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Transforming Plants - Basic Genetic Engineering TechniquesPamela Peters. "Biotechnology: A Guide to Genetic Engineering." Dubuque, IA: Wm. C. Brown Publishers, 1993.
Cloning of Plant Cells and Manipulation of Plant GenesPlant cells exhibit a variety of characteristics that distinguish them from animal cells. These characteristics include the presence of a large central vacuole and a cell wall, and the absence of entioles, which play a role in mitosis, meiosis, and cell division. Along with these physical differences, another factor distinguishes plant cells from animal cells, which is of great significance to the scientist interested in biotechnology: Many varieties of full-grown adult plants can regenerate from single, modified plant cells called protoplasts - plant cells whose cell walls have been removed by enzymatic digestion. More specifically, when some species of plant cells are subjected to the removal of the cell wall by enzymatic treatment, they respond by synthesizing a new cell wall and eventually undergoing a series of cell divisions and developmental processes that result in the formation of a new adult plant. That adult plant can be said to have been cloned from a single cell of a parent plant.Plants that can be cloned with relative ease include carrots, tomatoes, potatoes, petunias, and cabbage, to name only a few. The capability to grow a whole plant from a single cell means that researchers can engage in the genetic manipulation of the cell, let the cell develop into a completely mature plant, and examine the whole spectrum of physical and growth effects of the genetic manipulation within a relatively short period of time. Such a process is far more straightforward than the parallel process in animal cells, which cannot be cloned into full-grown adults. Therefore, the results of any genetic manipulation are usually easier to examine in plants than in animals.
A Cloning Vector that Works with Plant CellsNot all aspects of the genetic manipulation of plant cells are readily accomplished. Not only do plants usually have a great deal of chromosomal material and grow relatively slowly as compared with single cells grown in the laboratory, but few cloning vectors can successfully function in plant cells. While researchers working with animal cells can choose among a wide variety of cloning vectors to find just the right one, plant cell researchers are currently limited to just a few basic types of vectors.Perhaps the most commonly used plant cloning vector is the "Ti" plasmid, or tumor-inducing plasmid. This plasmid is found in cells of the bacterium known as Agrobacterium tumefaciens, which normally lives in soil. The bacterium has the ability to infect plants and cause a crown gall, or tumorous lump, to form at the site of infection. The tumor-inducing capacity of this bacterium results from the presence of the Ti plasmid. The Ti plasmid itself, a large, circular, double-stranded DNA molecule, can replicate independently of the A. tumefaciens genome. When these bacteria infect a plant cell, a 30,000 base-pair segment of the Ti plasmid - called T DNA - separates from the plasmid and incorporates into the host cell genome. This aspect of Ti plasmid function has made it useful as a plant cloning vector. The Ti plasmid can be used to shuttle exogenous genes into host plant cells. This type of gene transfer requires two steps: 1) the endogenous, tumor-causing genes of the T DNA must be inactivated and, 2) foreign genes must be inserted into the same region of the Ti plasmid. The resulting recombinant plasmid, carrying up to approximately 40,000 base pairs of inserted DNA and including the appropriate plant regulatory sequences, can then be placed back into the A. tumefaciens cell. That cell can be introduced into plant cell protoplasts either by the process of infection or by direct insertion. Once in the protoplast, the foreign DNA, consisting of both T DNA and the inserted gene, incorporates into the host plant genome. The engineered protoplast - containing the recombinant T DNA - regenerates into a whole plant, each cell of which contains the inserted gene. Once a plant incorporates the T DNA with its inserted gene, it passes it on to future generations of the plant with a normal pattern of Mendelian inheritance. One of the earliest experiments that involved the transport of a foreign gene by the Ti plasmid involved the insertion of a gene isolated from a bean plant into a host tobacco plant. Although this experiment served no commercially useful purpose, it successfully established the ability of the Ti plasmid to carry genes into plant host cells, where they could be incorporated and expressed.
A. Tumefaciens Infects a Limited Variety of Plant TypesThe fact that only certain types of plants were naturally susceptible to infection with the host bacterial organism initially limited the usefulness of the Ti plasmid as a cloning vector. In nature, A. tumefaciens infects only dicotyledons or "dicots" - plants with two embryonic leaves. Dicotyledenous plants, divided into approximately 170,000 different species, include such plants as roses, apples, soybeans, potatoes, pears, and tobacco. Unfortunately, many important crop plants, including corn, rice, and wheat, are monocotyledons - plants with only one embryonic leaf - and thus could not be easily transfected using this bacterium.
Overcoming the Limited Range of A. Tumefaciens InfectionResearch efforts in the past few years have reduced the limitations of A. tumefaciens. Scientists discovered that by using the processes of microinjection, electroporation, and particle bombardment, naked DNA molecules can be introduced into plant cell types that are not susceptible to A. tumefaciens transfection. Microinjection involves the direct injection of material into a host cell using a finely drawn micropipette needle. Electroporation uses brief pulses of high voltage electricity to induce the formation of transient pores in the membrane of the host cell. Such pores appear to act as passageways through which the naked DNA can enter the host cell. Particle bombardment actually shoots DNA-coated microscopic pellets through a plant cell wall. These developments, important in the commercial application of plant genetic engineering, render the valuable food crops of corn, rice, and wheat susceptible to a variety of manipulations by the techniques of recombinant DNA and biotechnology.
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