[Lecture Notes] Fundamentals of Biotechnology - L15: Plant biotechnology and transgenic plants

Plant tissue culture

Apical meristem cells in plants are totipitant, so the entire plant can be regenerated from just one of these cells. The line between somatic cells and germline cells in plants is blurry. Therefore cells can be cultured from a plant to regenerate an entirely new plant.

Callus cultures are those done on a Petri dish, and suspension cultures are those done in liquid solutions. Tissue/cells called the explant has to be removed from the plant and cultured to grow a new fully formed plant. Callus cultures can use a piece of apical meristem or an embryo, and suspension cultures use protoplasts, microspores (pollen) or macrospores (egg cells). In general, growth hormone needs to be added to induce good growth, such as auxin or cytokinin. Callus cultures form callus cells at the top of the growth medium (undifferentiated cells). These cells can be isolated and taken into a different environment with different hormones or a different concentration of hormine which then allows the cells to differentiate and form a plant shoot.

This is good for propagating rare plants if needed, they can be kept in tissue culture. One can also perform mutation breeding, undifferentiated cells (callus) can be exposed to a mutagen and then induced to form a plant shoot and the effects of mutagenesis studied.

Regenerated plants exhibit changes, such as blueberry plants which are shorter when grown in culture, but this effect doesn’t last forever. Epigenetic changes may occur, or true genetic changes may occur in this process as well. These can even be ploidity changes, transposon activation or chloroplast modifications. The rate of these mutations can be modulated by the nutrient and hormone composition of the growth medium.

Genetic engineering of plant cells

Glyophosate resistance can be conferred onto a plant by the addition of a single gene. This herbicide resistance means that plants survive while the surrounding weeds die.

Agrobacterium carry the Ti (tumour inducing) plasmid. This organism is important because it can transfer genetic information from itself to another organism (the plant). Compounds secreted by wounds in plants attract Agrobacterium to the site. Then two components, VirA and VirG are responsible for the integration of the plasmid into the plant genome. VirA is autophosphorilated in the presence of plant wound compounds causing VirG to begin the transcription of vir genes. The vir gene products clip T-DNA from the plasmid forming a ssDNA complex. The gap in the plasmid is repaired, and a protein coats the ssTDNA allowing it to inserted into the plant cell, through a pilus which forms a channel between the bacteria and the plant cell. Once inside the T-DNA enters the nucleus and is integrated into the genome. Then the genes in the T-DNA are expressed which forms auxin and cytokinin, causing tumour growth in the wound location. The T-DNA also carries opine genes, so when the infected cells begin to divide rapidly, opines are also created in large amounts which the Agrobacterium like to eat.

Scientists have exploited this system. The genes for opines and growth hormones have been removed from the plasmid and keeping only the genes involved with the transferral of T-DNA. This plasmid can then be worked with in E. coli and trans genes can be inserted into the MCS. In addition to the trans gene, a selectable marker such as antibiotic resistance or herbicide resistance is added. One can either use a constitutive promoter which ensures that the gene of interest is constantly switched on, or an inducible promoter, which is generally better as it allows the scientist to choose when the gene is on or off. A commonly used inducible promoter is the cab promoter which activates the gene in the presence of light, meaning that roots will not express the gene but leaves and other parts of the plant of interest will. The main function of the promoter is to express the gene only in areas of the plant that actually need the function of the trans gene.

In the lab, plants are grown in culture, and callus cells or protoplasts are exposed to Agrobacterium with the genetically engineered Ti plasmid, and incubated to allow the Agrobacterium to grow too. After a while the plant cells are harvested and re-cultured using the selectable marker (in media with herbicide or antibiotic), growth hormone added to induce plant shoot formation and then they can be screened to check for the expression levels of the chosen transgene.

Floral dipping is a similar technique but instead, the plant to be engineered is allowed to flower, the flowering buds are removed and once they start regenerating are dipped into a solution containing the modified Agrobacterium and a surfactant. The surfactant allows the Agrobacterium to stick to the surface of the bud, and because these buds are only just forming, the T-DNA enters the germline of the plant. Then this bud is allowed to continue growing and produce seeds, and these seeds are now genetically modified. This is advantageous because growing plants from single cells takes a long time, but growth from seeds is much faster. (naturally the seeds are grown first using selectable marker and then the seeds which grow are propagated).

Gene guns

Metal particles can be bound to DNA and blasted from a gun into plant cells. This is a non-specific technique meaning it can be done for plants which are not compatible with the Ti plasmid. The metal particles can break through the cell wall of plants and every now and then, some DNA will enter the nucleus and integrate into the genome. A callus or sample of cells from a leaf first need to be put into a vacuum chamber and gold beads are coated in transgene DNA. Bead accelaration can be done by high pressure gas or electrostatic charge. After an attempt has been made, the cells are transferred and selected using the selectable marker. This same technique has been applied to yeast, C. elegans and Chlamydomonas cells.

Detection

A reporter gene that can be engineered into the plant instead of a selectable marker is the luc gene which codes for luciferase, therefore if luciferin is added to the plant then the cells will glow in the dark. Luciferase has the advantage that it’s not stable in plants, and so its presence is directly correlated with the amount of expression. (Light intensity can be correlated with expression).

Removing the selectable marker

Removal of the selectable marker is important if one wants to do a second round of genetic engineering (reselect) or to alleviate the concerns (not based on any real evidence) of the public over there being antibiotic resistance in the food chain.

Bacteriophage P1 has the Cre/loxP system which allows for gene deletion. Cre is a recombinase enzyme which recognises specific loxP sites which are 34 base pairs long. A gene can be flanked by two loxP sites. Therefore when the Cre protein is expressed, it will find the two loxP sites and cut out the gene between the two of them, joining them back up after that. The gene is recombined out of the genome. One does not clone in the Cre gene system in the same plant that has the selectable marker. Instead once the plant with the selectable marker has been selected for, it is crossed with another plant that has the Cre system. The resulting plant is a plant with the Cre system and the selectable marker flanked by the loxP sites. The selectable marker gene is then cut out and no longer expressed. After this, to get a plant with only the trans gene but not the Cre gene, the plant can be crossed several times with wild type plants until by chance you get one with only the one gene. This allows for plants released to the public to ONLY have one trans gene in them without all the accompanying selectable markers and Cre genes.

Nuclease mediated genetic engineering

Creating double stranded breaks in DNA at exact locations allows non-homologous end joining (NHEJ) or homologous recombination at the site for integration of a transgene, or one can knock out a mutation by insertions or deletions within that gene.

In order to make cuts at very precise locations, one needs nucleases with extremely precise recognition sequences, This could be 3 zinc finger domains bound to a FokI as discussed earlier, or TALE nucleases. These cut the double stranded DNA. One can also use the CRISPR/Cas9 system. Using the Cas9 nuclease, one provides an RNA template, this system is easy to work with because the nuclease does not need to be modified, only the RNA template does, and it can be cloned in with a vector.

Herbicide resistance

Making a crop resistant to herbicides like glyophosate means that farmers can discriminate between weeds and crops when spraying. Glyophosate kills plants by blocking aromatic amino acid synthesis by blocking EPSPS. This enzyme isn’t found in animals so glyophosate isn’t harmful to us, and therefore we cannot synthesise these amino acids ourselves and must consume them. The herbicide targets the chloroplast (which we do not have), preventing the enzyme from synthesising these aromatics. One can isolate mutant strains of Agrobacterium which are resistant to glyphosate by using it as a selective marker, and transfer the mutated EPSPS gene aroA which is reistant to glyphosate to plants by the methods discussed earlier. An N-terminus peptide is required to be attached to the gene so that it can make its way into the chloroplast.

Insect resistance

Bt toxin is typically sprayed onto crops as an insecticide. Some Bacillus spores produce a crystal of Cry protein, which when eaten by the pest along breaks down in the gut and releases Bt toxin, punching holes in the intestines killing the pest. This cry gene has been inserted into some plants in an attempt to confer pest resistance. Codon optimisation and truncating the toxin so it’s produced more efficiently has significantly boosted the amount produced by plants, moreover using plant virus promoters allows plants to produce even more.

An alternative to incorporating Bt toxin into plants is to confer the ability to produce aphid alarm pheromones which signal danger to aphid predators. This has the advantage that resistance will not develop in insects and other endangered species of insects will not be harmed.

New traits

Golden rice is a strain of rice engineered to express beta-carotene, which is a vitamin A precursor in humans in order to reduce malnutrition in some parts of the world, similarly this is being attempted in bananas, and omega-3 in soybeans. Other strains of plants have been modified to reduce the amount of toxins found in them. Genes which confer resistance to fungi which produce aflatoxins on plants are being investigated.

Pharmaceutical manufacturing

Plants can post-translationally modify proteins, are low cost to grow, and their products can be isolated and purified in bulk (solubilise cells in detergent), moreover mammalian virus or cell contamination is not a risk. This makes them a good candidate for producing pharmaceuticals and this has already been done. There are risks with contamination of other plants though, if the strain were to get out into the wild.

Functional genomics

Functional genomics is the study of the entire genome of a plant at once instead of single genes. Arabidopsis, the model flowering plant is what most of this research is done on. One can use transposons or T-DNA which contains only a reporter gene. If this DNA integrates into a gene, it will change or delete its function and one can screen for phenotypic changes, and sequencing can identify exactly which gene was disrupted.

RNA interference is another way of knocking out plant genes with unknown function. Short interfering RNAs (siRNAs) are identified by the RISC complex and mRNA homologous to the siRNA is digested and so the gene is not expressed.

Neutron guns can be used in fast neutron mutagenesis whereby high energy neutrons cause deletions in the plant genome. The energy of neutrons can be controlled, and so the number of mutations can also be controlled. Seeds are treated with FNM, and each seed will have a unique set of deletions. The seeds can be grown and the DNA isolated, and PCR can identify where deletions have occurred by comparing fragment lengths (of which there will be two) to wild-type plants (where there will be one).

TILLING (targeted induced local lesions in genomics) creates point mutations by soaking M1 seeds in a mutagenic substance. Some of these seeds are saved and some are grown up. The DNA from the grown plants is isolated and placed into a large pool, and then smaller and smaller pools. PCR primers are designed to anneal to select regions on the genome and each primer has a different fluorescent label so that each end of the amplified product is a different colour. One takes wild-type DNA and mutant DNA and creates heteroduplexes by cooling slowly. An enzyme called CEL-1 cleaves at a mismatch, and therefore when analysing the fluorescent marker, only 1 will be seen. This indicates a point mutation in a particular strand and these can be cut out of the gel and sequenced to see where the point mutation occurred. This would not work for FNM because that creates deletions and heteroduplexes would not form.

References: my notes are made from, and follow the structure of my course textbook which is Biotechnology 2nd edition by David P. Clark, which can be found for purchase here.