Transformation 1 - Plant Tissue Culture

Overview

This lesson explains the technique of tissue culture as used in plant transformation. It discusses important issues, such as the use of selectable markers, genotype specificity, and tissue culture alternatives.

 

Overview and Objectives - Tissue Culture

Patricia Hain
Department of Agronomy and Horticulture at University of Nebraska-Lincoln, USA
Don Lee
Department of Agronomy and Horticulture at University of Nebraska-Lincoln, USA


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This lesson explains the technique of tissue culture as used in plant transformation. It discusses important issues, such as the use of selectable markers, genotype specificity, and tissue culture alternatives.


At the completion of this lesson, you should be able to:




Development of this lesson was supported in part by Cooperative State Research, Education, & Extension Service, U.S. Dept of Agriculture under Agreement Number 98-EATP-1-0403 administered by Cornell University and the American Distance Education Consortium (ADEC).
Any opinions, findings, conclusions or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture.

Introduction - Tissue Culture

Transformation is the step in the genetic engineering process where the new gene is inserted into a single plant cell.
Transformation is the step in the genetic engineering process where a new gene (transgene) is inserted into a single plant cell. The transformation step of producing a genetically engineered (GE) crop plant is a process controlled by poorly understood mechanisms. The process can be difficult because the genetic engineer must accomplish all of the following before they are successful.
  1. The new gene must be delivered into the nucleus of a cell and insert into a chromosome.
  2. The cells that receive the new gene must stay alive.
  3. The cells and plants that contain the new gene must be easilty identifiable (selectable markers).
  4. The transformed cell must divide and give rise to an entire plant.
  5. The location where the transgene inserts into the chromosome must not interfere with the expression of the gene.
  6. The new gene must not insert into an existing gene in the chromosome that influences survival of the plant cell or productivity of the entire plant.

Currently, geneticists have overcome these barriers by developing special techniques.

The first technique is tissue culture where clusters of undifferentiated plant cells are grown in culture, which allows them to be manipulated, and then induced to develop into whole plants.

The other technique is transformation where genetic engineers introduce the gene into these clustered cells using one of several possible methods including:
  • Agrobacterium tumefaciens
  • The gene gun (particle bombardment)
  • Electroporation
  • Microfibers.
This lesson will discuss the technique of tissue culture and how it is used in transformation. Transformation methods and results of the transformation process will be explained in the ’Transformation Methods’ and ’Events’ lessons.

Tissue Culture

The tissue culture process is used by crop genetic engineers because seeds and plants consist of billions of cells. It is not possible to put a new gene into every one of these cells. However, it is possible to put a new gene into one or a few individual tissue culture cells. Through tissue culture the transformed cell can then be regenerated into an entire plant with each cell containing the transgene. The gene has become a permanent part of the transgenic plant's genome and will be passed on to its progeny.

How is Tissue Culture Done?

Tissue culture is an important component of transforming plants with new genes.

During this procedure, plant cells can be removed from various parts of a plant and placed on media in petri plates. The media does not contain the growth hormones normally present in a plant that tell the cells which tissue to develop into. As a result, the cells do not differentiate and instead form a mass of cells called a callus that are not differentiated into at the tissue level.
Cells are taken from plants and grown into undifferentiated masses called callus.Immature embryos are removed from seeds and placed on media. Callus cells will then begin to grow from them.Callus are masses of undifferentiated cells.
Since plant cells are totipotent, growth hormones can be added to the media triggering the callus cells to develop roots, shoots and eventually entire plants. Plants regenerated from tissue culture will be clones genetically identical to the cell they originated from. The only animal cells that have this totipotent characteristic are fertilized eggs.
Single cells can be regenerated into entire plants.Growth hormones can be added to the media and the cells will begin to divide and differentiate into plants.Regenerated plants are then transferred into test tubes. Once they have reached a certain size, they will be transplanted into soil.

Genotype Specificity

The ability to obtain callus cells from a plant and then regenerate new plants from this callus is not easy to do. Some crop lines are genetically more equipped to handle the stress from tissue culture. Unfortunately, these lines are usually older lines or wild-type species, both of which are agronomically inferior to modern, high-yielding hybrids and have a significantly lower yield potential.

The nutrient and environmental requirements of callus can differ between lines. A certain amount of time must be invested up front to determine the optimal conditions for growing a particular callus line. There are so many lines developed each year, it would be impossible for a genetic engineer to develop a tissue culture program for each one. Genetic engineers can make their best progress if they develop methods that work reliably on a small group of lines and continually work with them over several years. The better the agronomic traits of those lines, the shorter the time period will be from identifying a transgenic plant to marketing a genetically engineered variety or hybrid.

Another limitation to tissue culture is the occurence of genetic changes it can induce. Previous to transformation, the original cell is genetically identical to the plant or seed it was sampled from unless a mutation occurs. In most instances, this tissue culture induced variation is not desired. However, it was desirable in the case of ALS herbicide resistant corn and high sucrose soybeans. These plants derived their traits from mutations that arose during tissue culture.

Transformation methods that use callus cells are consequently highly genotype dependent for their successful implementation. For this reason, genetic engineers try to develop transformation systems that introduce genes into cells of the explant that are already programmed to differentiate into a plant.

Alternative to Tissue Culture

This chimeric alfalfa plant has a natural mutation that prevents chlorophyl production in some of the cells in the leaves.
Some genetic engineers have bypassed the tissue culture process by producing chimeric plants with the transgene in the germline cells. This means that they introduced the new gene into some but not all of the cells in a young plant.

Some of the cells in the seedling that geneticists have transformed are those that divide and develop into the pollen or egg producing tissues in the plant (the germline). Sexual progeny from these chimeric plants will then inherit the new gene and have one copy of the transgene in every one of their cells.

Another transformation method that could potentially bypass tissue culture is the transformation of pollen. In a species such as corn, the transformed pollen could be used in a cross with the hope that some of the progeny will inherit the transgene. Research on this method has produced limited results.

The most promising method for germline cell transformation is to transform germ cells that are within the developing flowers. This process works in the model plant arabidopsis simply by dipping the plant’s inflorescence in a solution of agrobacterium. The hope is to adopt this strategy to crop species.

Selectable Marker Genes

After callus cells have gone through the transformation process, it takes weeks of recovery and growth in a petri dish before they can develop into plants. Thousands of cells are growing on a single petri dish, but only a few may actually have received the new gene(s). It would be very cumbersome to grow up each cell into a plant to test for the presence of the transgene. It is much more efficient for geneticists to determine which cells have been transformed while still at the cellular level. They can then select those few transgenic cells out, and grow them into entire plants. Therefore, geneticists need a way to determine transgenic cells from non-transgenic cells.

What if you tried looking for the trait the gene encodes?
This may work for a few traits, but many transgenes encode traits that are not easily detected, especially at the cellular level. For example, you could not test a cell in tissue culture for the expression of a trait that influences grain protein content.

What if you extracted the DNA from each cell and searched for the gene?
You would have to separate each cell, allow the cell to multiply into a pile of callus, and sample a portion of each callus pile and extract DNA. Then tests would have to be perforemed to identify the presence of the gene. This would be just as cumbersome as trying to grow each cell into a plant.

What is the solution?
Usually the desired transgene and the marker gene insert in the chromosome at the same location.
Callus cells that have been transformed with an anitibiotic resistance marker gene grow on selection media while non-transgenic cells around them die.
Selecting out transgenic cells is done by co-transforming the cells with the transgene plus an additional gene called a selectable marker gene. Selectable marker genes encode a trait that is easily detectable, even at the cellular level. The two most commonly used selectable marker genes encode the traits of herbicide and antibiotic resistance.

To select out the transgenic cells, all of the cells are grown on media containing the herbicide or antibiotic. Only those cells transformed with the resistance transgene can survive. Thus growing the cells on selection media with the herbicide or antibiotic will allow the scientist to regenerate only plants that are transformed.

A selectable marker used in the development of some Bt corn lines is the BAR gene or PAT gene that confers resistance to the herbicide called Liberty. Thus these plants will have European corn borer resistance and Liberty resistance.

Summary - Tissue Culture