**2.9 Mutation breeding in legumes improvement**

The basis for any crop improvement programme is the variations present in the concerned crop. For generation of new variations, mutation is a prerequisite. These mutations are caused by various factors and are broadly divided into two major categories: spontaneous and induced mutations. Natural causes like as ultraviolet (UV) irradiation, reactive oxygen species, and transposable elements may generate spontaneous mutations in nature. On the other hand, physical and chemical mutagens cause artificial mutations. Different mutagenesis techniques have been successfully utilized in molecular plant breeding to study gene functions. The alterations induced can be random or particular to the target. Chemicals and physical mutagens cause random mutations. Unfortunately, random mutagenesis is costly, time-consuming and also difficult to screen desirable mutants from a large, mutated population. In addition to conventional plant breeding and

GMO techniques, targeted mutagenesis has arisen as an alternative for improving crop plants. This approach relies on the use of nucleases that allow for precise double-stranded breaks to occur at certain sites within the genome. The specific methodologies for targeted mutagenesis include PCR-based techniques for in vitro mutation generation and analysis, transposon mutagenesis, RNA interference (RNAi), TILLING (Targeting Induced Local Lesion IN Genomes), and programmed meganucleases [also called homing endonucleases, site-directed nucleases (SDNs) or site-specific nucleases (SSNs)]. TALENs, ZFNs, and CRISPR/Cas9 are frequently used meganucleases.

### *2.9.1 TILLING*

Identifying a mutation in a particular gene and relating this mutation to the phenotypic alteration in the mutant organism is one of the most straightforward ways of determining gene function. TILLING (Targeting induced local lesions in genomes) is a non-transgenic, high throughput, general reverse-genetic strategy which aims to identify SNPs (single nucleotide polymorphisms) and/or INDELS (insertions/deletions) in a gene/gene of interest from a mutagenized population. TILLING has developed a few decades ago as an alternative to insertional mutagenesis in Arabidopsis thaliana. High-throughput TILLING provides a quick and cheap diagnosis method of induced mutations in artificially mutagenized populations. The important feature of TILLING is that it can be applied to any species, regardless of its genome size and ploidy level.

#### *2.9.2 Eco TILLING*

EcoTILLING (Ecotype Targeting Induced Local Lesions IN Genomes) is the modification of TILLING, which identifies natural genetic variations in populations in contrast to induced mutations in TILLING. This has been successfully used in animals and plants to discover SNPs and small INDELs. The classical method of Eco-TILLING is based on the enzyme endonuclease (Cel1, Endo1), which cleaves at the point of mutation by detecting mismatches in double-strand DNA. EcoTILLING is convenient for those plant species in detection of natural mutations where chemical mutagenesis is not suitable.

So far, TILLING and EcoTILLING have been implemented in many legume crops. In soybean, Tilling was used to screen more than 40,000 mutant lines and to create novel mutant alleles [57]. In chickpea, TILLING was also used to diagnose mutations in the M2 generation. Recently, in mungbean, five exon residing mutations were identified by TILLING and confirmed the potential role of each mutation in altering mungbean plant architecture to develop an ideal plant type [58].

#### **2.10 Transgenic approaches/genetically modified legumes**

Traditional breeding is tedious and success rate of obtaining desirable gene/ genes or gene combinations from a large number of crosses is very less. These limitations hamper the desirable changes in crop plants. Therefore, biotechnological approaches are complementary to traditional breeding methods for addressing global food demands. Today we have access to vast gene pools due to new biotechnological approaches, which can be utilized in food crops to add favourable features. In this way, Genetically-modified (GM) crops can contribute to satisfying the food demand by developing varieties which are high yielding, good in quality, nutrition-rich and different kinds of stress-tolerant. Genetically modified crops are plants in which one or more

#### *Recent Advancements in Genetic Improvement of Food Legume Crops DOI: http://dx.doi.org/10.5772/intechopen.106734*

genes have been introduced using genetic engineering techniques to produce desirable traits for agricultural purposes. Genetic engineering facilitates the direct gene transfer not only within the species and between the different plant species but also from unrelated organisms as well as also resolves the problem of linkage drag. Soybean was the first grain legume for which transgenic plants were developed [59]. Glyphosateresistant soybean was developed by transferring gene derived from Agrobacterium sp. strain CP4, which encodes a glyphosate-tolerant enzyme EPSP synthase. Genetically transformed other legume species have successfully developed glyphosate-resistant lines for example- narrow-leaf lupin (Lupinus angustifolius L.) [60]. This is an easy way of weed control, reduces the cost of production and has a positive impact on the environment. Water stress causes significant yield losses in soybean crops; to resolve this problem, transgenic soybean was developed by transferring a gene encoding an osmotin-like protein extracted from Solanum nigrum var. americanum [61].

Helicoverpa armigera, a food legume insect, causes significant yield losses in pigeonpea. To minimize the losses caused by Helicoverpa armigera; transgenic pigeonpea was developed by transferring two synthetic Bacillus thuringiensis insecticidal crystal protein genes, cry1Ac and Cry2Aa. The transgenic pigeonpea expressed Cry1Ac and Cry2Aa proteins exhibited 80–100% mortality of insect [62]. Chickpea crop often encounter terminal drought stress that affects its production. Desi chickpea variety C235 that has 120 days of crop cycle, a transcription factor DREB1A was transferred and observed better root and shoot partitioning as well as higher transpiration efficiency in transgenic chickpea under drought stress [63]. In storage, cowpea seeds are severely damaged by storage pests (Callosobruchus maculatus and C. chinensis). Introduction of the bean (Phaseolus vulgaris) α-amylase inhibitor-1 (αAI-1) gene into a commercial Indian cowpea cultivar (Pusa Komal) strongly inhibited the development of these insects [64].

Genetic engineering, however, has excellent potential to maximize crop performance coupled with conventional methods, even though there is somewhat risk related to the effects of transgenic crops on the environment and human health. To overcome these risks, each product should be critically examined. Appropriate biosafety and food safety measures should be strictly followed.

#### **2.11 Phenomics**

A better understanding of the biological processes is required to increase yield potential and multiple stress tolerance. Any crop for its improvement majorly depends on favourable genetic changes in the crop genome, but the current pace of crop improvement is incapable of meeting future food demands. Therefore, crop improvement requires introducing new approaches for genetic changes in crop plants and their breeding. Marker-assisted breeding/ molecular breeding gives more importance to genotypic information of a crop, but phenotypic information is also equally important. Plant phenotyping is now a bottleneck in advancing crop yield. To enhance the selection efficiency of crop plants, phenotyping is also important, along with genotyping. The rapid and accurate evaluation of the phenotype of breeding lines and different crop populations is required for new variety development.

Phenomics is the investigation of phenomes, which are the collection of phenotypes (physical and biochemical traits) that a given organism may generate during development and in response to environmental effects. Crop phenomics is a multidisciplinary approach which integrates agronomy, life sciences, information science, math and engineering sciences and combines high-performance computing and artificial intelligence technology. This technique provides non-destructive and non-invasive ways of imaging, including colour imaging, near-infrared imaging, far-infrared imaging and fluorescence imaging for different phenomena like; plant structure, biomass, leaf health, for measuring soil and tissue water content, canopy/ leaf temperature measurement etc. High-throughput phenotyping has been widely used, offering automated digital analyses of large data samples. The main benefit of high throughput phenomics approaches is the speed at which data can be collected: field data that could take several days to collect using conventional methods can be collected in a matter of hours using several sensors installed on a phenotyping platform. This saves time and allows several observations of a given plant/plot in a single day. These phenomics tools and techniques are making way for crop plant genetic improvement by using the potentiality of genomic resources.

#### **2.12 Rapid generation advancement approaches in legumes**

The biggest challenge for breeding higher-yielding and more resilient crops is the inability to complete more generations in lesser time. Generally, legume crops complete one or two breeding cycles in a year, so developing a new variety is timeconsuming. Speed breeding is a rapidly emerging method among plant breeders to develop new varieties in a short period of time. This technique greatly enhances breeding and research speed by reducing generation time. Plants are grown in controlled growth chambers or greenhouses with optimal light intensity and quality, as well as specific day length and temperature (22 h light, 22 °C day/17 °C night, and high light intensity), to speed up different physiological functions; especially photosynthesis and flowering, and thus reduce generation time. Under normal glasshouse conditions, 2–3 generations can be produced per year, while speed breeding can produce up to 4–6 generations per year. Chickpea was induced to flower early by Gaur *et al*. [65] using 24-hour photoperiod, which, with the aid of offseason nurseries, allowed the production of three generations per year. Similarly, early and late flowering genotypes of pea, chickpea, faba bean, lentil and lupin were grown by Croser *et al*. [66] in controlled environments under different light spectra (blue and far redenriched LED lights and metal halide). The time it took for the first seed to germinate was reduced significantly in half, and pollen viability was enhanced. In addition, costs for speed breeding are also reduced by combining this with genomics-based breeding and high-throughput phenotyping.
