Most methods of plant transformation applied to GM crops require that a whole plant is regenerated from isolated plant cells or tissue which have been genetically transformed.-This regeneration is conducted in vitro so that the environment and growth medium can be manipulated to ensure a high frequency of regeneration.-In addition to a high frequency of regeneration, the regenerable cells must be accessible to gene transfer by whatever technique is chosen.-The primary aim is therefore to produce, as easily
1. Introduction of Plant Tissue Culture-Most methods of plant transformation applied to GM crops require that a whole plant is regenerated from isolated plant cells or tissue which
have been genetically transformed.
-This regeneration is conducted in vitro so that the environment and growth medium can be manipulated to ensure a high frequency of regeneration.
-In addition to a high frequency of regeneration, the regenerable cells must be accessible to gene transfer by whatever technique is chosen.
-The primary aim is therefore to produce, as easily and as quickly as possible, a large number of regenerable cells that are accessible to gene transfer.
-The subsequent regeneration step is often the most difficult step in plant transformation studies.
-However, it is important to remember that a high frequency of regeneration does not necessarily correlate with high transformation efficiency.
-This chapter will consider some basic issues concerned with plant tissue culture in vitro, particularly as applied to plant transformation.
-It will also look at the basic culture types used for plant transformation and cover some of the techniques that can be used to regenerate
whole transformed plants from transformed cells or tissue.
-Plant tissue culture, the growth of plant cells outside an intact plant, is a technique essential in many areas of the plant sciences.
-Cultures of individual or groups of plant cells, and whole organs, contribute to understanding both fundamental and applied science.
-It relies on maintaining plant cells in aseptic conditions on a suitable nutrient medium.
-The culture can be sustained as a mass of undifferentiated cells for an extended period of time, or regenerated into whole plants.
-The starting point for all tissue cultures is plant tissue, called an explant. It can be initiated from any part of a plant - root, stem, petiole, leaf or flower - although the success of any one of these varies between species.
-It is essential that the surface of the explant is sterilised to remove all microbial contamination.
-Plant cell division is slow compared to the growth of bacteria and fungi, and even minor contaminants will easily over-grow the plant tissue culture.
-The explant is then incubated on a sterile nutrient medium to initiate the tissue culture.
-The composition of the growth medium is designed to both sustain the plant cells, encourage cell division, and control development of either an undifferentiated cell mass, or particular plant organs.
-The concentration of the growth regulators in the medium, namely auxin and cytokinin, seems to be the critical factor for determining whether a tissue culture is initiated, and how it subsequently develops.
-The explant should initially form a callus, from which it is possible to generate multiple embryos and then shoots, forming the basis for plant regeneration and thus the technology of micropropagation.
-The first stage of tissue culture initiation is vital for information on what combination of media components will give a friable, fast-growing callus, or a green chlorophyllous callus, or embryo, root or shoot formation.
-There is at present no way to predict the exact growth medium, and growth protocol, to generate a particular type of callus.
-These characteristics have to be determined through a carefully designed and observed experiment for each new plant species, and frequently also for each new variety of the species which is taken into tissue culture.
-The basis of the experiment will be media and protocols that give the desired effect in other plant species, and experience.
-History of Tissue Culture Techniques - The in vitro techniques were developed initially to demonstrate the totipotency of plant cells predicted by Haberlandt in 1902. Totipotency is the ability of a plant cell to perform all the functions of development, which are characteristic of zygote, i.e., ability to develop into a complete plant.
- In 1902, Haberlandt reported culture of isolated single palisade cells from leaves in Knop's salt solution enriched with sucrose.
-The cells remained alive for up to 1 month, increased in size, accumulated starch but failed to divide.
- Efforts to demonstrate totipotency led to the development of techniques for cultivation of plant cells under defined conditions.
- This was made possible by the brilliant contributions from RJ. Gautheret in France and P.R. White in U.S.A. during the third and the fourth decades of 20th century.
- Most of the modern tissue culture media derive from the work of Skoog and coworkers during 1950s and 1960s.
- The first embryo culture, although crude, was done by Hanning in 1904; he cultured nearly mature embryos of certain crucifers and grew them to maturity.
- The technique was utilised by Laibach in 1925 to recover hybrid progeny from an interspecific cross in Linum.
- Subsequently, contributions from several workers led to the refinement of this technique.
- Haploid plants from pollen grains were first produced by Maheshwari and Guha in 1964 by culturing anthers of Datura.
- This marked the beginning of anther culture or pollen culture for the production of haploid plants.
- The technique was further developed by many workers, more notably by JP. Nitch, C. Nitch and coworkers.
- These workers showed that isolated microspores of tobacco produce complete plants.
- Plant protoplasts are naked cells from which cell wall has been removed. In 1960, Cocking produced large quantities of protoplasts by
using cell wall degrading enzymes.
- The techniques of protoplast production have now been considerably refined.
- It is now possible to regenerate whole plants from protoplasts and also to fuse protoplasts of different plant species.
- In 1972, Carlson and coworkers produced the first somatic hybrid plant by fusing the protoplasts of Nicotiana glauca and N. langsdorfii.
- Since then many divergent somatic hybrids have been produced.
- A successful establishment of callus cultures depended on the discovery during mid-thirties of IAA (idole-3-acetic acid), the endogenous
auxin, and of the role of B vitamins in plant growth and in root cultures.
-The first continuously growing callus cultures were established from cambium tissue in 1939 independently by Gautheret, White and
Nobecourt.
-The subsequent discovery of kinetin by Miller and coworkers in 1955 enabled the initiation of callus cultures from differentiated tissues.
-Shoot bud differentiation from tobacco pith tissues cultured in vitro was reported by Skoog in 1944, and in 1957 Skoog and Miller proposed that root-shoot differentiation in this system was regulated by auxin-cytokinin ratio.
-The first plant from a mature plant cell was regenerated by Braun in 1959. Development of somatic embryos was first reported in 1958- 1959 from carrot tissues independently by Reinert and Steward.
-Thus within a brief period, the tissue culture techniques have made a great progress.
- From the sole objective of demonstrating the totipotency of differentiated plant -cells, the technique now finds application in both basic and applied researches in a number of-fields of enquiry.