Plant Biotechnology, Volume 2

Transgenics, Stress Management, and Biosafety Issues

Sangita Sahni, Bishun Deo Prasad, Prasant Kumar, Sangita Sahni, Bishun Deo Prasad, Prasant Kumar

• Publisher: Apple Academic Press
• Year: 2017
• eBook ISBN: 9781351819473
• Edition: 1
• Topic: Biological Sciences
• Subtopic: Botany
• Index: Biological Sciences

This volume is the second of the new two-volume Plant Biotechnology set. This volume covers many recent advances in the development of transgenic plants that have revolutionized our concepts of sustainable food production, cost-effective alternative energy strategies, microbial biofertilizers and biopesticides, and disease diagnostics through plant biotechnology. With the advancements in plant biotechnology, many of the customary approaches are out of date, and an understanding of new updated approaches is needed. This volume presents information related to recent methods of genetic transformation, gene silencing, development of transgenic crops, biosafety issues, microbial biotechnology, oxidative stress, and plant disease diagnostics and management.

Key features:

• Provides an in-depth knowledge of various techniques of genetic transformation of plants, chloroplast, and fungus
• Describes advances in gene silencing in plants
• Discusses transgenic plants for various traits and their application in crop improvement
• Looks at genetically modified foods and biodiesel production
• Describes biotechnological approaches in horticultural and ornamental plants
• Explores the biosafety aspect associated with transgenic crops
• Considers the role of microbes in sustainable agriculture

PART I

Genetic Transformation

CHAPTER 1

DIRECT AND INDIRECT METHODS OF GENE TRANSFER IN PLANTS

NANDLAL CHOUDHARY*, ARUNA JANGID, and SHIKHA DHATWALIA

Amity institute of Virology and Immunology, Amity University, Noida 201313, UP, India

* Corresponding author. E-mail: [email protected]

CONTENTS

Abstract

1.1 Introduction

1.2 Tissue Culture

1.3 Plants’ Genetic Transformation Methods

1.4 Future Prospects

Keywords

References

ABSTRACT

Transformation is a molecular biology method to alter the genetic material of cells by incorporation of desired foreign genetic material by direct and indirect methods. The Agrobacterium tumefaciens and A. rhizogenes mediated are the direct transformation methods whereas, the protoplast transformation, electroporation, particle bombardment, microinjection, sonoporation, lipofection, calcium phosphate, laser transfection, chloroplast transformation, and mediated transformation are the indirect methods. The plant transformation has become a versatile method for the incorporation of desired characteristics in the selected plant for the benefits of human society. Agrobacterium tumefaciens is the most successful and popular technique compared to other physical/mechanical techniques. Agrobacterium infection has been used for transfer of foreign DNA into a number of dicotyledonous species (utilizing its plasmids as vectors). Arabidopsis thaliana was stably transformed with high efficiency using T-DNA transfer by agrobacterium. Agrobacterium-mediated transformation using the floral dipping method is the most widely used method to transform Arabidopsis. Genetic transformation mediated by agrobacterium is a simple and comparatively less expensive than other methods of transformation. Transgenic crop obtained through agrobacterium-mediated genetic transformation have better fertility percentage.

1.1 INTRODUCTION

Plants are important sources of many important products, such as food, fibers, medicines, and energy, which fulfill the need of human beings. Humans have been cultivating these plants to meet desired products. These selective plants are being improved for better quality and quantity ofproduct by breeding to meet the needs of growing human population on Earth. The plant breeders are dependent on the existing gene pool and sexual compatibility of the plant species, which is a limitation.

In 1928, Griffith suspected the transfer of genetic material in an experiment when an nonpathogenic pneumococcus strain became pathogenic when mixed with heat-killed pathogenic pneumococcus strain. This was the first report of gene transfer but transforming substance was not identified (Griffith, 1928). After two decades, Hershey and Chase (1952) successfully demonstrated the transfer of genetic material DNA of bacteriophage into the Escherichia coli cells. This finding led the researcher to introducedesired DNA into wide variety of organisms. The genetic material of some species were altered by the incorporation of the selective foreign DNA following the molecular biology methods are called transformation. These transformation techniques if performed directly or indirectly in the plant cells are called plant genetic transformation. In 1981, the first successful gene transformation was demonstrated in tobacco plant using the soil bacterium Agrobacterium tumefaciens (Otten et al., 1981). Until now, more than 100 different plant species have been transformed with the desired foreign genes using A. tumefaciens or other available methods.

The plant genetic transformation has become a versatile method for the production of agricultural and medicinal value product for the benefits of human society (Campbell, 1999; Lorence and Verpoorte, 2004; Uzogara, 2000). The transfers of gene into plants cells are difficult because plant cells are impermeable which acts as a barrier to diffuse through the cell membrane. Because of diversity of plant species and their diverse genotypes, various gene transformation methods have been developed to overcome this barrier in plants.

1.2 TISSUE CULTURE

The capability of growing the plants from cells after gene transformation depends on the kind of selected. This is one of the most important steps for a successful gene transformation technology. The identification of correct cell types is difficult in plants because plant cells are totipotent which can be regenerated to become whole plant in vitro by theorganogenesis or embryogenesis process. But these processes may force a degree of genome stress which might lead to a somaclonal variation, if the whole plant is regenerated via callus phase. If the gene is transferred into pollen or egg cells to produce the genetically transformed gametes, and used for fertilization (in vivo), then they will rise to transformed whole plants. Similarly, gene is inserted intozygote by in vivo or in vitro, and then rescued embryos can also be used to produce transgenic plants. The individual cells in embryos or meristem can also be grown in vitro to produce the transgenic plants.

To generate successful transgenic plants, some kind of tissue-culture step depending on the plant species is necessary. In tissue-culture process, the explants or small piece of living tissue are isolated from the plant, grown aseptically on artificial nutrient medium into an undifferentiated mass known as callus. The explants, such as buds, root tips, nodal stem segments, or germinating seeds, are most preferred because they are rich in undetermined cells and are capable for rapid proliferation. The selected explant should first be disinfected by washing with the sodium hypochlorite or hydrogen peroxide before placing them on the medium because the medium also supports the growth of microorganism. The nutrient medium should contain the appropriate quantity of phytohormones to maintain the cells in an undifferentiated condition and develop into callus. The correct proportion of phytohormones, auxin, and cytokinins depends on species and explant types are important for the growth of callus culture. Thecy-tokinin is required for shoot culture and auxin for root culture, therefore, low auxin:cytokinin ratio favors the shoot growth, whereas high ratio leads to root growth in callus. Gibberellins, GA3, are required by some explant for their continuousgrowth, whereas abscisic acid boosts specific development actions like somatic embryogenesis. In addition to phytohormones, the nutrient medium also contains the macroelements and microelements, essential vitamins, amino acids, and sucrose. Some plant medium also containscasein hydrolysate, coconut water, yeast extract, and gelling agent. The plant material that can be manipulated in culture provides excellent opportunities for gene transfer methods and generation of transgenic plants.

1.3 PLANT’S GENETIC TRANSFORMATION METHODS

Various available plant transformation methods is discussed in this chapter such as biological methods: A. tumefaciens and protoplast-mediated transformation; and chemical methods: calcium phosphate, co-precipitation and lipofection, physical methods, electroporation, biolistics, agitation with glass beads, vacuum infiltration, silicon-carbide whisker, laser microbeams, ultrasound and shock-wave-mediated method.

Plant genetic transformations are classified into direct and indirect gene transfer methods.

1.3.1 INDIRECT GENE TRANSFER METHODS

For indirect gene transformation methods, two bacterial strains, A. tumefaciensand A. rhizogenes, have been discovered to transfer the desired gene into plant cells; however A. tumefaciens is widely used.

1.3.1.1 AGROBACTERIUM TUMEFACIENS

Among the various available vectors for plant transformation, the Ti plasmid of A. tumefacienshas been widely accepted. Agrobacteriumwas first discovered in grape plant in 1897 by Fridiano Cavara. In 1907, crown gall diseasewas reported in plants caused by T-DNA of Ti-plasmid of Agrobacterium (Smith andTownsend, 1907). Agrobacterium has been characterized as Gram-negative bacteria or soil phytopathogen that belongs to the rhizobiaceae family. Agrobacterium prefer to infect mostly the dicotyledonous plants and at the infection or wound site produces an unorganized growth of cells that is known as crown gall tumors. This bacterium harbors the tumor-inducing plasmid, known as Ti plasmids, which are exploited to transfer desired gene into target plant tissue. Agrobacteriumgot the natural ability to transfer the T-DNA, part of Tiplasmid, into the plants’ genome, and because of this unique ability, Agrobacteriumis known as natural genetic engineer of plants (Binns and Thomashaw, 1988; Nester et al., 1984). The unwanted sequence of T-DNA region of Ti-plasmid is the crucial region to replace with foreign desire gene.

1.3.1.2 MOLECULAR BASIS OF AGROBACTERIUM-MEDIATED TRANSFORMATION

1.3.1.2.1 Vectors for Gene Transfer

Vectors usually contain the selectable markers to recognize the transformed cells from the untransformed cells, multiple rare restriction sites, and bacterial origins of replication (e.g., ColE1). However, these features in vectors do not help in transfer of gene and integration into plant nuclear genome. Ti-plasmid of Agrobacterium has wide host range and capable to transfer gene that makes them preferred vector over other available vectors.

The T-DNA nucleotide sequence end are flanked by 25-bp direct repeat sequences known as left border (LB) and right border (RB) and both border sequences collectively known as T-DNA border. Plasmid DNA comprising the T-DNA with border sequencesis called mini- or micro-Ti-plasmid (Waters et al., 1991). Nucleotide sequences of T-DNA borders are essential and play an important role to transfer the T-DNA into the plant cell upon infectionif present in cis orientationbut border sequence itself does not get transferred. Any DNA sequence flanked by repeat of 25 bp in the correct orientation can be transferred to plant cells and similar attribute were exploited with Agrobacterium-mediated gene transfer to produce transgenics of higher plants. It was shown in the experiment that onlyRB sequence has been used and observean enhance sequence or sometimes refer overdrive sequence located upstream to RB sequence is also necessary for high-efficiency transfer of T-DNA (Peralta et al., 1986; Shaw et al., 1984). However, the left-border sequencehas little activity alone (Jen and Chilton, 1986).

1.3.1.2.2 Structure and Functions of Ti Plasmids

Agrobacterium harbor a large Ti-plasmid of 200–800 kbp which contains four main regions: T-DNA, vir region, origin of replication, region enabling conjugative transfer, and o-cat region (Hooykaas and Schilperoort, 1992; Zupan and Zambrysky, 1995). ...

Table of contents

• Cover
• Half Title
• Title Page
• Copyright Page
• Table of Contents
• List of Contributors
• List of Abbreviations
• Preface
• Acknowledgment
• Part I: Genetic Transformation
• Part II: Gene Silencing in Plants
• Part III: Transgenic Crops and Biosafety
• Part IV: Microbial Biotechnology
• Part V: Oxidative Stress
• Part VI: Plant Disease Diagnostics and Management
• Index