eChapters

Objectives

• Better understand the various types of gene interaction.

Introduction

Gene interaction is the phenomenon of two or more genes that affect each other’s expression in different ways in the development of a single character of an organism. It is the modification of normal phenotypic expression of gene due to interaction between alleles or non-alleles. In such situation, two or more than two genes may interact to give rise to a particular phenotype

Objectives

• Better understand the phenomenon of probability.

Introduction

The concept of probability is basis to many principles of classical genetics (segregation, independent assortment etc.) The principals of population genetics are based on the considerations of laws of probability. Probability is the likelihood of occurrence of an event.

Trial

A trial refers to an opportunity provided for an event to occur, e.g. tossing of a coin once constitute one trial as it provides an opportunity for head or tail to occur. A new borne can be either a boy or a girl.

Definition

Based on various standard sources, plant breeding has been defined in various ways. A few important and standard definitions are:

The art and science of changing plants genetically.

Plant breeding is the genetic adjustment of plants to the service of man.

Improvement of crop plants as the result of process of evolution directed by man for his own ends. The basic principles of plant breeding are thus the principles of evolution. Directed evolution as imposed in plant breeding differs from natural evolution chiefly in a greatly reduced time-scale, and in a more precise control over the factors which govern hybridization and selection.

The science and art of manipulating the heredity of plants for a specific purpose.

Plant breeding is a deliberate effort by human to nudge nature, with respect to the heredity of plants, to an advantage. The changes made in plants are permanent and heritable and the professionals who carry out this task are called plant breeders. This effort at adjusting the status quo is instigated by a desire of humans to improve certain aspects of plants to perform new roles or enhance existing ones. As a result, the term “plant breeding” is often used synonymously with “plant improvement” in recent years. Normally the term plant breeding connotes the involvement of sexual process in effecting a desired change; however, it is also true that modern plant breeding also includes the manipulation of asexually reproducing plants (plants that do not reproduce through the sexual process). Breeding is therefore about manipulating plant attributes, structure, and composition, to make them more useful to humans.

Gepts and Hanock (2006) have defined plant breeding as an applied, multidisciplinary science. It is the application of genetic principles and practices associated with the development of cultivars more suited to the needs of human than the ability to survive in the wild; it uses knowledge from agronomy, botany, genetics, physiology, pathology, entomology, biochemistry and statistics. Of particular importance is the ability to transfer, in addition to major genes, large suites of genes conditioning quantitative traits such as productivity and other traits of interest to humans. The ultimate outcome of plant breeding is mainly improved cultivars. Therefore, plant breeding is primarily an organismal science even though it is eminently suited to translate information at molecular level (DNA sequences, protein products) into economically important phenotypes. The traditional definition of plant breeding includes only those scientists who develop new cultivars and improved germplasm, however, many feel this definition should be expanded to include scientists who contribute to crop improvement through breeding research.

Self-Incompatibility

This is lack of self-fruitfulness and is a condition where the pollen from a flower is not receptive on the stigma of the same flower resulting into no seed setting. This happens despite the fact that pollen and ovule development both are normal and viable. This is caused by genetically controlled physiological hindrance to self-fertilization. This occurs widely among diverse families of flowering plants including the Leguminosae, Onagraceae, Rosaceae, Scrophulariaceae, Solanaceae, Compositae, Cruciferae, Papaveraceae and the Gramineae (now called as Poaceae). The incompatibility reaction is genetically governed by a single locus designated as S with multiple alleles whose number can go more than 100 in some species such as Trifolium pretense. East has estimated that this occurs in more than 3000 plant species distributed over 20 families. Lewis classified incompatibility systems into two main groups, namely, heteromorphic and homomorphic and homomorphic system has further been divided into gametophytic and sporophytic types as illustrated below:

Plant genetic resources (PGR) are the genetic material of plants which are of value for present and future generations of humankind. Often used as a synonym to plant germplasm, it can be defined as a seed, a plant or plant part including cell cultures, genes and DNA sequences that are held in a repository or collected from wild as the case may be and that are useful in crop breeding, research or conservation because of genetic attributes. The term is used to describe a collection of genetic resources for an organism or genetic material which forms the physical basis of inherited qualities. In brief, germplasm is the sum total of the hereditary materials in a species. Germplasm is the foundation and lifeblood of plant breeding without which plant breeding activities cannot be conducted. Germplasm is the genetic material that can be used to perpetuate a species or a population. The base germplasm can be improved for better performance of the crop. In the initial stage of a breeding programme, breeder evaluates the available germplasm lines and makes a selection from them for further test and final release to the farmers. Later on, the germplasm serves as a source of the parental lines which are used to initiate another phase of the breeding programme. Besides, using the base germplasm, breeders generate new variability using a variety of breeding methods such as crossing parental lines and developing new lines, induced mutations and more recently gene transfer. This new variability is again subjected to appropriate selection methods leading to identification and further evaluation of new and promising genotypes for release as cultivars. Generally, germplasm includes following types:

Heterosis or hybrid vigour is the condition where an F1 hybrid falls outside the range of the parents with respect to some character or characters. It is also defined as a manifestation of heterozygosity, expressed as increased vigour, size, fruitfulness and resistance to diseases, insects or climate extremes. It has been observed in nearly every crop including both self and cross-pollinated crops. It is converse of inbreeding depression. However, even the species that show little or no inbreeding depression, often benefit from crossing and resultant hybrids. Therefore, heterosis has been more emphasized than the inbreeding depression. It is defined in two basic ways:

Any phenotype is the result of interaction of genotype and the environment. Any genotype cannot express itself unless proper environment for its expression is made available. Likewise, environment cannot create any phenotype unless a genotype is there to be acted upon by the environment. However, the effect of genotype and the environment is not fixed, its keeps on changing depending upon the relative role of the genotype. Heritability therefore specifies the proportion of the total phenotypic variability that is due to genetic causes. In other words, heritability is defined as the ratio of genetic variance to the total phenotypic variance. Heritability is the degree to which the characteristics of a plant are repeated and expressed in the progeny. This can be expressed quantitatively as follows:

Heritability (h²) = Genotypic variance/Phenotypic variance

In a large random-mating population both gene frequencies and genotype frequencies remain constant from generation to generation in absence of migration, mutation and selection and the genotype frequencies are determined by the gene frequencies. These properties of a random mating population were first explained by G. H. Hardy and W. Weinberg independently in the year 1908 and are generally known as Hardy-Weinberg law. The operational understanding of this law involves three steps as follows:

1. From parents to the gametes they produce.
2. From union of the gametes to the genotypes in zygotes produced.
3. From genotypes of the zygotes to the gene frequencies in the progeny generation.

The gene and genotype frequencies in the original parent generation are assumed as follows:

Phenotype is the product of genotype and environment. In presence of genotype x environment interaction (differential performance of genotypes over the environments), phenotype is the product of genotype, environment and genotype x environment interaction. A stable genotype is one which interacts less with the environment or shows a minimum of g x e interaction. When g x e interaction is significant, one must investigate the specific types of g x e interactions such as genotype x location, genotype x year and genotype x location x year interactions by growing selected genotypes over multiple locations and years. Normally, the genotypes are evaluated for three years. A typical analysis of variance for g x e interaction is given in Table 7.1 (Allard, 1960).

The discovery of mutagenic effects of X-rays on the fruit fly (Drosophila) by H. Muller in 1927 and by L. J. Stadler in barley and maize in 1928 paved the way for researchers to experiment with its effects on various organisms. In 1928, H. Stubbe showed the use of mutagenesis in producing mutants in crop plants like tomato, soybean and others. The first commercial mutant was produced in tobacco in 1934. Reports by B. Sigurbjornsson and A. Micke mentioned 77 cultivars that were developed through mutagenesis prior to 1995. In 1995, this number was 484. This number has since been significantly increased covering a large number of crops, namely, corn, wheat, pea, chrysanthemum, poinsettia, dahlia, citrus, apple, peach and the modified traits include plant maturity, winter hardiness, lodging resistance, protein and lysine content and numerous ornamental mutants. By the end of 20th century, the number of mutant varieties rose to 2252 covering a range of crops like cereals, oilseeds, pulses, vegetables, fruits, fibres, and ornamentals. Majority of these mutants were released as direct mutants and the rest as a result of cross breeding with the mutants. Most of the mutant varieties have come as a result of using physical mutagens (X-rays, gamma rays, thermal and fast neutrons) with gamma rays alone accounting for more than 60% of the mutant varieties. By 2017, nearly 3250 mutant varieties belonging to 175 plant species have been developed and released where China, Japan, India, Russia, the Netherlands, Germany and USA are the leading countries. India with 345 commercialized mutants stands third after China (810 mutants) and Japan (481 mutants). Thus mutation breeding is a significant contributor to the development and release of commercial cultivars in several crops and for several traits.

Polyploid is any organism with other than two basic sets of chromosomes, that is, monoploid, triploid, tetraploid, and various aneuploids. Species that have their chromosomes as multiples of a basic number (x) are polyploids. Many of the species which have been developed most recently are polyploids. Autoploidy is not known to have significantly impacted the evolution of species. Autoploids of commercial importance include banana, a triploid which is seedless (diploid bananas have hard seeds not desirable for consumption). Other important autoploids are tetraploid crops, namely, alfalfa, peanut, potato and coffee. Spontaneous autoploids are important in horticultural crops where gigas feature has produced superior varieties of flowering ornamentals of narcissus, tulip, hyacinth, gladiolus, and dahlia among others. Triticale is the most common example of man-made cereal through allopolyploidy. A number of economically important crops are natural alloploids. These include wheat, oat, tobacco, cotton, sugarcane, strawberry and blueberry, wild mustard, black mustard, rutabaga/rape, etc.

Alinehomozygousatallloci, ordinarilyobtainedbysuccessive self-fertilization in plant breeding is known as pure-line. W. L. Johannsen (1903), a Danish biologist provided a sound scientific basis for selection in self-pollinated crops when he defined ‘pure-lines’ and described the genetic mechanism by which they are established through a series of experiments for seed weight selection in bean, variety Princess. Pure-lines in principle are supposed to be 100% homozygous but that hardly happens in practical plant breeding programmes and usually the so called pure-lines are ‘near pure-lines. Pure-lines tend to be uniform in the traits of interest. Genetic mutations, admixtures and limited out-crossing are the main causes of variation within pure-lines. This is a rapid and most in-expensive method. Pure-line theory may be summarized as follows:

This method has been first described by H. H. Lowe in 1927. This is the most widely used method to handle segregating generations following crosses between two parents to develop new cultivars combining desirable traits from both the parents. In this method, superior types are selected in successive segregating generations and a record is maintained of all parent-progeny relationships. The records help the breeder to advance only progeny lines with plants that exhibit genes for the desired traits. Selection begins in F2 generation where individual plants are selected which in judgment of breeder will produce the best progeny in F5/F6 when the selection is finally concluded. In F3 and F4 generations, many loci will have become homozygous and family characteristics begin to appear. By F5/F6 generation, most families are expected to be homozygous and uniform and hence selection within such families is no longer effective. At this stage selection emphasis shifts to the selection among families rather than within families.

Selection of parents is of great significance for the success of this method. Normally, this method is used to replace already established variety. Therefore, one parent should be selected on the basis of proven performance in the particular area where the breeding is in progress. The second parent should be such that it complements the first parent for the character under improvement. Sometimes, three or four parents may also be used to supply the desired character by way of three-way or double crosses.

Bulk population breeding developed by the Swede H. Nilsson Ehle in 1908 and later on supported and practiced by H. V. Harlan in barley breeding in 1940s is a breeding strategy in crop improvement in which natural selection is allowed to play its full role where the F2 generation is planted in a plot large enough to accommodate several hundreds of plants. Planting density and cultural practices are usually the normal ones followed for commercial crop planting. At maturity, the plot is harvested in bulk and the seeds are used to plant a similar plot in the next season. This process is repeated till at least F5 where the plants are space planted to allow individual plant evaluation for effective selection. During period of bulk propagation natural selection plays a key role in shifting gene frequencies in the bulk population. This role is of crucial importance as bulk handling is continued over a number of generations. Bulk population handling can be aided by artificial selection at any time during the period of bulk propagation. Aggressive and highly competitive but undesirable plants may be physically rogued out of the population to avoid increasing the frequency of undesirable genes. For example, determinate bush types of beans do not compete well against indeterminate, vine types. And if the breeder is interested only in determinate types, artificial selection against the indeterminate types is certainly called for. There is also good reason to apply artificial selection against seed colour, pubescence, awns or other characters that are undesirable in the variety to be developed but may be more or less neutral in survival. Bulk population handling often provides opportunities to make use of artificial aids to selection. For example, bulk harvesting when only part of plants are mature is an effective and inexpensive way to select for earliness. If particular sizes of seeds are required, screens or mechanical devices provide a rapid way of eliminating inferior types. Naturally occurring or artificially induced disease or insect infestation, of course, provide opportunities to rogue susceptible individuals from the bulk plot. In F6, the plants selected in F5 are progeny rowed and superior progenies are selected from where again few plants can be selected and seed bulked within a selected row. Depending upon seed requirement for yield trial in the following years, even the seed from selected entire row can be bulked. The follow up yield evaluation trials are as given in case of pedigree selection. This method is operationally best suited for seed crops such as small grains, beans and soybean and is almost totally unsuited for fruit crops and for most vegetable crops.

The application of back-cross breeding first proposed by H. V. Harlan and M. N. Pope in 1922 provides a precise way of improving varieties that excel in a large number of attributes but are deficient in a few key characteristics. F. N. Briggs too started an extensive back-crossing programme to develop bunt resistant varieties of wheat in 1922. As the name implies, the back-cross method of breeding makes use of a series of back-crosses to the variety to be improved during which the character or characters in which improvement is sought is maintained by selection. At the end of back-crossing, the gene (or genes) being transferred will be heterozygous. Selfing at the end of last back- cross produces homozygosity for this gene pair and coupled with selection will result in a variety similar to the well adapted and recurrent parent but superior for the particular characteristic for which improvement programme was initiated.

In back-cross method of breeding, the adapted and highly desirable parent is called the recurrent parent and the source of desirable character being transferred is called as donor parent. It is most effective and easy to apply when the missing trait is qualitatively inherited, dominant and produces a phenotype that is readily observed in a hybrid plant. The procedure for transferring a recessive trait is similar to that for dominant trait but involves an additional step of selfing. Back-crossing is also used to transfer entire sets of chromosomes in a foreign cytoplasm to create a cytoplasmic male sterile (CMS) genotype as has been practiced in maize, onion and wheat, etc. This is done by crossing the donor (of chromosomes) as male until all donor chromosome are recovered in the cytoplasm of the recurrent parent. Back-cross breeding has also been used to develop isogenic lines (lines that differ only in alleles at a specific locus) for traits like disease resistance and plant height, etc.

Initially, exploitation of heteosis was confined to corn and a few other open- pollinated crops but presently heterosis is being exploited practically in all species cutting across mode of reproduction. More and more evidences are emerging that heterotic expression in a cross is a function of genetic backgrounds and genes. Heterotic grouping would remain a key strategy in management of diverse genetic backgrounds and genes for full exploitation of heterosis. The commonly used methods in developing hybrids in self- pollinated crops are listed and explained as follows:

Manual emasculation and Pollination Method

In this method, female parent flower buds are emasculated manually and the emasculated buds are pollinated by the pollen grain collected from protected flowers of the male parent. This method is effective and successful where the crossed fruits produce plenty of seeds and the seed requirement for planting the commercial hybrid seed is much less. Consequently, this method is being used in a routine manner on a commercial scale in vegetable crops mostly by private sector seed companies in India and south-east Asian countries. The crops covered under this method of hybrid seed production are as follows:

Tomato, hot-pepper, sweet pepper, eggplant, okra, bottle gourd, bitter gourd, ridge gourd, smooth gourd, cucumber, pumpkin, water melon and muskmelon. Cucurbits are included here as although they are cross-pollinated but behave like self-pollinated crops in terms of almost no inbreeding depression.

Mass selection is the simplest, oldest and least expensive method of plant breeding as applicable to cross-pollinated crops. The method involves planting balanced bulk seed of the population to be improved, in isolation, inferior plants rogued out before pollination wherever possible and selecting and bulking superior genotypes that already exist in the population. A balanced bulk of selected ears, for example, in corn is made for planting the next cycle. Selection units are individual plants. Selection is surely on phenotypic performance. This method exploits additive gene effects plus additive x additive interactions. The efficiency of this method depends largely on the heritability of the trait under selection. Selection results using mass selection have been quite effective for less complex traits. Mass selection could be very effective for those traits that can be identified or selected prior to flowering, for example, curd/head shape, size, colour in cauliflower and cabbage, root shape, size, colour and quality traits in radish and carrot and bulb shape, size, colour and weight in onion. A few examples of such traits in maize could be flowering, leaf angle, prolificacy, resistance to thrips, fall armyworm, reduced plant and ear height, reduced anthesis-silking interval and barrenness and tolerance to pre-flowering diseases.

Lack of pollination control is major limitation of this method as both desirable and undesirable pollen will be involved in pollination of the selected plants. Further, if selection intensity is very high (small plant population size advanced), the possibility of inbreeding depression is increased. This selection is not very effective if the heritability of the trait under selection is low (less than 10% on individual plant basis). Mass selection is likely to be more effective for individual or a combination of few traits rather than a whole series of characters. Despite some limitations, mass selection can be very useful and relevant procedure in breeding of cross-pollinated crops in many situations characterized by resource limitations, non-availability of trained scientific manpower and additional test sites, lack of seed storage facilities even for short term and lack of continuity of technical personnel. Lack of control on soil variability also makes this method less effective.

The concept of recurrent selection grew out of need to increase the number of superior genotypes that could be obtained from breeding stock of corn. This could have been possible by increasing the frequency of superior genes in the gene pool and by increasing the chances for genetic recombination to occur. Both can be achieved by recurrent selection which is defined as “a method of breeding designed to concentrate favourable genes scattered among a number of individuals by selecting in each generation among the progeny produced by mating inter se of the selected individuals (or their selfed progeny) of the previous generation.” The original idea behind recurrent selection was proposed first by Hayes and Gerber in 1919 and independently by East and Jones in 1920. Jenkins (1940) was the first to describe the method in detail and Hull (1945) proposed the name ‘recurrent selection’. Each cycle of recurrent selection requires (i) evaluation and selfing of selections and (ii) crossing of the progenies of superior selfed plants in all combinations and bulking of equal amount of seed from each cross. The selection of plants before selfing is possible for maturity, plant height, ear/head characters, kernel characteristics and resistance to diseases and insects. In general, these are the characters which have high heritability. It is clear that the selection of plants before selfing is more for morphological traits and these evaluations do not include yield. Yield evaluations are done on the top cross progenies (crossing of selected plants to a tester). Such top crosses will be grown in the second year of the cycle during which time the selfed seed is held over until the evaluations for yield are over. Or the selfed seed may be grown out as S₁ lines which in turn are selfed. After yield evaluation is over, the selfed seed of superior plants is grown out for crossing. Progenies of selfed plants are crossed in all possible combinations. This is done manually or by growing progenies together in an isolated block. In isolation planting, random mating should be ensured. After manual crossing or random mating in isolation, equal amounts of seed of each are bulked and this bulk seed is used to start the second cycle of selection, selfing and crossing.

A variety produced by crossing inter se a number of genotypes selected for good general combining ability in all hybrid combinations with subsequent maintenance of the variety by open-pollination. According to Hayes and Garber (1919), the synthetic production of an improved variety by inbreeding and cross-breeding seems a reasonable plan. Synthetic cultivar is a commercial entity and a combination of number of inbred lines, sibbed lines, clones or other populations of cross-pollinated crops. The features of the components are as follows:

1. They will have been tested for their combining ability.
2. They will be preserved for future syntheses of the synthetic variety.
3. The component lines will be combined in a manner so as to allow random mating.

Synthetic varieties are normally used in cross-pollinated crops in those situations where hybrid development and organized seed production are in the preliminary stages. Inbred lines are common components of synthetics because they are easy to maintain and can be used again and again as per requirement of reconstituting the synthetics. When selfing is not possible, sibbing may be used. In case of perennials, clones are used as components. Combining ability of the component lines is determined in different ways. General combining ability is desired more than the specific combining ability because there are several components to a synthetic variety and each is required to combine well with the rest other lines. Random crossing of components of a synthetic variety is assumed and each line should contribute the same amount of seed to the next generation. Because a synthetic variety is a mixture of many genotypes, it might change under selection pressure during multiplication.

Mangelsdorf (1951) has opined that hybrid corn has been the most far-reaching development in applied biology. Hybrid cultivars have contributed enormously to world’s food and feed resources. Although dramatically successful at first in corn, hybrids have spread to several other crops both in cross-pollinated and self-pollinated species. Their use is likely to spread more in time to come. Hybrid varieties are those in which F1 populations are used to produce commercial crop. Parents of F1 may be inbred lines, clones, varieties, or other populations. Hybrid breeding is historically rooted in development of hybrids in corn and therefore most of the issues covered in this section relate to hybrid breeding in maize.

This chapter starts quoting first para of the book entitled Disease Resistance in Plants by Van Der Plank (1968). “In 1958 the American Phytopathological Society met to celebrate its Golden Jubilee and review the problems and progress in plant pathology during the 50 years, 1908 to 1958. J. C. Walker was elected to review the problems and progress in controlling plant diseases by host resistance, a fitting tribute to one who perhaps more than any other had contributed during those 50 years both to understanding of nature of resistance and to the practical application of resistance to problems in horticulture and agriculture. In his summary Walker wrote “By and large the development of resistant varieties must be looked upon as a continuing program. The potential variability of most pathogens (including viruses) will not permit any currently successful variety to remain so for an indefinite period”. This opening para tells the importance of breeding for resistance to diseases in crop plants on a continuous basis as the fight between host and the pathogen will go on and on.

When conditions in the plant growing environment are less than the optimum, the plants experience stress which is broadly termed as abiotic stress. Major abiotic stresses impacting crop yield significantly are as follows (Grover et al., 2004; Acquaah, 2007):

Drought: This is the environmental condition caused by lack of adequate rainfall. This is also termed as water stress or desiccation stress. Effects of drought on crop plants are complex and variable and are greatly accentuated by a number of interacting factors. The onset of drought in general has been observed to reduce germination, emergence, hypocotyl length, water uptake and the mobilization of dry matter reserves even at the early growth stages. Drought stress causes a marked reduction in leaf area. Soil moisture availability at the seed germination, anthesis and at post-anthesis growth stages determines as to what extent production potential of crops would be expected in drought-prone situations.

The yield losses in field crops may be attributed to inadequate water availability at the critical growth stages. Crops therefore differ in their susceptibility of growth stages to water stress. Stress exposed plants immediately lower down relative water content (RWC) of their leaves, leaf water potential, osmotic potential. Turgor pressure may also be decreased unless plants have finally adapted to drought by opening of stomata again and improving the water potential. It has been reported that low leaf expansion rate at the early stage and accelerated leaf senescence accompanied by mortality of older leaves at later stage decreases total leaf green area.

Spectacular productivity gains have been achieved in wheat and rice in south and south-east Asia and also several other developing countries in Latin America and central Asia due to introduction of dwarfing genes in these two crops purely through conventional plant breeding approaches and these developments were christened as “Green Revolution” (Reynolds and Borlaug, 2006). The wheat breeding progrmme was being spearheaded by Norman Borlaug at CIMMYT (International Maize and Wheat Improvement Centre, Mexico) supported by Sanjay Rajaram and the rice breeding programme was under leadership of Gurdev Singh Khush at IRRI (International Rice Research Institute, Manila, Philippines) coinciding with these exciting developments during mid-sixties to mid-seventies. In recognition of these quantum jumps in yield of wheat and rice, Borlaug was awarded Nobel Peace Prize for Peace in 1970. Later on World Food Prize was instituted by joint efforts of Borlaug and his colleagues to honour Agricultural Scientists and others on pattern of Nobel Prize who had made significant contributions to sustainable food production and food and nutritional security globally. M. S. Swaminathan happened to be Director General, Indian Council of Agricultural Research when Green Revolution was taking roots in India and because of his enormous contributions to provide effective leadership to ensure that Green Revolution happened in India, M. S. Swaminathan is credited as “Father of Green Revolution in India” and he was the first recipient of World Food Prize in 1987 for spearheading the introduction of high-yielding wheat and rice varieties in India. G. S. Khush along with Henry Beachell received 1996 World Food Prize for achievements in enlarging and improving the global supply of rice during a time of exponential population growth and Sanjay Rajram was the 1914 World Food Prize recipient for his scientific research that led to huge increase in world wheat production.

There is need to find better ways and solutions to mitigate future agricultural challenges toward increasing productivity across the crops and equip them with the desirable genes for resistance to biotic and abiotic stresses and to mitigate the impact of climate change and also to make them nutritionally superior. Here comes “Plant Genomics”—a newly evolved discipline of plant sciences—targeting to decode, characterize, and study the genetic composition, structures, organizations, functions, and interactions/networks of all plant genes in a genome-wide scale. Being evolved from plant molecular genetics, biology, and biotechnology, plant genomics represents the key sub- divisions of structural, functional, comparative, evolutionary, physiological, and genetical genomics. Its development and advances, however, are tightly interconnected with plant science sub-disciplines, such as proteomics, metabolomics, epigenomics, phenomics, metagenomics, transgenomics, breeding-assisted genomics, bioinformatics and system biology as well as modern instrumentation and robotics sciences.

Plant genomics is a recent convergence of many sciences. Simply stated “Genomics” is the new science that deals with the discovery and noting of all the sequences in the entire genome of a particular organism. The genome can be defined as the complete set of genes inside a cell. Genomics, is, therefore, the study of the genetic make-up of organisms. Determining the genomic sequence, however, is only the beginning of genomics. Once this is done, the genomic sequence is used to study the function of the numerous genes (functional genomics), to compare the genes in one organism with those of another (comparative genomics), or to generate the 3-D structure of one or more proteins from each protein family, thus offering clues to their function (structural genomics). In crop agriculture, the main purpose of the application of genomics is to gain a better understanding of the whole genome of plants. Agronomically important genes may be identified and targeted to produce more nutritious and safe food while at the same time preserving the environment. Genomics is an entry point for looking at the other ‘omics’ sciences. The information in the genes of an organism, its genotype, is largely responsible for the final physical makeup of the organism, referred to as the “phenotype”. However, the environment also has some influence on the phenotype. DNA in the genome is only one aspect of the complex mechanism that keeps an organism running – so decoding the DNA is one step towards understanding the process. However, by itself, it does not specify everything that happens within the organism. The basic flow of genetic information in a cell is as follows. The DNA is transcribed or copied into a form known as “RNA”. The complete set of RNA (also known as its transcriptome) is subject to some editing (cutting and pasting) to become messenger-RNA, which carries information to the ribosome, the protein factory of the cell, which then translates the message into protein.

Biotech crops or genetically modified (GM) crops or the transgenic crops are those crop plants into which certain genes have been introduced from unrelated sources beyond the boundary of sexual compatibility using techniques which bypass the normal sexual cycle. The transgenic production technology uses recombinant DNA technology where a recombinant DNA molecule is a vector into which the desired DNA fragment has been inserted to enable its cloning in an appropriate host. The recombinant DNA technology has opened up the flood gate of plant biotechnology to produce plants which are equipped with novel genes to produce products never heard of that a plant will do. Now transgenics in several crops have been commercialized globally. The importance and potential of GM crops can be realized from the fact that acreage under such crops at global level has reached to 189.8 million ha covering 24 countries in 2017. One can have a feel of this huge acreage under commercialized GM crops globally if it is compared with about total cultivated area of 143 million ha in India.

Over the last 30 years field of agricultural biotechnology has expanded rapidly due to greater understanding of DNA as the chemical double helix code from which genes are made. Genetic engineering is one of the modern biotechnology tools that are based on recombinant DNA technology. The term genetic engineering, often interchanged with terms such as gene technology, genetic modification, or gene manipulation, is used to describe the process by which the genetic makeup of an organism can be altered using “recombinant DNA technology.” This involves the use of laboratory tools and specific enzymes to cut out, insert, and alter pieces of DNA that contain one or more genes of interest. The ability to manipulate individual genes and to transfer genes between species that would not readily cross is what distinguishes genetic engineering from traditional plant breeding and opens up a huge opportunity to create crop plants equipped with desirable genes from the sources which cannot be sexually crossed. With conventional plant breeding, there is little or no guarantee of obtaining any particular gene combination from the millions of crosses generated. Undesirable genes can be transferred along with desirable genes or while one desirable gene is gained, another is lost because the genes of both parents are mixed together and re-assorted more or less randomly in the offspring. These are the major limitations of conventional crop breeding.

There have been lots of international efforts to establish crop based research centres primarily in the developing/under-developed countries to carry out concerted breeding and plant genetic resources management activities pooling germplasm from all over the world and also engaging scientists from different countries to do breeding work on the certain mandate crops of the particular region using all kinds of germplasm and technologies with strong financial support from a central agency known as Consultative Group on International Agricultural Research (CGIAR) for doing the stipulated work as per programme and deliver the products which can be used by different national plant breeding programmes either to use them directly as cultivars or to use them in their breeding programmes in various ways including parents in the crossing programme.

CGIaR (Consultative Group on International agricultural Research)

CGIAR System Organization headquartered in Montpellier, France is a global agricultural research partnership for a food secure future that was founded in the 1970s in response to the Green Revolution. There are presently 15 CGIAR agricultural research centres employing more than 8,500 researchers and support staff worldwide, with an annual budget of US$ 800 million.

CGIAR is a global research partnership for a food-secure future. CGIAR science is dedicated to reducing poverty, enhancing food and nutrition security, and improving natural resources and ecosystem services. Its research is carried out by 15 CGIAR Research Centers in close collaboration with hundreds of partners, including national and regional research institutes, civil society organizations, academia, development organizations and the private sector. The CGIAR Fund was established as a Financial Intermediary Fund (FIF) in 2010. It was subsequently closed and replaced by a new FIF in 2017, following the CGIAR System governance reorganization.

Statistics

Statistics may be defined as the mathematical analysis, based on the theory of probability, of experimental data in an attempt to summarize or to describe them so that conclusions can be drawn with respect to the phenomena that supply the data. It is the branch of mathematics concerned with collection, classification, analysis, and interpretation of numerical facts, for drawing inferences on the basis of their quantifiable likelihood (probability). Statistics can interpret aggregates of data too large to be intelligible by ordinary observation because such data (unlike individual quantities) tend to behave in regular, predictable manner. It is subdivided into descriptive statistics and inferential statistics. Statistics is also defined as a discipline that allows researchers to evaluate conclusions derived from sample data. Plural of statistic is also statistics. In practice, statistics refers to a scientific approach used to:

Plant breeding activities basically involve development of new and superior crop varieties to replace the old ones and this naturally needs comparison of several lines/cultivars for agronomically important traits which are often quantitative in nature. Therefore, lots of environmental influences are there on the mean performance and mere numerical comparison of mean data is not sufficient to draw valid conclusions regarding the superiority of one variety over the other. Great advances have been made during the last about 100 years in the area of experimental designs and analytical procedures to facilitate such comparisons. Most commonly used designs by the plant breeders are included here for better understanding by the students and the breeders and to handle the experimental data on their own when confronted with such issues. Before applying statistical procedures on the experimental designs, it must be well understood that proper use of field plot technique is of great value to have meaningful data for further statistical analysis and use.

Varietal release and notification comes under the purview of the Seeds Act which is intended to provide for regulating the quality of certain notified seeds for sale and for marketing and for the matters connected therewith. Released and notified varieties are offered for seed certification by the seed certification agencies which ensure that the varieties under certification meet certain prescribed field and seed standards failing which they stand to be rejected during the process of seed certification.

Purpose of Release

The purpose of release of cultivars is to introduce the newly evolved varieties to the public for general cultivation in the region in which it is suitable. It enables the farmers to choose cultivars for cultivation in a region. In other words, release of a cultivar is in the nature of a recommendation to the farmers for its adoption. Therefore, notification of a variety is linked to the release of a variety, though the release process itself does not have statutory cover.

Genetics and Gene

Genetics is defined as the branch of biology dealing with heredity and variation. The hereditary units which are transmitted from one generation to the next are called genes. Genes have been defined in various ways. These are units of inheritance located in linear order on chromosomes. The genes are considered as hereditary determinants of a specific biological function located in a fixed place on the chromosome. Chemically, gene is a segment of DNA (deoxyribonucleic acid) encoding one polypeptide and defined operationally by the cis-trans or complementation test. Gene is the functional unit of inheritance (a sequence of DNA) which is located on a certain position (locus, plural loci) on a chromosome. The locus is sometimes used interchangeably for gene. DNA, in conjunction with a protein matrix, forms nucleoprotein and becomes organized into structures with distinctive staining properties which are known as chromosomes. The chromosomes are located in the nucleus of the cell. Since genes are located on the chromosomes, in many ways behavior of genes is parallel to the behavior of the chromosomes. Two or more alternative forms of a gene are called as alleles. All genes on a chromosome are said to be linked and they all belong to the same linkage group.

Cell

Cells (small, membrane-bounded structures filled with a variety of chemicals in an aqueous solution that interact to give life) are the basic unit of life. Cells are capable of acquisition and use of energy, have the capacity to reproduce and respond to the environment and are able to carry out a variety of chemical reactions. They maintain a relatively constant internal environment despite changes in the external environment. There are two types of cells, namely, prokaryotes and eukaryotes.

The prokaryotic (pro = before, karyote = nucleus) cells are the simple form of cell types which include the various types of cells of bacteria and their relatives, all belonging to kingdom Monera. They possess a cell wall constructed of peptidoglycan, a substance unique to prokaryotes. Beneath the cell wall, there is a plasma membrane that encloses a cytoplasm containing DNA, RNA, proteins and various small molecules.

Chi-Square

The Chi-square test (c₂) proposed by Karl Pearson in 1899 is used to measure goodness of fit of observed and theoretical or expected data and is a continuous distribution.

Formula for Chi-Square

1. General Formula

The general formula for Chi-square is as follows

Historically the first and still the most conclusive evidence for the existence of genes comes from the phenomenon of segregation of traits observed in the offspring of hybrids between individuals or strains that differ in some recognizable respect. The principle of segregation was formulated by Gregor Johann Mendel in 1866 under such peculiar circumstances that the scientific world failed to recognize it until after a lapse of 34 years. In the first place Gregor Mendel was not primarily a biologist but a Monk in the Augustinian monastery at Bruenn, Austria (now Brno, Moravia in Czech Republic). During his childhood, Mendel worked as a gardener and studied beekeeping. He became part time monk because it enabled him to obtain an education without having to pay for it himself. From 1840 to 1843 he studied Practical and Theoretical Philosophy and Physics at the Philosophical Institute of the University of Olomouc. Upon recommendation of his teacher, Mendel entered the Augustinian St. Thomas Abbey in Brno and began his training as a Priest. Born Johann Mendel, he took the name Gregor upon entering religious life. Mendel worked also as a substitute High School teacher. In 1850, he failed the oral part of his examination and could not become a certified High School Teacher. In 1851, he was sent to the University of Vienna to study further so that he could get formal education. Mendel returned to his Abbey in 1853 as a Physics teacher. In 1856, he took the examination to become a certified teacher but again failed the oral part.

Phenotype is a result of gene products brought to expression in a given environment. Genes specify the structure of proteins. All known enzymes are proteins. Enzymes perform catalytic function, causing the splitting or union of various molecules. All of the chemical reactions which occur in the cell constitute the subject of intermediary metabolism. These reactions occur as stepwise conversions of one substance into another, each group being mediated by a specific enzyme. All of the steps which transform a precursor substance to its end product constitute a biosynthetic pathway. Several genes are usually required to specify the enzymes involved in even the simplest pathways. Before gene-enzyme relationship could be postulated, soon after rediscovery of Mendelian principles of inheritance, it was discovered that genes were not merely separate elements producing distinct individual effects, but they could interact with each other to produce completely novel phenotypes. The simplest example of gene interaction could be the fact that allele A could be characterized by phenotype A, and the allele B by the phenotype B and an organism having both alleles A and B at the same time might show phenotype C. The occurrence of such interactions means that the phenotype we observe is not present in the genes themselves but arises from a complicated developmental process resulting from a biosynthetic pathway as illustrated below (Stansfield, 1969)

In most of higher organisms including animals, the aspect of sexes has been normally confined to males and females which exist separately or even in the same individual. An individual possessing both male and female reproductive organs is usually referred to as hermaphrodite. In plants, where staminate (male) and pistillate (female) flowers occur on the same plant, the term used is monoecious. The most common plants under monoecious form are cucurbits. However, majority of the flowering plants have male and female parts within the same flower and this situation is referred to as hermaphrodite. There are relatively less numbers of plants having male and female flowers located on different plants and this is termed as dioecious condition. Among the common cultivated crops known to be dioecious are asparagus, date palm, hemp, hops and spinach.

Linkage

When two or more genes are located on the same chromosome, they are said to be linked. Genes on different chromosomes are distributed to the gametes following the law of independent assortment. However, genes located on the same chromosome, tend to stay together during gamete formation. For example, results of test-crossing dihybrid individuals will produce different results depending upon whether the genes are linked or are located on the same chromosome. This is illustrated as follows:

When there is indication of linkage from F or testcross data through significant deviation from 9: 3: 3: 1 and 1: 1: 1: 1 ratio, it is implied that independent assortment is not taking place and this is taken as an indication of linkage. The inverse of linkage strength is the recombination value and is often symbolized by ‘p’ whose maximum value is 50% because out of four strands of a tetrad, two are always parental types and two are recombinant types when all the cells show crossing over.

Translocations

Chromosomes occasionally undergo spontaneous rupture or can be induced to rupture in high frequency by ionizing radiation. The broken ends of such chromosomes behave as though they were ‘sticky’ and may rejoin into non- homologous combinations giving rise to translocations. The most common type of translocation is reciprocal translocation which involves the exchange of segments between two non-homologous chromosomes. Translocations change only the arrangement of genes in the chromosomes, not the quality or quantity of genes. For this reason, they are sometimes referred to as chromosomal rearrangements. Individuals carrying such rearrangements should be phenotypically entirely normal unless the relations of a gene or genes to adjacent genes affect the phenotypic expression (position effect). The first translocation was discovered by Bridges in 1923 in D. melanogaster.

The genetic techniques for detecting and studying translocations will be more easily understood when the cytological phenomena produced by translocations are known. Suppose two chromosomes having respectively the genes ABCDEF and GHIJKL exchange sections and give rise to translocation chromosomes ABCJKL and GHIDEF. The individual thus formed receives from one of the parents the normal and from the other parent the translocation chromosome. Such an individual is a translocation heterozygote (Fig. 36.1).

So far, we have seen the examples in which mode of inheritance of a particular character is tied to the behaviour of the nucleus or more specifically to the chromosomes. In other words, transmission of a character and its appearance in the individuals can be predicted from the knowledge of chromosome segregation and independent assortment. This analogy gets further strengthened on the ground that the DNA has been considered as the basic genetic material and practically all the cellular DNA is localized in the chromosomes. It has also been seen that DNA does not produce an organism alone but obviously depends upon an already existing medium in order to function. Thus, genotypic effects may considerably get modified by the environment. In the cell itself, one important source of environmental effect is the cytoplasm immediately surrounding the nucleus. The components of the cytoplasm may show wide variation between the individuals and therefore it cannot be ruled out that a genotype in one cytoplasm may function somewhat differently than the same genotype placed directly into another cytoplasm. It is well known fact that the cytoplasmic contribution of one of the parents in form of an egg is considerably larger than the cytoplasmic contribution of the other parent in form of sperm. For example, cross-fertilization between two strains capable of producing both sperm and eggs, leads to contribution of ‘A’ genotype and ‘a’ egg cytoplasm in one case and of ‘B’ genotype and ‘b’ egg cytoplasm in the other case. Thus, the cross-fertilized diploid zygotes have two possible genotypic-cytoplasmic combinations, namely, AB-a, and AB-b depending upon which strain provides the egg. If the egg is contributed by the A parent, then the genotype of the progeny shall be AB-a, and if the egg is contributed by the B parent, then the genotype of the progeny shall be AB-b. If each such cytoplasm produces a unique phenotype, then AB-b would have the same b phenotype as BB-b individuals. Such effects produced by the egg cytoplasm are termed as maternal effects.

In 1868, Johann Friedrich Miescher, a young Swish medical student got excited with an acidic substance that he isolated from pus cells obtained from bandages used to dress human wounds. To obtain large quantities of pus for his experiment, Miescher began purchasing used bandages from surgical clinics in Tuebingen, Germany. He separated pus cells from bandages and treated the cells with pepsin, a proteolytic enzyme that he isolated from stomach of pigs. After pepsin treatment, he recovered an acidic substance that was named as “nuclein” by him. This nuclein was unusual in the sense that it was rich in nitrogen and phosphorous, two elements known at that time to coexist only in certain types of fat. Miescher’s paper on this investigation was published in 1871. At that time, importance of Miescher’s nuclein was not fully appreciated. Even the existence of polynucleotide chains, the key component of the acidic material in Miescher’s nuclein was not documented until 1940s. The role of nucleic acid in storing and transmitting genetic information remained un-established until 1944 and the double-helix structure of DNA was not discovered until 1953 by James Watson and Francis Crick. Even in 1953, many geneticists remained skeptical to accept the role of nucleic acid as the genetic information carrier rather than proteins as nucleic acids exhibited less structural variability than the proteins (Snustad et al., 1997).

The search for the chemical basis of genetic material began about one and half century ago. Soon after discovery of Mendelian principles of segregation and independent assortment in 1865, Friedrich Miescher (1844-1895) published a method for separating cell nuclei from the cytoplasm and from these cell nuclei, he extracted an acid material termed as “nuclein” which had unusually large amount of phosphorous. Miescher concluded that this substance was unique and was just not comparable with any other group known at that point of time. Later on nuclein, now named as “nucleic acid” was found to be associated with various proteins and the combined entities were termed as “nucleoproteins”.

Watson and Crick based on their double helical DNA model, offered to explain DNA replication by a method where each single strand of DNA is template or mold for its complement and a new helix has one old strand and one newly synthesized strand. This type of DNA replication is called as semi-conservative as in this method one old strand is retained and one new complementary strand is synthesized. In contrast to semi-conservative mode of DNA replication, two more modes of DNA replication were proposed. One is conservative type where two new strands are synthesized in form of a double helix while the old double helix remains unchanged. The third type of DNA replication proposed has been dispersive mode of DNA replication which amounts to breaking down of double helical strands into small pieces along the length, their replication and random assemblage with newly synthesized pieces to form a patchwork single string of dispersed old and new pieces. These three modes of replication of DNA molecule are shown in Fig. 40.1.

According to the central dogma of molecular biology, genetic information flows (i) from DNA to DNA during its transmission from generation to generation and (ii) from DNA to protein during its phenotypic expression in an organism (Fig. 41.1).

In transcription, it has become clear as to how there is the transfer of genetic information stored in the sequence of nucleotides-pairs in DNA to the sequence of nucleotides in m-RNA molecules which carry that particular information from the nucleus to the sites of protein synthesis (ribosomes) in the cytoplasm. Further, there is the requirement of the process by which genetic information stored in the sequences of nucleotides in m-RNAs is used to specify the sequences of amino acids in the polypeptide gene products. This process known as translation takes place in the cytoplasm on complex workbenches called as ribosomes. With exception of water, proteins are by far the most prevalent component of living organisms in terms of total mass.

Proteins (= enzymes = genes) are composed of polypeptides and every polypeptide is encoded by a gene. Each polypeptide has a long sequence of amino acids linked together by covalent bonds. There are 20 different amino acids (glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, cysteine, serine, threonine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine) present in most proteins. All amino acids except proline contain a free amino group (NH₂) and a free carboxyl group (COOH) as illustrated below:

Biochemical reactions are mediated by enzymes and all enzymes are proteins. Proteins are polymers of subunits (monomers) called amino acids which are often termed as ‘residues’. Each amino acid has an amino group (NH ) at one end and a carboxyl group (COOH) at the opposite end. 20 different kinds of amino acids occur naturally in proteins. Each enzyme consists of a certain number of amino acids in a precisely ordered sequence. The blueprint for making proteins is coded in the nucleotide sequence of DNA. The number of nucleotides which code for an amino acid is termed a codon. There are 20 common amino acids but only four different nucleotides. Obviously a singlet code (one nucleotide coding for one amino acid) could code only for four amino acids. A coding sequence of two nucleotides for one amino acid or a doublet code would produce 4₂ = 16 possible coding combinations or codons and this arrangement is not sufficient to take care of 20 amino acids. A codon size of three nucleotides for one amino acid or triplet code seems more likely as it produces 4₃ = 64 possible codons as illustrated in Table 43.1 (Strickberger, 1968).

Regulator Genes

Bacterial cells are capable of producing many kinds of enzymes in relatively large quantities but the kinds and amount of enzymes actually produced at any given time are usually those which are required at that particular time in response to the prevailing environment. Inducible enzymes are ordinarily absent or present in very small quantities until a specific compound appears. This compound is called as inducer. A typical example of this is production of b galactosidase (an enzyme that splits lactose into galactose and glucose, Fig. 44.1) once the lactose is introduced into the medium on which E. coli cells are grown. Here lactose acts as inducer for the enzyme b galactosidase.

The concept of gene has undergone several refinements since its discovery in 1866 by Mendel. Most genes encode one polypeptide and can be operationally defined by the complementation test. The complementation or cis-trans test provides an operational definition of the gene. It is used to determine whether mutations are in the same gene or in different genes. Intragenic complementation may occur when a protein is a multimer containing at least two copies of one gene product. The concept of gene has evolved from a bead on a string, not divisible by recombination or mutation to a sequence of nucleotide pairs in DNA encoding one polypeptide chain. The unit of genetic material not divisible by recombination or mutation is the single nucleotide pair. Historically several experimental developments have occurred to facilitate understanding of the structure and function of gene and these are briefly described in a chronological order as follows to have better understanding of the gene concept as it evolved. However, before discussing evidences supporting various gene concepts, it is better to summarize important stages in the evolution of the gene concept.

Abstract

Pigeonpea (Cajanus cajan) is an important pulse crop of India and its production is greatly affected by wilt disease. Pigeonpea wilt is a very destructive soil borne disease caused by Fusarium udum. In the experiments, ten treatments were applied viz.; Neem leaf extracts (T₁), Garlic extracts (T₂), Tulsi leaf extracts (T₃), bio-agents Trichoderma viride (T₄), Pseudomonas fluorescens (T₅), Polymer coating (polycot) @ 4 ml diluted in 5 ml of water per kg of seed (T₆), Polymer coaating + Trichoderma viride @ 6.0 g per kg seed (T₇), Polymer + Pseudomonas fluorescens @ 6.0 g per kg seed (T₈), Polymer coating (polycot) 4 ml diluted in 5 ml of water per kg seed + Vitavax power @ 3g per kg seed (T₉) and T₁₀ served as untreated. The observations were recorded six times at 10 days intervals and started from 40 days. These polymer alone @ 4.0ml per kg and in combination with bioagents and fungicides were applied to seed were tested against Fusarium udum. The highest per cent disease control and per cent wilt disease incidence were recorded 96.09 to 64.09 and 2.74 to 33.32, respectively in T₉ (Polymer + Vitavax power @ 3g per kg seed) at 10 days intervals followed by T₈ (Polymer + P. fluorescens) gave 94.46 to 53.06 per cent inhibition, T₇ (92.39 to 52.74) and T₆ (90.97 to 49.73) however, the lowest per cent disease control was noticed as 76.19 to 40.13 per cent in T₁ (Neem leaves extract) amongst all treatments except untreated.

Abstract

Pigeon pea is an important pulse crop of India and its production is greatly affected by wilt disease. Pigeon pea wilt is a very destructive soil borne disease caused by Fusarium udum. In present research work plant extracts (Garlic, Neem, Tulsi), bio-agents (Trichoderma viride and Pseudomonas fluorescens) and fungicides (Vitavax, Bavistin and Thiram + Carbendazim) were tested against Fusarium udum. Among them fungicides were found most effective in inhibiting mycelial growth of Fusarium udum recorded 100% inhibition followed by garlic bulb extract (52.12 to 56.33%). Among bioagents, Pseudomonas fluorescens inhibit the growth up to (46.00 to 51.79%) followed by T. viride (44.32 to 48.79%) amongst all treatments except untreated.

Keywords: Pigeon pea, Fusarium udum, bio-agent, fungicide

Pigeon pea (Cajanus cajan L. Millsp.) is one of the major pulse crops grown in the tropics and subtropics belonging to family Leguminaceae. It has wide adaptability and low input requirements mostly grown in kharif season.

Abstract

Lentil is an important pulse crop and play very important role in the supply of the protein to under nourished vegetarian population of the country. Fusarium wilt of lentil caused by Fusarium oxysporum f. sp. lentis is one of the wide spread and destructive diseases in India under rice-based cropping system. Use of synthetic fungicides has led to the emergence of several problems like environment pollution, residual effect in grains and killing of non-target organism(s). Hence, for minimizing the losses caused by Fusarium wilt, four plant extracts such as Garlic, Neem, Eucalyptus, Tulsi and three antagonistic bio-agents viz. Trichoderma viride, Trichoderma harzianum and Pseudomonas fluorescence were evaluated in vitro. The results clearly showed that all plant extracts were more or less inhibitory to mycelial growth of Fusarium oxysporum f. sp. lentis. The effectiveness of extracts increased with increase of their concentration and maximum growth inhibition was obtained in Eucalyptus followed by Garlic and Tulsi. While minimum per cent inhibition was recorded in Neem. Among bio-agents, T. viride showed maximum growth inhibition of Fusarium oxysporum f. sp. lentis followed by T. harzianum at both 4 and 7 days incubation period.