Genome Size and Genetic Homogeneity of Regenerated Plants: Methods and Applications

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)- CRISPR-associated protein (Cas)-mediated genome editing is a recently developed gene editing technology, which has transformed functional and applied genomics. This technology is precise, cost-efficient, and rapid than other previously developed genome editing tools such as Meganucleases (MNs), Zinc-Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs). The CRISPR-Cas9 system is widely exploited for developing plants with enhanced tolerance towards various environmental stresses, resistance against pathogens, improved yield and nutritional superiority. The method is robustly applied to alter both DNA and RNA at specific target regions. The availability of well annotated genome sequence and an efficient genetic transformation system may open numerous possibilities to gain desirable traits in crop plants employing CRISPR-Cas-mediated genome editing technology. In this chapter, we summarized the basics of CRISPR-Cas technology, various kinds of CRISPR systems and their associated Cas proteins, application in generating abiotic and biotic stress tolerant crops, and bottlenecks of CRISPR-Cas systems. 

The study of somaclonal variation is a growing research area that has allowed to identify several biological processes involved in genetic instability during plant tissue culture. These changes may be undesirable during the micropropagation of elite plants or desirable during plant breeding programs. There are different molecular techniques that allow to analyze this somaclonal variation. Due to the progress that has been made in the manipulation and analysis of DNA, the number of molecular markers has increased to achieve this objective. These methods have been increasing in number, while some of them have been widely used since their development [simple sequence repeat (SSR), inter simple sequence repeats (ISSRs), amplified fragment length polymorphism (AFLP), and random amplified polymorphic DNA (RAPD)], others, such as retrotransposon amplification, SSR-markers derived from expressed sequence tags, targeted region amplification polymorphism (TRAP), transcribed sequences (RNAseq). Whole genome sequencing is increasing their use and they complement each other by providing more information, allowing to link genetic markers with specific phenotypes in somaclonal variants. The aim of this chapter is to highlight the methodology of the most commonly used molecular markers to assess somaclonal variation during plant tissue culture. 

Valeriana is an important genus due to its immense medicinal properties. This plant contains over 150-200 chemical constituents, which make it useful as a herbal remedy for various ailments. Conventionally, these plant species are cultivated through seeds; however, poor seed setting coupled with low germination rate restricts its cultivation in the wild as well as poses a problem for its cultivation. Due to irregular grazing and excessive harvesting by local people for herbal drugs, the wild population of Valeriana species are at a high risk of rapid elimination and extinction. Plant tissue culture is one of the most important methods used for the effective conservation of many rare, endangered and exploited plant species. However, the induction of genetic variability in regenerants may limit the purpose of micropropagation. Assessing the clonal fidelity of in vitro derived regenerants is highly essential to know whether plants are true to type or not. The development and utilization of molecular markers for the identification of plant genetic diversity is one of the most important progresses in the field of molecular genetics studies. Molecular markers are a prevalent tool, due to their stability, cost-effectiveness and ease of use for a variety of applications in the field of molecular genetics. Several molecular markers have been efficaciously employed to evaluate the clonal fidelity of the Valeriana clones so that only the elite, genetically identical plants are propagated. This chapter highlights the biology, pharmacology, need for micropropagation and application of DNA molecular markers in clonal fidelity assessment of the in vitro propagated Valeriana species. 

A genetically modified (GM) poplar (Populusnigra) that overexpresses a key enzyme in the plant hormone gibberellin (GA) synthesis system was produced by the Agrobacterium method. Gibberellin is known to control the elongation and growth of higher plants. GA20-oxidase is a key enzyme in the biosynthesis of active GAs. The major gibberellin biosynthetic pathway involving the participation of GA20-oxidase is shown and described along with other genes and enzymes like GA20ox, GA3ox, GA 3beta-hydroxylase, GA2ox, and GA 2beta-hydroxylase. There are six clades in the GA oxidase gene subfamily. In this chapter, the production of genetically modified (GM) poplar overexpressing a known tobacco GA 20-oxidase gene, NtGA20ox and a poplar gene PnGA20ox, classified under the same GA 20-oxidase gene, is described. This genetic variant and recombinant poplar showed enhanced above-ground growth and stem biomass production. In addition, GM poplars with altered expression of genes controlling flower bud formation. This successfully reduced the long juvenile phase period to less than one year. Genetic modification has made it possible to control tree growth and reproduction. 

Coffee is one of the most preferred beverages consumed by millions of people throughout the world. It is cultivated in more than 80 countries in tropical and subtropical zones of Asia, Africa, and Latin America and provides livelihood to 125 million people worldwide. Among 125 coffee species known so far, only two coffee species, Coffea arabica (arabica coffee) and Coffea canephora (robusta coffee), are commercially cultivated for beverage production. Coffee is a perennial plant and therefore subjected to constant environmental stress. However, during the last few decades, sustainable coffee cultivation has been threatened by unprecedented climate change. This calls for unified efforts, including cutting-edge research and modified management practices. Although conventional breeding efforts have been developed to address some issues, emerging biotechnology research, especially in-vitro propagation technology, could augment the coffee cultivation landscape. Despite the tangible progress made in coffee tissue culture, there were some grey areas, such as the level of somaclonal variation and the genomic changes associated with somatic embryogenesis in coffee, which needs to be addressed imminently. This chapter provides detailed progress on coffee tissue culture and addresses some of the critical issues associated with the genetic homogeneity of tissue culture plants.

The chapter presents the knowledge accumulated on the recent investigation of somatic embryogenesis (SE) in genera Medicago. The role of 2,4-D in the process of induction of embryogenic potential in diploid Medicago and its transport by the combined action of auxin transporters or diffusion of dissociated molecules is discussed. Among the many methods for studying the process, this chapter is focused on cellular and molecular tools – flow cytometry, assessment of expression level of SE related transcripts of key genes of auxin inducible process and different PCR techniques. Our recent studies on the process of SE in M. truncatula are focused on the role of the two genes MtLAX3 (an auxin transmembrane transporter) and a transcriptional factor MtARF-B3 (an auxin response factor, containing a B3-binding domain). The transcription profiles of these genes are evaluated and their expression patterns are assessed during indirect somatic embryogenesis – steps of callus formation, embryogenic zone formation and the stages of globular, torpedo and cotyledonary embryos. The localization of expression during the process of SE is traced by the β-glucuronidase reporter gene (GUS) under the control of the promoters of these genes. Inverse PCR (IPCR) and Transposon display (TD) are techniques which evaluate transposition and new retrotransposon copies in the investigated mutant lines, and we used these methods as markers for the efficiency of the induction phase of the process of SE. The use of all these methods turns light on a better understanding of the process of somatic embryogenesis in the model species Medicago truncatula and other annual medics.

Polyploidy is the condition of having more than two sets of chromosomes. The mechanism of polyploidy helps in deriving special traits like an increase in biomass, an increase in the size of various organ systems, and secondary metabolite content for the progeny. Various chemical compounds (colchicine, trifluralin, and oryzalin) that have the capacity to alter the mitotic cycle were used for the purpose of inducing polyploidy. Various techniques, such as counting of chromosome number, chloroplast number, determination of pollen diameter, and estimation of leaf stomatal density and size, were developed to analyze the polyploidy of the plants. However, these methods are not reliable for their regular use. Thus, of all the above-mentioned approaches, the estimation of ploidy level by flow cytometry (FCM) has been the most popular over the last few decades. Flow cytometry is now extensively used for the verification of haploidy, aneuploidy, and polyploidy. The ease of sample preparation, fast acquisition, and accurate measurements have made the method popular in the domains of plant cell biology, systematics, evolution, genetics, and biotechnology. The current chapter discusses the induction of polyploidy and its importance in plant breeding. It also emphasizes the importance of FCM in the analysis of polyploidy and enumerates the various polyploidy studies involving the application of FCM.

Somaclonal variation is generally undesirable in woody plant tissue cultures when the main aim is In Vitro micropropagation or transformation of selected material, however, it could sometimes be useful for the production of new and valuable varieties. Thus, the determination of somaclonal variation is very important for the genetic fidelity of the micropropagated woody plant species. Molecular markers are generally used in the identification of plant species, analysis of qualitative and quantitative trait loci, determination of the genetic distance between genotypes, detection of stable, high yielding and qualified varieties for variety registration and certification. Moreover, molecular markers are also very useful for the evaluation of the genetic fidelity of micropropagated cultures. Among many markers, ISSRs, SSRs, AFLPs and MSAPs are found to be very efficient for the assessment of genetic stability of micropropagated different woody plants since they are easy to apply, quick to use, and more reliable due to their efficiency and repeatability. In this context, the aim of the present book chapter is to review the advantages of molecular markers together with the summarization of the studies on the determination of genetic stability of micropropagated woody species using this technique in the last decade and causes of somaclonal variation. 

Orchids occupy a significant position in the international floricultural market because of their spectacularly beautiful flowers with varied sizes, forms, patterns, and colorations. Apart from their high ornamental values, they are known for therapeutic application in the traditional medicinal system. However, natural orchid resources are quickly depleting because of excessive unregulated commercial collection and mass habitat destruction. Orchid production through conventional propagation methods cannot meet the present demands for these ornamental plants. Micropropagation of orchids through plant tissue culture provides an excellent opportunity to propagate true-to-type quality plants on a large scale rapidly. However, somaclonal variation may appear in the in vitro clones producing undesired plants with phenotypic and molecular defects. It is obligatory to test the genetic integrity of the propagated plants to ensure the production of identical quality orchids. Genetic stable orchids are produced by evaluating the fidelity of the regenerants using molecular markers. The present chapter highlights the genetic stability assessment of several micropropagated orchids using molecular markers and the flow cytometry method.

Flow cytometry is one of the sophisticated tools with its applications in different biological disciplines. It is potentially efficient in the characterization of mixed populations of cells present in biological samples, including blood cells, lymphocytes, microorganisms, sperms, cancer cells, metabolites, antibodies, DNA/RNA content, proteins, toxins, plant spores, etc. Flow cytometry is widely applied in the determination of cellular characteristics and cellular components profiling like cell size, intracellular pH, DNA, RNA, proteins, surface receptors, membrane potential, calcium, and others. Currently, flow cytometry is pragmatic in basic as well as applied plant research and plant industrial applications like plant breeding. Flow cytometry has been considered a reliable, rapid, efficient, and accurate tool for analysis of ploidy level and nuclear genome size estimation. It is also subjected to taxonomy to study population/subpopulation dynamics. Gender determination from pollen grain is also possible due to flow cytometry. 

In tissue culture, plants are genetically identical to native plants. Using methods such as flow cytometry, cytogenetic analysis, and molecular markers such as AFLP, ISSR, RAPD, RFLP, and SSR, we can detect the genetic uniformity of plants. Among these techniques, flow cytometry (FCM) is a fast, easy, cost-effective, and accurate method for screening the genetic stability of propagated plants. FCM involves measuring the fluorescence light of cell nuclei with a flow cytometer after separation and staining with a chemical with fluorescence properties related to DNA. There is a computer with software for receiving, storing, further processing, and displaying result information. The information is presented in an uncomplicated diagram. FCM is used to determine the genome size and ploidy levels of plants produced In Vitro. FCM also stimulates cell cycle function and replication rate in various plant organs and tissues. It was used to study plant organs in greenhouse/field conditions and laboratory conditions (anther culture, eggs, and protoplasts). Plant materials grown in tissue culture are unstable due to somaclonal diversity, especially in their DNA content, and therefore, the use of the FCM method is very effective.

Most of the medicinal, aromatic and other commercially important crops are poor rooters, and some of them are sessile in terms of seed production; hence these plants are very difficult to propagate either through stem cuttings based vegetative propagation or through seedlings based sexual propagation. During the last two decades, plant tissue culture has emerged as an alternative technique for the propagation of plants with commercial importance. Majorly, the somatic tissues, viz., leaf, node and shoot tip, are being used as explants for the production of genetically similar plantlets through tissue culture studies. Recently, abnormalities with respect to ploidy level and genetic fidelity have been reported in In Vitro regenerated plantlets. This is mainly due to the usage of synthetic chemicals or artificial plant growth regulators in In Vitro culture studies, the fragile nature of callus and exposure of cultures to artificial light sources. In order to ensure the commercial production of genetically true clones of commercial plants, nowadays it has become an obligatory step to assess the ploidy level and genetic fidelity of regenerated plantlets with that of mother plants. This book chapter focussed on different molecular techniques which are in use for the detection of ploidy level and genetic fidelity of In Vitro micro propagated plantlets.

The flow cytometry technique has currently been employed in various fields of research, especially in measuring the 2C DNA of plants. The technique is also used in modern biosystematics, speciation, evolutionary studies and in molecular breeding. A large number of tissue culture raised ornamental and medicinal plants’ DNAs are currently made and compared with field grown plants. Various factors influence the quality of active nuclei isolation, which determines the success of accurate DNA estimation. The importance of extraction buffer, reference standards, fluorochrome dyes, and the process of gating is highlighted in order to understand various steps of flow cytometry in measuring DNA. An array of compounds act as inhibitors to disrupt fluorochrome binding to DNA, causing errors in estimating nuclear DNA content; these compounds with their families are presented and summarized. Micropropagation using shoot tips and nodal stems produces true-to type plants, while callus regenerated plants show somaclonal variations – a process showing altered DNA. The role of flow cytometry in investigating the genetic homogeneity of tissue cultured plant population is therefore reviewed. The 2C DNA and genome size of a few medicinal and ornamental plants such as Catharanthus, Allium, Rawolfia, Gladiolus, Caladium, Zephyranthes from authors’ laboratory were measured and described. The intra-specific and inter-specific genome size and chromosome number variation with reference to gene duplication and DNA sequence loss are discussed. The present chapter, in general, discusses the applications of flow cytometry in field and tissue culture grown ornamentals and medicinal plants.