Hairy Roots

Agrobacterium rhizogenes induces hairy root disease in plants. The neoplastic (cancerous) roots produced by A. rhizogenes infection, when cultured in hormone free medium, show high growth rate and genetic stability. These genetically transformed root cultures can produce levels of secondary metabolites comparable to that of intact plants. Several elicitation methods can be used to further enhance the production and accumulation of secondary metabolites. Thus, hairy root culture offer promise for high production and productivity of valuable secondary metabolites in many plants. Hairy roots can also produce recombinant proteins from transgenic roots, and thereby hold immense potential for pharmaceutical industry. Hairy root cultures can be used to elucidate the intermediates and key enzymes involved in the biosynthesis of secondary metabolites, and for phytoremediation due to their abundant neoplastic root proliferation property. Various applications of hairy root cultures and potential problems associated with them are discussed in this chapter.

Molecular Markers

Conventionally, establishment of relationship between the genotype and phenotype through genetic analysis was considered as key to success in plant breeding. The discovery of molecular markers has changed the entire scenario of genome analysis. Coinheritance of a gene of interest and a marker suggests that they are physically close on the chromosome. A marker must be polymorphic in nature for their identification and utilization. Such polymorphism can be detected at three levels: phenotype (morphological), difference in biomolecules (biochemical), or differences in the nucleotide sequence of DNA (molecular). These markers act as a versatile tool and find their importance in taxonomy, plant breeding, gene mapping, cultivar identification, and forensic science. They have several advantages over the conventional methods of plant breeding for developing new varieties with higher rate of success. This chapter covers the basic principles and applications of various types of markers with special emphasis on molecular markers.

Concepts of Molecular Plant Breeding and Genome Editing

Traditional plant breeding depends on spontaneous and induced mutations available in the crop plants. Such mutations are rare and occur randomly. By contrast, molecular breeding and genome editing are advanced breeding techniques that can enhance the selection process and produce precisely targeted modifications in any crop. Identification of molecular markers, based on SSRs and SNPs, and the availability of high-throughput (HTP) genotyping platforms have accelerated the process of generating dense genetic linkage maps and thereby enhanced application of marker-assisted breeding for crop improvement. Advanced molecular biology techniques that facilitate precise, efficient, and targeted modifications at genomic loci are termed as “genome editing.” The genome editing tools include “zinc-finger nucleases (ZNFs),” “transcription activator-like effector nucleases (TALENs),” oligonucleotide-directed mutagenesis (ODM), and “clustered regularly interspersed short palindromic repeats (CRISPER/Cas) system,” which can be used for targeted gene editing. Concepts of molecular plant breeding and genome editing systems are presented in this chapter.

Software Tools to Assist Breeding Decisions

Plant breeders are usually faced with the problem of predicting the performance of new individuals with untested gene combinations. Therefore, it is important to follow an integrated breeding approach by combining molecular tools, molecular mapping, and MAS. It is also required to develop tools for modeling and simulation analysis by utilizing all pre-existing and newly generated data. Several software tools have been developed that integrates breeding simulations and phenotype prediction models using genomic information. Reliable phenotype prediction models for the simulation were constructed from actual genotype and phenotype data. Such simulation-based genome-assisted approach to breeding will help optimize plant breeding in all important agricultural crops. Software tools have also been developed for designing target sites or evaluating the outcome of genome/gene editing system. This chapter provides an overview of the key software support tools that will assist the plant breeders in decision making during the process of conducting various breeding program.

Genome Editing

Targeted editing of the genomes of living organisms not only permits investigations into the understanding of the fundamental basis of biological systems but also allows to improve productively and quality of crops. This includes the creation of plants with valuable compositional properties and with traits that confer resistance to various biotic and abiotic stresses. Recently, several novel genome editing systems have been developed, which include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALNEs), and clustered regularly interspersed short palindromic repeats/Cas9 (CRISPER/Cas9). These exciting new methods have proved themselves as effective and reliable tools for the genetic improvement of plants. The genome editing systems can also be used to exploit the genetic diversity present in the semi-domesticated and wild relatives of the cultivated crops by targeting homologous domesticated genes through allele-mining. In this chapter various tools available for gene editing, their merits, and demerits have been discussed.

Gene Cloning

The discovery of two naturally occurring biological molecules, plasmid DNA and restriction enzymes, with remarkable properties have made possible the development of methods to isolate and manipulate specific DNA fragments. Through this technology, a DNA fragment, even an entire gene and its controlling elements, can be isolated and rejoined with a plasmid or phage DNA, and the hybrid DNA molecule can be inserted into a bacterium. The foreign DNA insert can be multiplied inside the bacterial host and induced to express or synthesize the protein product of the foreign DNA. The entire process through which this can be achieved is called recombinant DNA technology or genetic engineering. The recombinant DNA technology has been extended to animal and plant cells. In this chapter, methods for isolation, modification, rejoining and replication of genomic DNA, and production of new or enhanced protein products within a host cell have been described.

Genomics, Proteomics, and Metabolomics

Genomics could be viewed as the study of the randomness of DNA sequences. It may be possible to predict the structure of a gene product from the nucleotide sequences and thereby predict its function. The terms “structural genomics” and “functional genomics” were coined to denote the assignment of structure and function to a gene product, respectively. Proteomics focuses on the products of gene, which are basically proteins. Proteins are responsible for the development of phenotype, and proteomics is the bridge between genotype and phenotype. The transcribed mRNAs and their abundance are called transcriptome. Proteomics also deals with the interaction between proteins called intractomics. Metabolomics is concerned with identification, abundance, and localization of all the molecules excluding lipids and polysaccharides in the cell. In this chapter, the basic concepts and analysis of the genomic, proteomic, and metabolomics data for their practical utilization are discussed.

Molecular Markers for Plant Variety Identification and Protection

The identification of varieties of crop plants is important for their registration, breeding, seed production, and trade. The traditional approach to variety identification involves analysis and recording of their morphological characters, which is less informative, highly influenced by environmental factors and time consuming. Availability of molecular markers in large number in all the major crops has opened new avenue for their utilization in plant variety identification and protection. Molecular markers have the advantage of not being influenced by the environment and thus stable. Development of software to analyze and characterize the molecular markers has enhanced the process significantly. It also helps in protecting Plant Breeders Right. The establishment of genome and transcriptome sequencing projects for crops has generated a huge wealth of sequence data that could find much use in identification of plants varieties. In this chapter molecular basis of variety identification and their protection has been discussed.

Molecular Markers for Phylogenetic Studies and Germplasm Conservation

Application of molecular markers in phylogenetic studies has become increasingly important in recent times. Availability of fast DNA sequencing techniques and robust statistical analysis methods provided new momentum to this field. Different nuclear encoded genes (16S rRNA, 5S rRNA, 28S rRNA), mitochondrial encoded genes (cytochrome oxidase, mitochondrial 12S, cytochrome b, control region), and few chloroplast encoded genes (rbcL, matK, rpi16) have been used as molecular markers. This method allows researchers to obtain new evidence concerning their phylogeny and biodiversity. Measurement of genetic diversity is important for development of strategies for effective germplasm management. The DNA-based technologies can overcome all the limitations of traditional methods used for the estimation of genetic diversity. This chapter deals with historical developments of molecular phylogeny, use of molecular markers in phylogeny, and evolution of phylogenetic tree building methods.

Marker-Assisted Breeding

Advancement in sequencing technologies has contributed towards identification and development of different types of molecular markers. Molecular plant breeding has contributed to a more comprehensive understanding of molecular markers and their role in identifying the genetic diversity within the crop plants. Marker-assisted breeding is basically the application of molecular markers, in combination with linkage maps and genomics, to alter and improve plant traits on the basis of genotypic assay. Several modern plant breeding strategies were developed which include marker-assisted selection (MAS), marker-assisted backcrossing (MABC), marker-assisted recurrent selection (MARS), and genome-wide selection (GWS) or genome selection (GS). The selection of right type of molecular markers is usually dependent on the breeding objectives. Similarly, selection strategies of molecular markers for qualitative and quantitative characters may differ. The procedure followed for marker assisted selection under various breeding objectives and conditions, for qualitative and quantitative traits are discussed in this chapter.