Overview

Genetic transformation in plants means changing the plant's DNA with either an increased copy of the specific gene(s) or reduction/blocking the function of a gene(s). Here is a general explanation of how it works:

1. Choosing the Trait: Scientists decide what new trait they want the plant to have, like resistance to pests or better transportation or resistance to abiotic stress.
2. Finding the Gene: They identify a gene that encodes the trait. This gene can be from either the same plant or another plant, an animal, or even bacteria.
3. Inserting the Gene: Through an experimental process, the gene is introduced into the plant for integration in the DNA. There are a few common ways to do this
   • Using Bacteria: A special bacteria called Agrobacterium can naturally insert genes into plants. Scientists put the new gene into the bacteria, and then let the bacteria transfer into the plant.
   • Particle Bombardment: Scientists coat tiny particles with the specific gene and shoot them into plant cells. This specific gene then becomes part of the plant's DNA.
4. Growing the Plant: The plant cells with the inserted gene are grown into whole plants. These plants now have the new trait.
5. Testing: Scientists check the new plants to make sure they have the trait and that it works as expected.

This process allows plants to have new and useful characteristics that they didn't have before.

BASIC COMPONENTS FOR SUCCESSFUL GENE TRANSFER TO PLANT CELLS

Before the mid-1980s, improving plants was mainly done through traditional breeding. This involved crossing plants and looking for desired traits in the offspring. This method involves combining thousands of genes at once, making it slow and not a directed process.

Site directed gene transfer requires a careful insight into overcoming the barriers in the entry as well as gene integration. The plant cell has several barriers, including the cell wall and membranes, that need to be carefully breached to insert the gene without damaging the cell too much. The methods like using bacteria (Agrobacterium) or shooting DNA-coated particles into the cells help deliver the gene to the nucleus. Once the gene reaches the nucleus, the plant’s natural DNA repair processes help integrate the new gene into the plant’s DNA.

A. DNA Delivery

Delivering DNA into plant cells to make transgenic plants involves overcoming several barriers.

Barriers to DNA Delivery

1. Cell Wall: - Think of the cell wall like a tough, flexible scouring pad made of cellulose fibers. It is rigid and held together by materials like pectin. - There are small holes, but these do not allow DNA to pass through easily. - To facilitate the entry of DNA inside the cell, scientists need to make small breaks in the cell wall without damaging the cell too much.
2. Cell Membrane: - After getting through the cell wall, the DNA must also cross the cell membrane.
3. Nuclear Membrane: - Finally, the DNA needs to enter the nucleus where the plant's DNA is stored.

Steps to Improve DNA Delivery

• Reduce Cell Pressure:
  • Plant cells have internal pressure that keeps them firm.
  • Scientists can temporarily reduce this pressure by drying the tissue or using sugars, making it easier for DNA to enter the cell without too much disruption.

Delivery Methods

• Physical Methods:
  • DNA can be delivered near or directly into the nucleus using methods like particle bombardment (shooting DNA-coated particles into the cells).
  • This DNA is often unprotected, so it needs to quickly reach the nucleus.
• Biological Methods:
  • Using bacteria like Agrobacterium, the DNA is coated with proteins that protect it and guide it to the nucleus.

Inside the Nucleus

• Integration Process:
  • The plant’s natural DNA repair machinery helps integrate the new DNA into the plant’s own DNA.
  • This involves the plant’s DNA being accessed, modified, and reassembled, allowing the new DNA to be stitched into the plant’s genome.

B. Target Tissue Status

To successfully create transgenic plants, scientists need to target plant cells that can grow into whole plants. This ability is called totipotency. The fertilized egg is naturally totipotent, meaning it can grow into a whole plant. While most plant cells have this potential, it has not been achieved for all cell types.

Key Points:

1. Target Cells:

• Not all plant cells can easily grow into whole plants, so scientists focus on specific cell types that are easier to work with.
• For a few plants, many different cell types can be used to grow whole plants through a process called tissue culture.

2. DNA Delivery:

• Successful production of transgenic plants requires coordinating DNA delivery with the ability of the targeted cell to grow into a whole plant.

Ideal Targets:

• Fertilized Egg and Pollen:
  • These are the best targets, but they are not responsive in most plants, except for a model plant called Arabidopsis thaliana.
• Shoot Meristem:
  • This is another suitable target, as it gives rise to the above-ground parts of the plant.
  • However, the shoot meristem is complex, and the best target cells are buried deep inside, making them hard to reach.

Common Practice:

• Rapidly Growing Cells:
  • Scientists usually target specialized plant cells that are rapidly growing and can form whole plants.
  • These cells should be:
    • Physically accessible: Easy to reach.
    • Actively dividing: Actively replicating DNA to help integrate the new DNA.
    • Totipotent: Able to grow into whole plants.
    • Resilient: Strong enough to survive the intrusion of new DNA.

In summary, to create transgenic plants, scientists target specific plant cells that can grow into whole plants, focus on cells that are easy to access, actively growing, and resilient. The process requires careful coordination to ensure successful DNA integration and plant development.

C. Selection and Regeneration

As the process of introducing DNA into plant cells is difficult, only a small number of cells actually get the new DNA. Most cells do not, so scientists need a way to find the cells that have an uptake of DNA. This is done using a process called selection.

How Selection Works

1. Adding a Resistance Gene: - Along with the gene of interest, scientists also add a gene that gives resistance to an antibiotic or herbicide.
2. Exposing Cells to Toxins:

• The mixture of transformed (with new DNA) and non-transformed cells is exposed to the antibiotic or herbicide.
• Only the cells with the resistance gene will survive and grow.

Types of Selection

• Negative Selection:

  • The most common type.
  • Cells that survive in the presence of a toxin (antibiotic or herbicide) are the ones that have the new DNA.
  • Example:
    • Neomycin phosphotransferase: Provides resistance to kanamycin.
    • Hygromycin phosphotransferase: Provides resistance to hygromycin.
    • Bar gene: Provides resistance to glufosinate and bialaphos herbicides.

• Positive Selection:

  • Uses nutrient sources that only the transformed cells can use.
  • Example:
    • Phosphomannose isomerase gene allows cells to convert mannose to fructose, a usable form of sugar. Cells with this gene can grow on mannose, while others cannot and will starve.

Identifying Transformed Cells

• Reporter Genes:
  • These genes give transformed cells a unique characteristic, like a new color or fluorescence.
  • Example:
    • Green Fluorescent Protein (GFP): This makes cells glow green under blue or UV light. Scientists can see and separate these glowing cells.

To identify the few plant cells that have successfully taken up new DNA, scientists use selection methods. They add genes that give resistance to toxins or allow cells to use specific nutrients. Then, they expose the cells to these conditions, so only the transformed cells survive. They can also use reporter genes to make transformed cells visually stand out. Once identified, these cells can be grown into whole plants.