9.2: Applications of "Green" Biotechnology

Genetically Modified Crops & Foods

Genetically-modified (GM) crops are classified as Genetically-Modified Organisms (GMOs) - organisms whose genetic material has been altered using biotechnology. In botany, genetically-modified crops are used for multiple purposes, including to resist herbicides (e.g., "Roundup Ready" soybeans), prevent spoilage (e.g., Flavr Savr® tomatoes), and enhance nutrition (e.g., Golden Rice with added vitamin A).

When making a GMO, the "host" organism whose genome will be altered can range from something as simple as unicellular bacteria and yeast, to multicellular, complex organisms such as animals and plants. To read about how genetically-modified animals are created and used, go to Chapter 8: Animal Biotechnology. For plants, the introduction of a new gene is often done using a bacteria called Agrobacterium tumefaciens, a rod-shaped Gram-negative soil bacterium. In plants, A. tumefaciens causes "crown gall" disease, in which tumors, called "galls" form within the infected plant. These galls, often at the base of the stem or roots, can disrupt nutrient and water transport, weakening the plant and potentially leading to its death. As such, A. tumefaciens is a serious pathogen of many commercial crops like walnuts, grape vines, stone fruits, nut trees, sugar beets, and rhubarb. The persistent nature of the tumors caused by the disease make it particularly harmful for these crops.

The tumors caused by A. tumefaciens are due to the presence of a tumor-inducing plasmid, known as the Ti plasmid (or T-plasmid). This plasmid contains all of the genes required for the bacteria to be virulent and efficiently infect plants cells. Once within the cell, the Ti plasmid incorporates a segment of its DNA, called T-DNA, into the genome of the host cell. This makes T-DNA an efficient gene delivery method that can be exploited in the laboratory. Today, Ti-based plasmids are used by researchers for the transfer and insertion of their desired gene into a plant genome (Figure \(\PageIndex{2}\)). A. tumefaciens-infected cells with the Ti plasmid can be selected and used to regenerate new plant tissue. Because plant cells are "totipotent", a single plant cells can be used to recreate an entirely new plant. This means that the genetically-modified cells created using Agrobacterium tumefaciens can easily be grown into genetically-modified plants that are clones of the originating cell.

Certain plants, like wheat and corn, do not respond well to Agrobacterium-mediated transformation. As an alternative, botanists can use a "gene gun" to introduce their gene of interest into these plants. A gene gun, also known as a biolistic particle delivery system, can be used to deliver genetic material (like DNA or RNA) into prokaryotic or eukaryotic cells, but is typically used on plant cells (Figure \(\PageIndex{3}\)). For this, the plant cells are plated onto a surface and placed into the gene gun. Tiny metal particles (usually gold or tungsten) are coated with DNA, containing the desired gene. The particles are loaded onto a plastic membrane or cartridge and placed inside the gene gun. The gene gun is aimed at the cells. A burst of compressed gas (like helium) or a high-voltage electric discharge is applied to the membrane, propelling the particles at high speed into the cells. The particles shoot through the cell walls and membranes, carrying the DNA into the cell interior. Some of the cells will then integrate the DNA into their genome for gene expression. Antibiotic resistance is used to identify those cells containing the integrated DNA. Following development under highly controlled conditions and genetic testing, the organism may be considered a GMO.

While the introduction of genetic material into cells is typically done using liposomal-mediation transfection (see Lab Protocol 17.9 Liposomal transfection into mammalian cells), plant cells, with their rigid cell walls made of cellulose, make transfection with liposomes difficult. As such, botanists will use a gene gun to improve their transformation efficiency. This is because the gene gun is capable of "firing" genetic information through the cell wall and plasma membrane. In addition, the gene gun can introduce genetic material into a few plant cells, a single plant tissue or a specific plant organ (e.g. a leaf) in order to observe the effects of genetic manipulation without creating an entire genetically-modified plant.

GMOs like genetically-modified crops have helped address food security and resource sustainability. However, they also raise ethical and environmental concerns. Some have expressed concerns about the potential impacts of GMOs on biodiversity, long-term human health effects, and the monopolization of food resources by biotech companies, making GMOs a complex and often controversial area of modern science.

Biofertilizers and Biopesticides

Once of the focuses of green biotechnology is in the creation and use of biofertilizers and biopesticides that are eco-friendly and sustainable. A biofertilizer is a natural fertilizer that contains living microbes that help plants grow by increasing the supply or availability of nutrients in the soil. The types of microbes found will depend on the biofertilizer but can contain beneficial bacteria, fungi, or algae. One important function of biofertilizers is in nitrogen fixation - the process by which atmospheric nitrogen gas (N₂) is converted into ammonia (NH₃) or other nitrogenous compounds (Figure \(\PageIndex{4}\)). These nitrogenous compounds are used by the plants to synthesize proteins.

Biofertilizers are easily applied to seeds, soil, or to plant roots. The seeds can be coated with the biofertilizer prior to planting. This is common for legumes like beans and peas. Alternatively, the biofertilizer can be mixed with compost materials or carrier material, like peat, and the spread into the soil. Other techniques include root "dipping" in which the roots of the plants are dipped into a solution containing the biofertilizer and the spraying of a biofertilizer solution onto leaves. Table \(\PageIndex{1}\) lists some commonly used biofertilizers.

Biopesticides are biologically-based substances that control pests, such as insects, weeds, fungi, and bacteria. Biopesticides tend to be safer for humans, animals, and the environment. They don't pollute the soil or water and break down quickly in the environment. Biopesticides are target-specific as they usually affect only the pest and not beneficial organisms. Moreover, the targeted pests are less likely to develop resistance to the biopesticide. However, biopesticides can be slower-acting than chemical pesticides and may need specific conditions in order to work effectively.

There are several types of biopesticides, including:

• Microbial biopesticides: contain living organisms, like bacteria, fungi, viruses
  • e.g. Bacillus thuringiensis (Bt)
  • Bt produces toxins that kill insect larvae
• Botanical pesticides: pesticides extracted from plants
  • e.g. neem oil
  • disrupts insect hormones and feeding
• Biochemical pesticides: natural substances that affect pest behavior
  • e.g. insect sex pheromones
  • interfere with mating to reduce pest populations
• Entomopathogenic fungi: fungi that infect and kill insects
  • e.g. Beauveria spores infect and grow inside insect hosts

One of the most widely used biopesticides in both organic and conventional farming is the soil bacteria, Bacillus thuringiensis (Bt). Bt bacteria are used to control the populations of several moth and butterfly species, in addition to beetles, flies and mosquitoes. During sporulation, Bt cells produces crystal (Cry) proteins, which are also known as δ-endotoxins (Figure \(\PageIndex{5}\)). When ingested by certain insects, the alkaline environment of the gut processes and activates the toxin. The activated form of the toxin binds to specific receptors expressed by the epithelial cells lining the mid-gut portion of the digestive system. Receptor activation results in the formation of pores in the epithelial lining. The insect stops feeding and dies within days to a few weeks. Because the receptors to the activated Cry proteins are found only in certain insect species, Bt is safe to beneficial insects, plant cells, and animals. In organic farming, Bt spores and toxins can be directly sprayed onto crops. In addition, genetically-modified crops, such as corn, cotton, and soybeans, have been engineered to produce the Bt toxins themselves. This genetic engineering involved use of a gene gun to introduce the relevant Bt genes.