Rethinking Cannabis: Part 2
By Jay Roller of Alphakronik Genes Seeds
For many growers, the choice between growing from seed or from clones can be a difficult one. Many new growers will often go with clones to help prevent pollination of their crops due to inadvertent males showing up in the garden, or to ensure the end result of their hard work and dedication is up to the standard of quality they are expecting from previous results with the mother plant. Other growers choose to go with seeds due to the fact that they want genetic variance, new growth vigor, or are looking for a male to make hybrids with.
Over the past few years there has been quite a lot of discussion going on in the cannabis industry concerning whether “feminized” seeds (self-pollinated) or seeds that are created the old fashioned way with a male and female plant are best. While many will argue until they are blue in the face on either side, new technology and research is making it possible that we won’t have to continue to argue for long.
For the first time in history, plants have now been cloned from seed. Researchers from HHMI (Howard Hughes Medical Institute) and their international collaborators have taken a major step towards making hybrid crop plants that retain the best traits from generation to generation in the form of cloned seeds. "Agricultural companies and farmers around the world have a tremendous interest in this method," says Vielle-Calzada, a plant researcher at the Center for Research and Advanced Studies of the National Polytechnic Institute in Irapuato, Mexico. "It would allow them to simplify the labor-intensive cross-hybridization methods they now use to produce hearty seeds with desirable traits."
Sexual reproduction in plants involves the generation of male and female gametes that each carry half of the organism's genes. Flowering plants exhibit the most advanced form of sexual plant reproduction, producing pollen-derived sperm cells that join with egg cells to produce seeds. Each seed, then, is genetically unique. There are several types of asexual reproduction in plants, but all produce the same result: genetically identical daughter plants.
Ville-Calzada began to develop an asexual seed 10 years ago, after investigating apomixis, a specific type of asexual reproduction. Many species of plants use apomixis to produce viable seeds without the use of sperm and eggs. This method of asexual reproduction results in the formation of seeds that are essentially clones of the main plant and has great potential for crop improvement.
In apomixis, reproductive cells retain the full complement of chromosomes, rather than losing half their genes via meiosis, as happens in sexual reproduction. About 350 families of flowering plants rely on apomixis to reproduce, but nearly all plants used for food reproduce sexually. "We've been trying to induce apomixis in a species that doesn't practice it," he says. Using the Arabidopsis thaliana, a small flowering mustard plant with a compact and well understood genome, he homed in on a reproductive structure of Arabidopsis called the ovule. Each tiny ovule produces a single female gamete, which, when fertilized, grows into a seed. The team used a genetic screen to identify genes that are active in the ovule - reasoning that measuring gene activity would lead to important insights into which proteins are essential for guiding asexual reproduction.
Of all the interesting genes in their screening, one in particular caught their attention, Argonaute 9. The large family of Argonaute proteins has gained widespread attention among researchers because the proteins control which gene products—either RNA or proteins—a cell makes. Argonautes do this by slicing up messenger RNA before it can be translated into proteins. The identification of Argonaute activity in the ovule was all the more interesting, says Vielle-Calzada, because Argonaute proteins had never been seen in Arabidopsis reproductive cells before.
Vielle-Calzada and his colleagues mutated the Argonaute 9 gene and watched what happened next. Instead of producing a single gamete, most of the ovules with the disrupted Argonaute gene produced several gametes, which were abnormal. Instead of carrying half of the species' chromosomes, they carried the full complement of genetic material— implying that they had not undergone meiosis.
"By cutting off the function of Argonaute, we caused a 'schizophrenic' reaction of the cells in the ovule, which were not supposed to become gametes," Vielle-Calzada says. "It looks like Argonaute normally prevents those cells from being transformed into gamete precursors." That suggested that Argonaute 9 prevents the initiation of apomixis in Arabidopsis.
The finding raises the possibility that many—or maybe even all—plants have the ability to reproduce through apomixis, but that potential is suppressed by Argonaute 9. "It's possible that plants have a very old memory that allows them to reproduce asexually," Vielle-Calzada says.
The team then searched inside the ovule to look for the pieces of RNA that Argonaute 9 degraded. They found that Argonaute chewed up 2,600 snippets of RNA. The experiment "was a complete tour de force for the lab," Vielle-Calzada says. "It required a lot of ovules and a lot of fiddling."
After mapping those RNA sequences back to the Arabidopsis genome, the team discovered that more than half were produced by transposons. Transposons, also called "jumping genes," are mobile genetic elements that copy and insert themselves throughout the genome. Their function remains somewhat mysterious, although some evidence suggest they are important in controlling gene expression.
"It seems that Argonaute 9 silences transposons in the ovule of Arabidopsis," Vielle-Calzada says. "The open question now is, 'Why?'" His working hypothesis is that squelching the transposons prevents apomixis, but his lab is working to prove the connection. "These results are exciting because they suggest for the first time that transposons could be controlling early development in plants," he says.
Though he has made great progress, Vielle-Calzada is still working toward creating a fully asexual Arabidopsis plant. His current mutants do not develop completely asexual seeds. But by highlighting the role of Argonaute 9 in plant reproduction, Vielle-Calzada has moved a step closer to a slew of agricultural possibilities. "Now we just need to discover how to trigger the second and final step of making sexual plants asexual," he says.
That is where Simon Chan steps in. Assistant Professor of Plant Biology at UC Davis and author of new science paper that has expanded upon Vielle-Calzada’s work on the Arabidopsis plant. "We're trying to make a hybrid that breeds true," Chan said, so that plants grown from the seed would be genetically identical to one parent. Some plants, especially fruit trees and cannabis, can be cloned from cuttings, but this approach is impractical for most crops. Other plants, especially weeds such as hawkweed and dandelions, can produce true seeds that are clones of themselves without sexual reproduction -- using apomixis. The new discovery gets to the same result as apomixis, although by a different route, Chan said.
Normally, eggs and sperm are haploid -- they have half the number of chromosomes of the parent. The fertilized egg and the adult plant it grows into are diploid -- containing a full complement of chromosomes, half contributed by each parent.
Chan and his colleagues focused their work on the laboratory plant Arabidopsis, which has certain genetic mutations that allow it to produce diploid eggs without sexual recombination. These eggs have the same genes and number of chromosomes as their parents. But those eggs cannot be grown into adult plants without fertilization by sperm, which adds another parent's set of chromosomes.
Last year, Chan and UC Davis postdoctoral researcher Maruthachalam Ravi showed that they could breed haploid Arabidopsis plants that carried chromosomes from only one parent. They introduced a genetic change so that after the eggs were fertilized, the chromosomes from one of the parents were eliminated. Such haploid plants would reduce the time needed to breed new varieties.
In the new study, Chan's lab, with colleagues from India and France, crossed these Arabidopsis plants programmed to eliminate a parent's genes with either of two mutants that can produce diploid eggs. In about one-third of the seeds produced, the diploid eggs were successfully fertilized, and then the chromosomes from one parent were eliminated, leaving a diploid seed that was a clone of one of its parents.
Ravi described the result as a step on the way towards artificial apomixis. The team hopes to produce crop plants, such as lettuce and tomatoes that can fertilize themselves and produce clonal seeds. Applications for provisional patents on the work have been filed.
If you have been torn between deciding between clones, seeds, and feminized seeds for your cannabis garden, don’t worry. Science is working hard at finding the ultimate solution, so you don’t have to.
(Vielle-Calzada, March 7, 2010) (Chan, Feb 18, 2011)