Plant Biotechnology: Part 2

Plant Biotechnology – In vitro Propagation and Genetic Transformation of Plants (Part 2)

The main purpose of Modern Agriculture’s Plant Biotechnology series is to explain and elaborate on… well… exactly what genetic engineering is and how it is accomplished. The first part of the series was meant to get our readers acquainted with biotechnological terms (some of them), general rooting and regeneration media, and why sterilization is a key component of genetic transformation in any setting.

In the second part of our series, we’ll be going over the basics of in vitro propagation and genetic transformation:

Let’s Recap

Plants can be regenerated in vitro from various parts and tissues (cool, right?) using defined growth media containing appropriate salts, nutrients, and growth regulators (aka hormones). This technology has been successfully used as an alternative to producing plants from seeds for the rapid propagation of commercially important ornamental and crop plants, for “rescuing” endangered species, for improving agronomic traits in various crops, and for numerous other applications. Handling of the plant tissue requires an aseptic environment (sterilized!) that may be created using Laminar Flow Hoods and other sterilization equipment including autoclaves.

The Plant Regeneration Part

Regeneration refers to the development of a whole plant from a single cell or a group of cells. Generally, two different regeneration pathways are recognized: somatic embryogenesis and direct organogenesis. Somatic embryogenesis is the development of embryos from somatic cells (non-reproductive cells such as leaf cells). Like embryos that form following fertilization in seeds (regular embryos), somatic embryos develop roots and shoots simultaneously. However, unlike regular embryos, somatic embryos are not surrounded by endosperm or a seed coat and hence require special care on defined medium in order to survive. Organogenesis is referred to the direct in vitro development of shoot parts (i.e., apical meristem, leaves, and stem) from somatic cells. The regenerated shoot can then be treated to produce roots, and eventually form a whole plant.

Plant regeneration has a tremendous value in basic research concerning cell and molecular biology, genetics, and biochemistry. The technology has also direct commercial applications in several areas including production of large numbers of identical plants, production of industrial compounds in cultured plant cells, production of pathogen-free plants, crossing distantly related species (using protoplast fusion), and genetic transformation of plant. The latter allows scientists to introduce genes of any source into the plant genome, imparting novel traits.

The ratio of plant growth regulators auxin and cytokinin included in the media is an important factor that determines type of organ induced. Higher auxin/cytokinin ratio or the use of auxins alone tends to produce roots, while inclusion of cytokinin alone in the medium most often induces shoot formation.

The Plant Transformation Part

Plant transformation (genetic engineering) is the deliberate modification of the genetic makeup of a plant by introducing new genes (the transgene) into its genome. Two types of transformations are known: stable and transient. Stable transformation involves the insertion of a gene into the genome of an organism in such way that the transgene is transferred to future generations like other genes. In the case of plants this is easily achieved as plants can be regenerated from a single somatic cell under the right conditions. Briefly, upon treatment with a plant growth regulator (a cytokinin), somatic plant cells divide rapidly and produce a callus, which is a mass of undifferentiated cells. Some cells then undergo organogenesis to form a shoot, which can be nurtured to produce a whole plant. If cells are transformed before the proliferation process begins, the resulting callus, and hence the regenerated plants, are ‘stably’ transformed. Although production of stable transformed plants is time consuming and can take several months, the technology is now routinely used to impart novel traits including disease resistance, stress tolerance, and improved nutritional value to crop plants.

Transient expression involves localized temporary expression of a transgene (for example a reporter gene) in a specific cell type, tissue, or organ. As there is no need for plant regeneration, transient expression can be achieved in 2 to 10 days depending on the plant being studied. This technology cannot be used to improve plants. However, it can facilitate various studies such as protein targeting, promoter functions, and so on.

The Most Common Way of Transforming Plants: Agrobacterium mediated transformation

One of the most common ways of transforming plants involves the use of the Agrobacterium tumefaciens, a Gram negative soil bacterium that attacks plants at a site of injury causing tumours known as crown galls. Crown galls form because the Agrobacteria can insert a few genes needed for the formation of the tumour into the plant genome. The transformed cells proliferate in an uncontrolled manner, forming crown galls. The genes responsible for the tumour formation are found on a large plasmid (a genetic structure in a cell that can replicate independently of the chromosomes), where they are clustered near one another. The region of the plasmid encoding these genes is known as the T-DNA (transferred DNA), and is flanked by two 20-nucleotide long stretches of DNA known as the right and left borders (RB and LB). A number of proteins [virulence proteins, encoded by virulence (vir) genes] present outside of the T-DNA region on the plasmid are involved in the excision at the borders, and transfer the T-DNA to the plant cell.

Any DNA inserted between the RB and LB of the T-DNA will be transferred to plant cell. Indeed this is the basis for “Agrobacterium-mediated plant transformation”. However, the naturally occurring Ti plasmids are very large and rather hard to manipulate. To eliminate this problem “binary vectors” were developed. These vectors are much smaller, do not have the vir genes, and contain a modified T-DNA.

The modified T-DNA, which does not carry any of the genes found in naturally occurring T-DNA, contains multiple cloning sites (MCS) for cloning various genes. It also carries a gene that confers resistance to an antibiotic (or some other form of selection), and is flanked by sequences corresponding to the right and left borders (RB and LB) of the Ti plasmid. A gene of interest (to be transferred into a plant) is inserted into the MCS between the RB and LB of the vector, which is then introduced into an Agrobacterium strain (e.g., EHA105) that carries the vir genes on a separate “disarmed” Ti plasmid. Upon infecting, all genes between the RB and LB region of the binary vector are transferred to the plant cell through the of vir genes from the disarmed Ti plasmid. The T-DNA usually contains an antibiotic resistance gene to assist selection of transformed plant cells.

Plant Biotechnology Part 3

Phew. It took me a while to understand that the first time around. The next step in the genetic engineering of our lavender plants is to see if our transferred DNA was successful in… well… it’s transformation. Our next article will explain how we quantitatively analyze our DNA!





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