Zain, S.M.
Faculty Agro-technology and Food Science, Universiti Malaysia Terengganu, Malaysia E-mail: [email protected]
Abstract
Crop diversity is the difference of cultivated plants used for producing food, fodder, fibre, fuel, pharmaceuticals, etc. Nearly 2,500 plant species have been cultivated worldwide and about 120-130 of them have been considered as the commercial crops. Among these, only 30 cropsprovide 95% of human food and just four of them, i.e. rice, wheat, maize and potatoes provide 60% food for the mankind. Crop plants evolved from the wild species through domestication bringing about genetic, physiological, morphological, and biochemical changes which render the plant differs from its wild form. Nowadays, a wide range of genetic and phenotypic diversity, called plant genetic resources (PGR), exists in the crop species. PGR provides the basic genetic material for improving crop varieties.
Knowledge on the collection, documentation, characterization, evaluation and conservation of PGR are important to ensure that effort to save PGR from becoming extinct and make it always available to plant breeders are continued and enhanced. Variety improvement is a process of changing the genetics of plants using plant breeding technique in order to produce plants with characteristics desired by man. Techniques of plant breeding including conventional technique of selection and cross breeding, and the modern techniques of controlled pollination, hybridization, embryo rescue, recombinant DNA, doubling chromosome set/ number will be described in this paper.
Keywords:crop improvement, diversity, plant genetic resources
Introduction
Biodiversity or biological diversity refers to the variability among living organisms (plants, animals, fungi, microbes, lichens, etc) and the ecological systems in which the organisms live. Biodiversity involves ecosystem, species and genetic diversity.Ecosystem diversity refers to the differences of life forms in a given territory or area and the ecological processes that make them function. Species diversity encompasses the variety of different species within a geographical area. Genetic diversity denotes the variation of genes or the functional units of heredity within any species.
Variety improvement of a crop is an effort to improve the characteristics through reconstructing the genetic composition of the crop, so that it became more desirable agronomically and economically. Improvements can include things like better disease resistance, drought tolerance, fruit size and rapid growth comparing to the previous one.
Plant genetic diversity is the only materials useful for variety improvement including the development of new cultivars.
The objective of this paper is to explain the origin, role and assessment of crop genetic diversity, and to share several points on the techniques of crop variety improvement for sustaining agricultural production.
Crop Diversity
Crop diversity is the difference of cultivated plants used for producing food, fodder, fibre, fuel, pharmaceuticals, etc in any particular area or region. The example of crop diversity is an orchard garden where a large number (40 – 50) of different crop species could be present in one or two hectares of farmer’s land (Table 1) (Hawkes, 1983).
Table 1: Crop diversity with different species of economic important in an orchard garden in the Southeast Asia.
Source: Hawkes, 1983
Table 2. The world’s top 30 food crops (Ford-Llyoyd and Jackson, 1986).
Nearly 2,500 plant species have been cultivated worldwide for various reasons. This number includes about 850 food species which is covering 370 fruits, 280 vegetables, 60 edible flowers, 60 seeds/nuts/grains and 80 root and tuber crops (PROSEA, 1993-1999;
Malik and Singh, 2006). Among these only 230 species are widely cultivated in which about 120-130 species are considered as the major or commercial crops. However, only 30 crops (Table 2) provide 95% of human food energy needs and just four of them, i.e. rice, wheat, maize and potatoes provide 60% food for the mankind (Ford-Llyord and Jackson, 1986).
Genetic Diversity
Although the number of major crops is significantly small but the genetic and phenotypic diversity within a crop are very great. For example, the number of distinct varieties of rice (Oryzasativa)worldwide is estimated more than 40,000 varieties. Also, the number of land races variety of potato (Solanum tuberosum) in Andes, South America was more than 175 varieties. Similarly, a great genetic diversity was also found in other major crops such as wheat, barley, maize, sorghum, millet, etc. (Harlan, 1975; Hawkes, 1983; Ford-Llyord and Jackson, 1986; Hasan, et al, 2008; Hasan & Abdullah, 1997).
Domestication Increases Crop Variability
The cultivated variety of a plant has evolved from the wild species through a process of domestication (Lester and Hasan, 1991; Simmonds, 1984; Hawkes, 1983). Since the beginning of agriculture about 10,000 years ago wild plants from natural population have been taken into cultivation by man (Harlan, 1975). During the course of cultivation, man consciously and unconsciously selects plants with useful characters and bringing about genetic, physiological, morphological and biochemical changes which render the plant differs from its wild form (Hawkes, 1983). Subsequent selection pressure occurred continually on the crops through the differentiation-hybridization cycle. In this cycle, cultivars were separated for a while by gene flow barrier due to geographical, ecological or morphological differences that allow crop establishes new sub-populations or cultivars.
Soon or later, crossing between two cultivars results in a release of great variability within the crop. Selection pressure provided by different culturing practice, soil fertility, irrigation, etc accelerated the increase and distribution of variation in a crop (Ford-Llyord and Jackson, 1986).
Centre of Genetic Diversity
Genetic diversity of our major food crops mostly occurred in one of the eight mega-diversity of the world (Ford-Llyord and Jackson, 1986) (Table 3). It is the place or region where the crop shows the greatest variability ranging from new cultivars to landraces, weedy and wild relative forms. These regions were considered as the areas in which the domesticated plants have taken place and in some cases, were also considered the places of origin of the crop (Hawkes, 1983).
Plant Genetic Resources (PGR)
Now, a range of genetic and phenotypic diversity, called plant genetic resources (PGR) existed in the cultivated species ranging from the wild to fully cultivated state. Such diversity is an important plant genetic resources that provides the basic genetic material for improving crop varieties. PGR or germplasm is a living tissue from which the new plant can be grown. PGR is grouped into; i, primitive cultivars or landraces, ii, obsolete cultivars, iii,
modern or advanced or elite cultivars, iv, breeding lines, v, genetic stocks, vi, weedy types and vii, wild relatives (Hawkes, 1983).
Table 3. List of the world important crop in its centre of diversity and possible place of origin (Ford-Llyoyd and Jackson, 1986).
Erosion of PGR
The existing of the present day plant genetic resources is seriously endangered. In nature, PGR has been reduced due to the industrial development, deforestation, climatic changes, changing in agricultural practices, disease and natural disasters. The diversity of local and well-adapted landraces has long been replaced by modern cultivars, particularly in
monoculture system where a single high-yield variety has been used, such as in rice and oil palm industries.
Figure 1. Diversity of tuber morphology (size, shape and branching patterns) in traditional cultivars or land race of yam (Dioscorea alata) (a) and eggplant (Solanum melongena) (b) found in Malaysia (Hasan, et al., 2008; Hasan & Abdullah, 1997)
Figure 2. a. Crossing polygon showing diversity in cross-ability relationship between cultivated S. melongena weedy form (E), landraces (F), obsolete cultivar (G) and elite cultivar/ black beauty (H), and its putative wild relatives S. incanum, groups A, B, C and D (Hasan, 1989).
b. Schematic diagram of primary gene pool (GP1), secondary gene pool (GP2) and tertiary gene pool (GP3) in eggplant genetic resources (Hasan and Lester, 1992).
Management of PGR
Proper management including collection, characterization, evaluation and conservation of PGR is needed to ensure that the genetic resources of a species are saved from becoming extinct and are always available to the breeders and other users. Collection involves
gathering samples of a species from populations in the field or naturalhabitats for conservation and subsequent use. Characterization refers to recording of characters which are highly heritable, easily identified (usually qualitative), and are expressed in all environments. Evaluation refers to documentation of additional characters (often quantitative traits relating to yield and symptom of diseases) which are thought desirable by the users or breeders of the crop (Hasan, et al., 2008; Hasan & Abdullah, 1997).
Conservation involves maintaining PGR whether in the natural habitats where they occur (in-situ conservation), or outside the native habitat (ex-situ conservation) such as seed storage in gene-bank which capable to maintain the integrity large number of the germplasms over prolonged periods of time (Figure 3).In vitro conservation through tissue culture techniques is also considered as an ex situ conservation (Figure 4a). In vitro conservation can maintain the materials in a pathogen-free environment and are not subjected to environmental disturbances. They fall under two categories: (i) slow growth procedures (Figure 4b) and (ii) cryopreservation (Figure 5).
Figure 3. Seed storage in National Genebank at Tsukuba, Japan (Visited by the author in 1994)
Figure 4. a. In-vitro storage of pineapple germplasm in standard tissue culture needed sub-culturing at every three months of culturing (Hasan and Nursuraya, 2007).
b. Reduced growth storage of yam (Dioscorea alata) germplasm in medium culture supplemented with osmotic agent of mannitol (Hasan and Lasim, 1996)
a b
Figure 5. Shoot-tip tissue culture encapsulated in alginate-bead (a) and kept in cryo-tube (b) for cryopreservation test in liquid nitrogen at -160° C (Hasan and Takagi, 1995)
Estimating Genetic Diversity
Estimating genetic diversity is essential for effective use of genetic resources in variety improvement. The level of genetic variability would determine the PGR either having a wide or narrow genetic base. Presently, beside agro-morphological traits, bio-chemical such as protein and molecular DNA (RAPD, RFLP, ISSR, etc) are also used as the markers to assess the extent of genetic variability present in the PGR collection (Figs. 6 and 7) (Hasan, et al.
2010, 2009, 2008, 2006; Hasan & Isa, 1998; Hasan & Abdullah, 1997).
a b
Figure 6. Diversity of seed protein profile (banding pattern) in eggplant (Solanum melongena) genetic resources collected in Malaysia, where it was rather uniform in population of advanced cultivar (a) and quite diverse in old or obsolete cultivar (b) (Hasan and Isa, 1998)
Assessment of diversity is made by comparing the differences of the markers between individuals or populations using statistical analysis (Dunn and Everitt, 1982). Comparison of mean and variances among the populations are the simple way to estimate the diversity (Hasan and Abdullah, 1997). Calculating the similarity coefficient is another way to assess the PGR diversity (Table 4) (Hasan, et al, 2010 and 2009). Recently, multivariate analysis of principal component analysis (PCA) and cluster analysis (CA) using SPSS computer program is commonly used in diversity study. In this analysis, similarity or dissimilarity index of
individuals is used to separate the individual on the PCA and also to cluster the individuals in order to construct the hierarchical relationship among the individuals (Figs. 8a & 8b). The PCA is often used to find out the most variable characters in the population, while cluster analysis (CA) is used to elucidate the relationship among individual in the PGR collection (Hasan, et al. 2010, 2009, 2008 & 2006)
OPG-02 primer
Figure 7. RAPD banding patterns of twenty seven (27) D. alata accessions based on a primer OPG-02. Each lane represents an accession marked above. Lane M is a marker of 100bp DNA ladder plus
Table 4. The similarity coefficient among twenty seven (27) accessions of yam (Dioscorea alata) genetic resources in Peninsular Malaysia after analyzed data from RAPD marker using Jaccard’s similarity coefficient (Hasan et al., 2006)
Gene Pools Concept
The ease of crossing among the groups of PGR also varies (Figure 2a). This variation is used to classify PGR according to the gene pools concept (Figure 2b) (Hasan & Lester, 1992) in order to assist breeders to select particular genes or variant for use in crop improvement. In this concept PGR is classified into four gene pools (GP) (Ford-Llyord and Jackson, 1986;
Harlan, 1975).
GP1: The crop itself and wild relatives that are easily inter-crossed and produce fertile progeny.
GP2: Species that cross to GP1 but with difficulty and produce progeny with reduced viability
GP3: Species that cross to GP3 only using advanced techniques (embryo rescue, tissue culture) and produce progeny with low viability
GP4: Other species that do not cross to GP1 at all. Molecular genetics and gene cloning techniques must be used to transfer these genes into species in GP1.
a
b
Figure 8. a. Two-dimensional plot of the first two principal components (PC-1 and PC-2).
Accessions that are encircled by the indented lines and marked as groups A, B, C and D in the cluster analysis (Figure 9b ) (Hasan, et al., 2008).
b. The hierarchical dendogram showing the relationships among seventy accessions of D. alata collected in Malaysia (Hasan, et al., 2008).
Euclidean dissimilarity distance
0.88 2.77 4.65 6.54 8.42
B A
A1
A2
D
A A A
C
A
B
a b
Figure 9. Artificial cross-pollination in eggplant (a), and growing progeny of eggplant for selection (b). The work had been conducted by the author in 1990 – 1993 at UPM, Serdang, Malaysia (Hasan, 2005; Hasan & Abdullah, 1997).
Figure 10. Germination of hybrid seed or embryo rescue in medium of tissue culture Variety Improvement
Variety improvement is a process of plant breeding, that is a technique of changing the genetics of plants in order to produce plants with characteristics desired by man. Basically, the process started with the searching and identification of desirable trait from the gene pools and this greatly depends on the genebank collection. Then followed by creation of variation that normally done through the hybridization process (Figure 10a). The best plants are selected from the derived progeny and spread into a large population (Figure 10b).
These plants are allowed to cross-pollinate, and seed is harvested from them. The process is repeated with many following generations, until the desired characteristics are exhibited on the general population. The new variety is then released and dispersed to the public.
Aspects of Variety Improvement
The main aspects of crop variety improvement are :
- Improved yield: To increase and stable crop yield production.
- Improved quality: To produce better size, colour, shape, taste and nutritive value in food.
- Enhance biotic and abiotic resistance:To increase resistance against pest and disease and environmental stress.
- Shorter in maturity duration: To reduce duration and synchronous maturity. Early maturity can make the crop double or triple cropping system and reduce the cost of production.
- Modifying agronomic traits:To change the agronomic characteristics of crops such as plant height, tillering, branching, determinate growth, etc.
- Photo and Thermo insensitivity: To develop varieties insensitive to light and temperature to permit crops grown in areas, which might not have been ideally suited for their growth (Example Strawberry in Cameron Highland)
- Wider Adaptability: To develop varieties with wider adaptability and thus help stabilize crop production under unfavourable environmental conditions (Hawkes, 1983).
Methods of Plant Breeding
Plant breeding can be accomplished through many different techniques ranging from simply selecting plants with desirable characteristics to more complex molecular techniques for improving crop variety (Allard, 1960). Any one technique adopted, however, is depending on the reproductive system of the crop and objectives of the improvement.
Based on the reproductive system, the crops are divided into self-pollinated/ inbreeding and cross-pollinated/ outbreeding groups. The major different between the two groups is related to their influence on the genetic structure of the populations (Ford-Llyoyd and Jackson, 1986).
Conventional Plant Breeding
Conventional plant breeding is a breeding method practiced by the breeders since 1900 when the Mendel's laws of genes inheritance have been adopted as the scientific basis for plant breeding. As all traits of a plant are controlled by genes located on chromosomes, conventional plant breeding can be considered as the manipulation of the combination of chromosomes. In general, there are two main procedures in conventional breeding are used to manipulate chromosome combination for improving the crops.
1. Selection
Selection is the basic procedure in variety improvement. It generally involves first, selection large number (mass selection) of plants from the genetically variable original parent population. Second, the progeny from the individual selected plants are grown and the progenies are selected again and grown for over several generations. The final selected progeny are also tested for selection under different environmental condition. Thirdly, the new varieties are then compared to the existing commercial varieties in their yield performance and other aspects of agronomic importance. There are two selection methods are applied in self-pollinated crop, pure-line selection and mass selection. Also two
selection methods are applied in cross-pollinated crop, mass selection and recurrent selection (Allard, 1960).
2. Hybridization/ Cross-pollination
Hybridization is an effort to bring together the desired traits found in different plant lines into one plant line via controlled or artificial cross-pollination (Figure 9a) (Hasan, 2005). The first step is to generate homozygous inbred lines. This is normally done by controlled self-pollinating (Figure 9a) where pollen from male flowers is pollinated on the female flowers from the same plants. Once a pure line is generated, it is then out-crossed with another inbred line. The segregating F2 progeny is then selected for the desired traits (Figure 9b). If a trait from a wild relative of a crop species, such as resistance against a disease, is to be brought into the genome of the crop, the undesired traits (like low yield, bad taste, low nutritional value) are then removed by repeated back-crossing with the crop parent.
Modern Technique of Hybridization
In general, there are three types of hybridization: varietal (narrow crosses), inter-specific and inter-generic hybrids (wide crosses). Beyond this biological boundary, hybridization cannot be accomplished due to sexual incompatibility (incongruity), which limits the possibilities of introducing desired traits into crop (Hasan, 1990; Hasan & Lester, 1990). Nowadays a number of techniques were developed to overcome these barriers and that allowed plant breeders to hybridize distantly related species.
Bridge Cross: When a direct cross between two species is not possible, an intermediate crossing with a third species, which is compatible with both species, is used to break down the crossing barriers.
Pollination using sub-optimal age of stigma: In order to overcome incompatibility barriers posed by the stigma or style, mature pollen is placed on a stigma that is immature to prevent presence of active factors that inhibit pollen tube growth.
Pollination with chemical application; Plant growth regulator (hormones) such as auxin, cytikinin and gibberellins are applied to flowers to promote pollination.
Pollen mixture: A mixture of incompatible pollen with temperature pre-treated compatible pollen is used in pollination. The compatible pollen is able to germinate and penetrate the style, thereby clearing the way for the incompatible pollen.
Style treatment: Heat treatment of stigma, style or the whole flower before pollination.
Heat treatment can reduce the incompatible reaction of the stigma surface, the style or the whole flower before pollination.
In-vitro Methods: This technique can be considered as a high level of sophisticated tools applied in plant breeding. The technique is conducted under the aseptic condition. Some of the methods have been used as a routine tool and some others are still at the experimental stage in plant breeding.
a. Pollination after manipulation of style: Removal of the stigma or shortening of the style to remove the factors that inhibits pollen tube growth.
b. Graft style method: a style with pollen germinated on a compatible stigma is cut and put on an ovary of the other incompatible parent where the stigma has previously been removed.
c. In vitro pollination: Pollen is applied on the stigma under aseptic condition. The flower bud is removed from the plants and after dissection of flower parts, the bud is put on a growth medium. Fertilized ovules are then subsequently isolated and further cultured on fresh medium. Pollen and ovule can also be brought into direct contact for fertilization. Pollen can also be placed on the placenta of the ovary.
d. In vitro culture of excised ovaries: After pollination, the ovary is excised from the flower and cultured aseptically on a suitable medium.
e. Embryo rescue; If fertilization is possible between two species or genera, the hybrid embryo may abort before maturation due to incompatibility with the endosperm or surrounding maternal tissue of the ovary. If this does occur the embryo resulting from an interspecific or intergeneric cross can sometimes be rescued and cultured on media to produce a whole plant. Such a method is referred to as Embryo Rescue (Figure 10) (Hasan & Lester, 1990).
f. Induced mutation or mutation breeding: The mutation is induced by chemical mutagens like EMS and DMS, radiation (beta or gamma ray) and transposons to generate new variances with desirable traits and used to bred with other cultivars.
The mutant hybrids are tested and further selected for desired traits. Because the great majority of mutants carry undesirable traits, this method has not been widely used in breeding programs.
g. Somaclonal variation: Plant breeders also generate genetic diversity within a species by exploiting a process of somaclonal variation, which occurs in plants produced from tissue culture, particularly plants derived from callus.
h. Somatic Hybrid Plants: Somatic hybrid plants are plants derived from the fusion of somatic cells. Cell fusion is done by making contact a number suspension cells after stripped of their cell walls, or referred as protoplasts. Methods such as particle bombardment, electroporation and polyethylenglycol permeabilisation are used in chloroplast fusion breeding. Since, protoplasts from distinctly unrelated species can be fused, the technique have been used to overcome sexual incompatibility of the cross species. The technique is uneconomic to practice, but contributes important knowledge in understanding somatic hybrid in plant.
i. Genetic Engineering: The incompatibility problem to transfer genes can be overcome by using the methods of genetic engineering or recombinant DNA technology, which in principle allow introducing valuable traits through carrier organism (other plants, bacteria, fungi, animals, viruses) into the genome of any plant. Recently, transgenic plants have been obtained using Agrobacterium-mediated DNA-transfer and direct DNA-transfer. When a desirable trait has been bred into a species, a number of crosses to the favored parent are made to arise the new plant similar to the favored parent as possible. The new plant is called transgenic plant or genetic modified organism (GMO).
3. Doubling the chromosomes set / Polyploidy/ Haploidization
Most plants contain two sets of chromosomes number and called diploid plant. Plants with three or more sets of chromosomes are also common and are referred to as polyploids. The increase of chromosomes sets can be artificially induced by applying a mitotic inhibitor, colchicine, which leads to a doubling of the chromosome number. The sterile hybrid can be retrieved and producing fertile progeny.
Haploid plants can be arisen in tissue culture from pollen (anther) culture or ovule (ovary) culture. The regenerated haploid plants are diploidized to obtain doubled haploids (homozygous) plants by using colchicine.
Conclusion
Regional centre of genebank should be established to conserve a vast amount of plant genetic resources present in this region. The PGR is extremely valuable genetic materials for the development of new variety and improving the existing one, particularly new variety with high resistant against biotic and abiotic stresses and high yield.
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