Tài liệu Plant tissue culture

Plant tissue culture (19 Pages)

Introduction
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 (gene transfer methods are described in Chapter 3). 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
Practically any plant transformation experiment relies at some point on tissue
culture. There are some exceptions to this generalisation (Chapter 3 will look
at some), but the ability to regenerate plants from isolated cells or tissues in
vitro underpins most plant transformation systems.
Plasticity and totipotency
Two concepts, plasticity and totipotency, are central to understanding plant
cell culture and regeneration.
Plants, due to their sessile nature and long life span, have developed a
greater ability to endure extreme conditions and predation than have animals.
Many of the processes involved in plant growth and development adapt to environmental
conditions. This plasticity allows plants to alter their metabolism,
growth and development to best suit their environment. Particularly
important aspects of this adaptation, as far as plant tissue culture and regeneration
are concerned, are the abilities to initiate cell division from almost any
tissue of the plant and to regenerate lost organs or undergo different developmental
pathways in response to particular stimuli. When plant cells and tissues
are cultured in vitro they generally exhibit a very high degree of plasticity,
which allows one type of tissue or organ to be initiated from another type. In
this way, whole plants can be subsequently regenerated.
This regeneration of whole organisms depends upon the concept that all
plant cells can, given the correct stimuli, express the total genetic potential of
the parent plant. This maintenance of genetic potential is called ‘totipotency’.
Plant cell culture and regeneration do, in fact, provide the most compelling
evidence for totipotency.
In practical terms though, identifying the culture conditions and stimuli required
to manifest this totipotency can be extremely difficult and it is still a
largely empirical process.
The culture environment
When cultured in vitro, all the needs, both chemical (see Table 2.1) and physical,
of the plant cells have to met by the culture vessel, the growth medium and
the external environment (light, temperature, etc.). The growth medium has to
supply all the essential mineral ions required for growth and development. In
many cases (as the biosynthetic capability of cells cultured in vitro may not
replicate that of the parent plant), it must also supply additional organic supplements
such as amino acids and vitamins. Many plant cell cultures, as they
are not photosynthetic, also require the addition of a fixed carbon source in the
form of a sugar (most often sucrose). One other vital component that must also
be supplied is water, the principal biological solvent. Physical factors, such as
temperature, pH, the gaseous environment, light (quality and duration) and
osmotic pressure, also have to be maintained within acceptable limits.