lies in the rapid production of haploid plants which are of great value in plant breeding and genetics. An unlimited number of haploid plants can be produced within short time; success has been achieved in barley, rice, wheat, potato, tomato, etc.
Mutants of any nature can be detected easily as allelic interactions are non-existent. Another culture avoids natural loss of inbred lines due to excessive inbreeding depression. The non-viable gene-combinations causing sterility are promptly exposed, so selection is automatic.
Somaclonal Variations:
From the observation of Larkin and Scowcroft (1981) it is obvious that natural variability in tissues, i.e., somaclonal variation can be utilised at selection level. Somaclonal variation can be generated through tissue culture technique and the selected clone can be produced in mass scale. There are various reports in many crops where the different somaclones have been reported.
Gene Transfer Techniques in Plant Breeding:
In plant breeding, techniques involving gene transfer through sexual and vegetative propagation are well established. The aim being to introduce genetic diversity into plant population and to select superior plants carrying the desired traits and to introduce some new characters into the cultivar, with the rapid improvement of genetic engineering techniques based on the knowledge of gene structure and function, plant breeding method has been changed.
The directed desirable gene transfer from one organism to another and the subsequent stable integration and expression of foreign gene into the genome is referred as genetic transformation. The gene is called transgene and the changed plants carrying the stably integrated desirable gene are transgenics.
In vitro gene transfer technique allows transferring desirable genes across taxonomic boundaries into plant from other plants, animals, microbes or any artificial, synthetic or chimeric gene also. The techniques presently rely on natural plant vectors as well as vector-less systems, which include directed physical and chemical methods for delivering foreign DNA into plant cells.
One of the most important thing of these techniques is the potentiality of the recipient ceil to express the introduced gene. Only a small fraction of cell get transformed i.e., DNA gets stably integrated into the chromosome of the cell.
The DNA introduced into majority of the cells is lost with cell division. Stable transformation occurs when DNA is integrated into the plant nuclear or plastid genomes, expression occurs in regenerated plant and is inherited in subsequent generations (Fig. 7.1).
Different Transfer Techniques:
(a) Agro-infection or Agrobacterium mediated gene transfer method is widely used with engineered Ti plasmid (modified T-DNA) in case of most dicot plants as well as monocot.
(b) Direct or physical transfer method is commonly used in case of cereals as these are naturally reluctant to Agrobacterium infection.
There are many types of delivery systems like:
(i) Biolystic or Particle bombardment,
(ii) Electroporation,
(iii) Microinjection,
(iv) Pollen transformation,
(v) Liposome mediated transfer,
(vi) Silicon carbide fibre (SCF) mediated transfer,
(vii) PEG mediated transfer.
All these different techniques have been applied in different plant materials using different kinds of genes for many desirable traits of agronomic values e.g., the genes for stress tolerance i.e., drought, salt and temperature stress (physical or abiotic stress); genes for disease resistance such as resistance against any pathogen by producing toxin, PR protein, plantibodies, or RNA mediated resistance gene; herbicide resistance, insect resistance; genes for development of male sterile line and also the restorer line; genes for better nutritional quality as in many cereal crops (rice, wheat, maize), oil seed crops (Brassica), pulses and vegetables (potato, tomato); etc. Many transgenic plants bearing the desirable traits have already been released as variety.
Few of the major achievements have been listed below:
Molecular Breeding Technique (Use of DNA Markers in Plant Breeding):
Molecular breeding using DNA markers often provide a wide array of applications in the field of plant improvement. Molecular markers are used for the analysis of genetic variation in germplasm available for plant improvement.
Molecular marker aided breeding strategy involves the potentiality of molecular markers in plant breeding, particularly helps in marker assisted selection procedure which speeds up the whole breeding process. Molecular markers are DNA sequences whose inheritance pattern can be established.
Some of the unique features of molecular markers are:
(i) They exhibit polymorphism,
(ii) They show co-dominant inheritance which helps in distinguishing homozygous from heterozygous,
(iii) They are easy for detection, and
(iv) They are distributed frequently throughout the genome.
Different molecular markers in use are:
(a) RFLP (Restriction Fragment Length Polymorphism)
(b) RAPD (Randomly Amplified Polymorphic DNA)
(c) AFLP (Amplified Fragment Length Polymorphism)
(d) VNTR (Variable Number Tandem Repeats)
(e) STS (Sequence Tagged Sites)
(f) SCAR (Sequence Characterised Amplified Region)
(g) SNP (Single Nucleotide Polymorphism)
Molecular marker development and its implementation in breeding programme has made the whole breeding exercise less time consuming and offers selection of desirable combination of traits. This approach is done by establishing linkage between molecular marker and traits to be selected.
In this process the whole breeding procedure can be conducted in laboratory not waiting for the phenotypic expression in field, e.g., resistance property to plant pathogen can be evaluated in the absence of disease.
Marker Assisted Selection (MAS):
(a) Mapping of Plant Genome:
Among several crop plants, rice has been the most wanted target plant. RFLP markers from closely related species are good markers for constructing gene map.
(b) Linkage of Molecular Marker to Desired Trait:
Identification of genes responsible for useful trait may be established by a linkage analysis with markers on a genetic map of plant genome. Polymorphic markers are generally used to identify linked markers.
Bulked segregate analysis (BSA) helps to detect the polymorphism between two species. Then the segregating population is tested, generally the polymorphism between the species is likely to be linked to genes for the trait.
(c) Accelerated Back-Crossing:
Marker assisted selection accelerates the back crossing and selection for desired trait which help in earlier release of improved variety. Instead of several back-crossing and then selection which requires lots of time, molecular markers facilitate selection of individuals with more of the recurrent genome at each generation.
Applications of MAS:
Markers have been used in the breeding of desirable disease resistance property against virus and fungal pathogen. For introducing this trait into cultivar variety, MAS helps in the following ways for the new breeding strategy.
Identification of the Breeding Line:
Molecular markers are used to identify the breeding line among the large number of germplasm available.
Identification of Hybridity:
Using RAPD analysis the somatic hybrid or the hybrid nature of self-pollinated crops can be easily identified.
Purity of Breeding Lines:
Cross contamination and seed harvestation may lead to contamination of breeding lines. Molecular markers can be used to assist establishment of pure breeding lines and check contamination of breeding.
Prediction of Hybrid Performance (Heterosis):
Genetic distance between possible parents can be estimated by employing molecular markers. RFLP microsatellite markers are selected as useful marker for these predictions.
Identification of Germplasm:
Identification of several useful genetic resources of possible parents for use in breeding requires suitable molecular technique. RAPD marker is useful tool for the survey of germplasm. Survey of rice germplasm using RAPD shows linkage between the presence of specific marker and QTL for novel character.