Biotechnology, as an aid to crop improvement, became scientifically established following developments in our understanding of the genetics that took place in the early 1900s. These developments led to the production of high yielding crop varieties that substantially boosted global agricultural output throughout the twentieth century. However, developments in cell and molecular biology that have taken place since the early 1970s have permitted scientists to shift their attention from yield to pest control.
The main biotechnological methods that can be used to produce genetically modified organisms that are of relevance to pest control can be split into three (not mutually exclusive) categories, i.e.:
(1) Those that involve tissue culture techniques,
(2) Those that involve the use of recombinant DNA technology, and
(3) RNA Interference.
These techniques have been extensively developed in relation to the production of crop plants while the latter have been employed with plants, pests and natural enemies. In many cases, both of these techniques have been used to produce genetically modified organisms. For example, tissue culture techniques are often used to regenerate plants that have been produced using recombinant technology.
Technique # 1. Tissue Culture:
Tissue culture is a technique of growing plant tissues on synthetic medium under controlled and aseptic conditions. A German plant physiologist, G. Haberlandt, is considered to be the father of plant tissue culture who in 1902 conceived the idea of totipotency, which refers to the capability of a cell to give rise to a complete plant under suitable cultural conditions.
Such a property of the cell has far reaching implications to manipulate plant cells for rapid multiplication of plants, to cross plants at the level of somatic cells by overcoming the limit of cross-ability, and also to regenerate adult plants after modifying the DNA molecule at the cellular level.
One of the principle advantages of using tissue culture is that the desirable traits can be selected in the laboratory and the screening process can, therefore, be completed relatively quickly in contrast to consider whole plants in a field-based situation. Plant tissue culture includes several specialized areas, like micro-propagation, somaclonal variation, protoplast and anther culture.
(I) Micro-Propagation:
Micro-propagation involves the production of plants from very small (0.2-1.0 mm) plant parts through tissue culture techniques. Micro-propagation of selected plant species is one of the best and most successful examples of commercial application of tissue culture technology for mass multiplication of plants that has four district advantages, viz.-
(i) It is independent of seasonal constraints and hence ensures year-round rapid propagation,
(ii) Micro-propagated plants are generally true to type,
(iii) Micro-propagated plants are disease free,
(iv) Micro-propagated field grown plants usually exhibit vigorous growth, better quality and higher yields.
(II) Somaclonal Variation:
Somaclonal variation refers to variation among tissues or plants derived from the in vitro somatic cell cultures, i.e. callus and suspension cultures. Historically, it was accepted that all plants arising from tissue culture should be exact copies of the parental plants.
However, phenotypic variability was observed among regenerated plants, which was usually ignored. Plants regenerated from stem callus have been referred to as calliclones and those from protoplasts as protoclones. A general term somaclone has been proposed for plants derived from any form of cell culture and the variation among such plants termed as somaclonal variation.
The somaclonal variation may be genetic or it may result from culture induced epigenetic changes. The epigenetic changes are expressed at cell culture stage but usually disappear when plants are regenerated or they reproduce sexually. Variation arising out of anther/pollen culture is more precisely known as gametoclonal variation and that through protoplast culture is called protoclonal variation.
The major steps involved in the isolation of somaclones for insect resistance are:
(i) Growing calli or cell suspension cultures for several cycles from a high yielding and well-adapted susceptible variety,
(ii) Regeneration of plants from such long-term cell lines, and
(iii) Evaluation of large population of regenerated plants for insect resistance.
Using this procedure, somaclonal variants of sugarcane and sorghum have been obtained with good levels of resistance to sugarcane borer, Diatraea saccharalis (Fabricius) and fall armyworm, Spodoptera frugiperda (J.E. Smith), respectively.
(III) Protoplast Culture and Somatic Hybridization:
Somatic hybridization is an effective approach to hybridize the sexually incompatible species. Complete fusion of the nuclei and cytoplasms of somatic cells from both species leads to the formation of somatic hybrid cell and plant. Likewise, the fusion of cytoplasm from two species and nuclear genes from any one leads to the development of cybrid. The plant cells are surrounded by a thick cell wall which does not allow the two cells to fuse to get somatic hybrid cell/plant.
However, the protoplast can be easily fused and employed in several experiments aiming at the genetic modification of the plants. Protoplast is a naked cell without cell wall surrounded by plasma membrane and potentially capable of cell wall regeneration, growth and division. The techniques of isolation, culture and regeneration of protoplasts have been established in more than 100 plant species including major field, vegetable and fruit crops.
(IV) In Vitro Production of Haploids:
In self-pollinated crops, an inordinately long period is required to recombine desirable gene combinations from different sources in homozygous form. Generally, it takes 8-10 years to develop stable, homozygous and ready to use material from a fresh cross of two or more lines. Likewise, due to inbreeding depression in cross-pollinated crops, it is difficult to obtain vigorous inbreeds for hybrid seed production programmes.
In this regard, haploids possessing gametic chromosome number are very useful for producing instant homozygous true breeding lines. Besides, haploids constitute an important material for induction and selection of mutants particularly for recessive genes. The period required to develop homozygous true breeding line is less through haploid breeding as compared to conventional breeding.
Technique # 2. Recombinant DNA Technology:
One of the most significant breakthroughs in modern science is the development of techniques to transfer genes from unrelated sources into crop plants. Until recently, scientists could manipulate only the primary and secondary gene pools of the cultivated species for crop improvement. However, recent advances in molecular biology have made it possible to introduce genes from diverse sources such as unrelated plants, bacteria, viruses, fungi, insects, higher animals and even from chemical synthesis in the laboratory.
The recombinant DNA technology involves the use of genetic engineering so that the modified plants carry the functional foreign genes. These novel genes either reinforce the existing functions or add new traits to the transformed plants. These developments have provided the opportunity to develop crops with novel genes for insect resistance.
The whole process of introduction, integration and expression of the foreign gene(s) in the host is called genetic transformation or transgenesis. In fact, transgenesis has emerged as a novel tool to carry out single gene breeding or transgenic breeding of crop plants. Unlike conventional plant breeding, in this case only the cloned gene(s) of agronomic importance are introduced into the plants without the co-transfer of other undesirable genes from the donor.
The recipient genotype is least disturbed thereby setting aside the need for any backcross. This approach has the potential to serve as an effective means to remove certain defects of an otherwise well adapted cultivar, which are not easily manageable through conventional breeding approaches. Foreign genes can be transferred through vector- mediated or direct DNA transfer methods using protoplasts or other tissues. In vector-mediated approach, the transgene is combined with a vector, which takes it to the target cells for integration. In direct gene transfer, on the other hand, the gene is physically delivered to the target tissue.
(I) Vector-Mediated Gene Transfer:
A vector is a vehicle that transports the foreign genes into the recipient cells, protoplasts or intact plant. It is a DNA molecule, capable of replication in a host organism, into which a gene is inserted to construct a recombinant DNA molecule. This method is also called indirect method of gene transfer.
A vector could be either a DNA virus such as caulimovirus or geminivirus or could be plasmids such as tumor inducing (Ti) and root inducing (Ri) plasmids of Agrobacterium tumefaciens and A. rhizogenes, respectively. A plasmid is usually a circular piece of DNA, primarily independent of host chromosome often found in bacterial cells. The cells to be used for transformation must be replicating DNA, which is available in wounded or dedifferentiated cells or protoplasts.
(i) Agrobacterium-Mediated Gene Transfer:
Agrobacterium tumefaciens is a gram negative soil bacterium which infects a wide range of dicot plant species causing the crown gall disease. It has been demonstrated that a virulent bacterium, in addition to its chromosomal DNA, carries Ti plasmid. The Ti plasmid has two major regions of interest in transformation, i.e. T-DNA and the vir region. During infection, Ti plasmid transfers a protein of its transfer DNA (T-DNA) into the plant cell, which becomes integrated into the chromosomal DNA of plant.
T-DNA segment of Ti plasmid carries a number of genes encoding enzymes for the synthesis of phytohormones such as cytokinins and auxins, which stimulate the growth and division of the plant tissue resulting in the formation of characteristic tumors and production of specific metabolites called opines such as octopine and nopaline.
Foreign genes inserted within the T-region of the Ti plasmid are transferred to and stably integrated into the plant genome. Agrobacterium-mediated transformation has been a method of choice in dicotyledonous plant species, where plant regeneration systems are well established. However, there are several reports of successful transformation of cereals using Agrobacterium.
(ii) DNA Viruses as Vectors:
Due to their ability to cause systemic infections, the viruses have been investigated as vectors for gene transfer in plants. Genetic engineering of the genomes of DNA and RNA viruses has been accomplished with the introduction of foreign DNA sequences. The foreign genes replace a part of the viral genome and create a defective viral particle that can infect the target plant only in the presence of a helper virus.
The genomes of caulimoviruses such as cauliflower mosaic virus (CaMV) and geminivirus such as tomato golden mosaic virus (TGMV) are double stranded DNA which makes these viruses as potential transformation vector. However, viral vectors have not been developed to a stage where these can be routinely used for plant transformation.
(II) Direct Gene Transfer Methods:
Since the host range of Agrobacterium has been largely limited to dicotyledonous plant species, some other methods of transfer of genes to monocotyledonous plants such as cereals have been developed.
Many grain legume species and woody plants are also not amenable to Agrobacterium- mediated transformation. For genetic transformation of such recalcitrant crops, techniques of direct gene delivery have been attempted. Direct gene transfer methods make use of chemical, physical or electrical methods to introduce DNA into the cells.
Some of the methods are explained below:
(i) Direct uptake of DNA:
This method is based on the ability of the protoplasts to uptake the foreign DNA from the surrounding solution. An isolated plasmid DNA (vector) is mixed with the protoplasts in the presence of polyethylene glycol (PEG), polyvinyl alcohol and calcium phosphate, which enhance the uptake of DNA by protoplasts.
After 15-20 minutes of incubation, the protoplasts are cultured in the presence of appropriate selective agent. Upon treatment with PEG, the permeability of the plasma-membrane is increased, leading to transient formation of pores, thus facilitating the uptake of DNA by protoplasts. Foreign gene of interest can be as naked DNA such Ti or E. coli plasmids or can be encapsulated into liposomes.
Although the frequency of integration of DNA into plant genome is relatively low, regeneration of transferred protoplasts selected for the presence of the particular selectable marker, yields transgenic plants. This method, however, depends on the plant regeneration ability of the protoplasts and has been successfully used to produce transgenic plants in brassica, strawberry, lettuce, rice, wheat and maize.
(ii) Electroporation:
In this technique, a high voltage current is applied in a pulsed manner, which creates tiny holes in the plant cell membrane. These holes are large enough for DNA molecule to diffuse into the cell. The cells recovered from the electric shock can be regenerated into whole plants. Generally, protoplasts are used since they have exposed plasma membrane.
Protoplasts are suspended in buffered saline solution containing plasmid DNA in a cuvette and an electrical impulse is applied across two platinum electrodes in the cuvette. The protoplasts are then cultured to regenerate the plants. Electroporation has been successfully used for obtaining transgenics in tobacco, maize, rice, wheat and sugarcane.
(iii) Micro-projectile Bombardment:
This method is also called biolistic approach and has been a major development in direct gene transfer of DNA. It has enabled transformation of many plant species that were not amenable to Agrobacterium or protoplast based gene transfer techniques. The method is considered independent of genotype and the tissue.
This method consists of delivering DNA into cells of intact plant organs, cultured tissues or cell suspensions via projectile bombardment. High-density particles, usually gold or tungsten (1-2 µm in diameter) coated with plasmid DNA containing the foreign gene are accelerated to high velocity by a particle gun apparatus. These dense particles thus acquire sufficient kinetic energy to penetrate the membranes, and thus to deliver the DNA into the cells.
The transgenic plants have been recovered in several important crops including banana, barley, bean, canola, cassava, cotton, maize, papaya, peanut, poplar, rice, soybean, squash, sugarbeet, sugarcane, sunflower and wheat, by using more regenerable tissues/organs such as embryogenic calli, immature embryos and shoot meristems.
(iv) Microinjection:
This method involves the injection of DNA directly into the plant cell nucleus or intact plant organ. The foreign DNA is delivered into protoplasts using a glass micro-pippette having an orifice diameter of less than one µm. It is a useful technique for the precise delivery of DNA into target cell.
In this method, the location of DNA delivery can be controlled with micromanipulator and the volume of injection can be controlled by the microinjection apparatus. It is a direct and precise method of delivery of DNA into specific cell compartments. Microinjection of DNA into the nucleus can give high efficiency of transformation.
This method raises the possibility of microinjecting a variety of materials such as chromosomes, and even chloroplasts and mitochondria can also be transferred by microinjection. This technique has been successfully used in alfalfa, tobacco and brassica crops.
Technique # 3. RNA Interference:
RNA interference (RNAi) is a method of blocking gene function by inserting short sequences of double-stranded ribonucleic acid (dsRNA) that match part of the target mRNA sequence, thus no proteins are produced. It is a cellular process in which exogenous dsRNA with a strand complementary to a fragment of such mRNA causes degradation of the complementary mRNA, i.e., knock down the expression of genes.
This technology is proving a prized functional genomics tool in insects to ascertain the function of the many newly identified genes accumulating from genome sequencing projects. RNAi can unveil the functions of new genes, lead to the discovery of new functions for old genes, and find the genes for old functions.
The dsRNA, introduced into the cell, is cleavage into short 21-25 nucleotide RNAs, by an enzyme called dicer that has RNase III domains, which are known small interfering RNAs or short interfering RNAs (siRNAs). These siRNAs are unwound and the antisense siRNA strand couples to RNA-induced silencing complex (RISC).
This RISC complex finds the complementary sequence of a target messenger RNA molecule and induces cleavage by Argonaute, the catalytic component of the RISC. The cleaved target mRNA fragments are released from the RISC complex and are subsequently degraded by cytoplasmic nucleases.
RNAi-related phenomenon was initially elucidated in plants (Petunia) and was named post- transcriptional gene silencing (PTGS). In animals, the first clue of RNAi is demonstrated in Caenorhabditis elegans Maupus in 1995. Drosophila melanogaster Meigen was the first among the insects in which the RNAi was used in vivo to study the functions of genes frizzled and frizzled 2.
Later, RNAi has been used to study the function of various genes in number of insect pests like Tribolium castaneum (Herbst), Oncopeltus fasciatus (Dallas), Periplaneta americana (Linnaues), Blattella germanica (Linnaues), Spodoptera litura (Fabricius), Helicoverpa armigera (Hubner),Plutella xylostella (Linnaues), Bemisia tabaci (Gennadius), etc.
Three techniques, viz. direct injection, feeding and egg soaking have been adopted to deliver the dsRNA into different test organisms. RNAi technology holds significant importance in functional genomics. The first successes of development of transgenic plants expressing dsRNA against insect pests have opened new vistas for pest management. Thus, RNAi technology is emerging as an alternate biotechnological tool in the ongoing task of developing pest-resistant crops.