Molecular Approaches to Crop Improvement (2)

DNA Markers

It is estimated that each chromosome has 10⁸-10¹⁰ base pairs but only about 10% of the genome is actively coding with the result that a bulk of the genetic material remains unnoticed through phenotypic analysis. Furthermore, not much of the coded information may be easily observable that restricts the mapping of only a small fraction of the genome. The assessment of variation at the DNA level provides, at least in principle, mapping of each and every point of the entire genome. Specific spots on DNA molecule both in coding as well as non-coding regions can be identified as markers. In fact, the detection of naturally occurring DNA sequence polymorphism is the most attractive application of molecular biology for the welfare of mankind. A DNA marker is a small region of DNA showing sequence polymorphism in different individuals within a species or group of individuals. The interest in DNA based markers started from surprisingly highl level of variation for sequence  changes in DNA from different individuals.

Random Amplified Polymorphic DNAs (RAPDs)

This is a PCR based technique where a single short oligonucleotide primer which binds to many different loci, is used to amplify random sequences from a complex DNA template such as a plant genome. For most plants the primers that are 9-10 nucleotide long are expected to generate 2-10 amplification products (amplicon). The primers are generally of random sequence, biased to contain at least 50% GC content and to lack internal repeats. The products are easily separated by standard electrophoretic techniques and visualized by UV illumination of ethidium bromide stained gels. Pollymorphism results from changes in either the sequence of the primer binding site (e.g. point mutations) or from changes which alter the size or prevent the amplification of target DNA (e.g. insertions, deletions, inversions). In inheritance studies, the amplification products are transmitted as dominant markers (Waugh and Powell, 1992).

Molecular Approaches to Crop Improvement

MOLECULAR MARKERS

The progress in plant breeding is easy and quick for qualitative characters controlled by major genes with easily identifiable effect on the phenotype. But there are a number of plant characters which are difficult to be observed and where final phenotype does not give a reliable composition of the underlying genetic constitution. An individual may be carrying genes for resistance to say an insect but shall be known after it is exposed to insect attack. Similarly a male sterile plant cannot be identified before flowering stage and grain quality may not be known until before maturity of the crop. The identificatio of desirable genotypes for the quantitative characters like yield, quality, earliness, adaptation etc. is still more complicated. In all such cases the breeder attempts to select the desirable plants on the basis of some other easily observable attribute as a marker for the genes associated with the character of interest. Plant breeders have the convention of using many morphological characters as 'proxy' for the genes affecting complex characters. But the available number of such markers is too limited to serve as an index of each and every segment of DNA of the entire genome which may be controlling especially the quantitative characters. The available mutants can be assembled through breeding into the stocks under investigation but it requires additional efforts only for the purpose of finding association of genes with markers. The polymorphism at the DNA level provides a unique type of markers to serve as an index of genetic worth of the entire genome. An exact location of genes especially the loci governing quantitative traits (QTL) is basic and of paramount importance for map based cloning of these genes. Such markers can also be used to determine the nature of genetic variation in wild and cultivated types. But the most important use of these markers lies in the indirect manipulation of desirable genes in the form of marker-assisted selection (MAS). The following three classes of markers are available for use in plant breeding:

1. Morphological markers

2. Protein markers

3. DNA markers

Tissue Culture in Crop Improvement

Haberlandt?

Tissue culture is a technique of growing plant tissues on synthetic medium under controlled and aseptic conditions. G. Haberlandt, a German plant physiologist is considered to be the father of plant tissue culture who conceived the idea of totipotency in 1902 which refers to the capability of a cell to give rise to a complete plant under suitable cultural conditions. Such a property of cell has far reaching implications to manipulate plant cells for rapid multiplication of plants, to cross plants at the level of somatic cells by overcoming limits of crossability, and also to regenerate adult plants after modifying the DNA molecule at cellular level. Suitable explants i.e. organs excised from plants such as roots, hypocotyls, cotyledons, leaves, shoot apices, nodal segments, anthers, embryos, and seeds are surface sterilizedwith a disinfectant like sodium hypochlorite (10-50% w/v; 10-30 minutes) or with mercuric chloride (0.1%, w/v; 5-10 minutes), thoroughly washed with sterile (autoclaved) water and then aseptically cultured on a synthetic medium in the culture vessels like test tubes, jars and petridishes. The cultures incubated at 25±1℃ exhibit growth in 1-3 weeks depending upon the plant species, nature of explant, type of culture medium, kind and concentration of the growth regulators (hormones) used in the medium and the light intensity in the incubation room.A large number of media have been developed for plant tissue culture but the most commonly used include: Murashige and Skoog, 1962 (MS-1962), White (1963), Gamborg et al. (1968) (B5) and Chu (1978) (N6) medium. 

Tissue culture medium contains major (macro) elements, micro elements, vitamins and amino acids, carbohydrattes (sucrose) and growth regulators (auxins, cytokinins). Auxins such as Indole acetic acid (IAA), Indole butyric acid (IBA), Naphthalene acetic acid (NAA) at concentrations ranging from 0.1-5.0 mg/l favour cell elongation and rooting, whereas 2,4-Dichloro-phenoxyacetic acid (2,4-D) at concentrations of 0.5-4.0 mg/l usually induces callus i.e. homogenous mass of undifferentiated cells. Likewise, cytokinins such as 6-furfuryl amino purine (kinetin) and benzyl amino purine (BAP) at concentrations (0.1-2.0 mg/l) cause rapid cell divisions and development of shoot buds/shoots. The solidification of the medium is achieved by adding chemically inert powdered gelling agents such as agar before autoclaving. The medium is poured into culture vessels such as agar before autoclaving. The medium is poured into culture vessels and sterilized using autoclave at temperature 121 ℃, 15 lbs/sq. inch pressure for 20-25 minutes. Inculation of explants in the culture vessels is done in Laminar Air Flow Cabinet fitted with HEPA filters (pore size 0.2-0.3 µm).

Approaches to Germplasm Conservation

Germplasm resources is a very wide term that covers all the allelic resources spread in types ranging from most primitive wild progenitors to the highly bred cultivated varieties and strains. The scope and procedures of collecting, conserving and utilization of these resources depends upon their biological status. The germplasm may fall in either of the following categories:
(a) Wild relatives
These are the types which contributed directly or indirectly to the evolution of a crop plant but still survive only in the wild stage. Being strictly the product of nature, these types carry useful genes to protect plants against all types of stresses e.g. diseases, pests and are endowed with hardiness, tolerance to heat, frost, drought, excessive water etc. On account of the requirements of their survival without human care, these types have characteristic behaviour for germination, growth patterns, reproductive behaviour that makes their use in plant breeding bit difficult.
(b) Weedy relatives
There is a strong chaing of transformation from wild to weedy and then to cultivated form for each crop where weedy relatives serve as a bridge for transfer of genes. Wild species survive under situations that are undisturbed by man for cultivation. The weedy relatives are, therefore, easy to grow under normal crop condition and serve as a vehicle of transferring genes from wild to cultivated types.
(c) Primitive cultivars or land races
These types represent the product of nature or the effect of human selection under cultivation particularly developed over a prolonged period of time. The original material might have been selected by each farmer according to his requirements and the environmental factors splitting it into different types. These primitive types survive for such a prolonged period only on the best source of genes for resistance. The fact that these are farmer's varieties avoids all the problems associated with collection, maintenance and use in plant breeding.
(d) Modern cultivars
This type of germplasm represents the assembly of useful genes whose survival is dependent upon human interference. Such populations carry a greater bulk of useful allelic resources for most of the economically important characters.
(e) Advanced breeding lines
This represents the most advanced product of plant breeding activity which combines useful genes wherever from these are available. Elite genetic stocks, as these usually are called, serve as the most useful type germplasm which can be easily maintained and used for further breeding.

Baffled

A storm of laughter broke loose when it finally occured to the class that Mr.Harno had whipped me.
On another occasion.
A silence began when I start reading a poem.