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KEN MUDGE: There's been a lot of controversy lately about human cloning and related issues, like test tube babies. But have you ever heard about plants in test tubes? Welcome to the world of plant propagation.
This is the last of the asexual propagation methods or approaches that I'd like to talk about in this Cyber Tower room. Micropropagation is a rather general term that refers to any of a number of different approaches to plant tissue culture where the objective is to multiply plants, to bring about propagation.
All these different methods have in common three features that are characteristic of most micropropagation systems. First of all, you start with a small explant. Image on the right of the screen shows a very small apical meristem that's only about a tenth of a millimeter in diameter. That's the kind of explant that can be used to start a plant tissue culture.
Second characteristic is aseptic culture. We work in a germ free environment where we exclude microorganisms, and the only living part of this system is the plant itself. Because of the sugar and the nutrient media, microorganisms would overtake the entire system.
And the third characteristic is heterotrophic nutrition. In other words, these plants are not dependent on photosynthesis for production of sugars. But instead, we provide the sugars in this artificial gel nutrient medium along with mineral nutrients, vitamins, and plant hormones.
So I'd like to show you two different micropropagation systems. One is organ culture and the other is somatic embryogenesis.
Organ culture depends on proliferating shoots and culture, and then using hormones to induce adventitious root formation. So basically, we're dealing with cuttings at the end of the process, but they're micro cuttings.
Somatic embryogenesis is a variation on the thing we considered earlier of asexual embryo formation. So let's go over to the sterile work area and take a look at it.
Well, OK. In the case of organ culture, or shoot organ culture in this case, the strategy is to take a small explant, either a actively growing shoot or even a dormant bud, surface sterilize it so the culture remains aseptic. Get rid of bacteria and the fungi that are on the explant.
And then place that bud into a culture medium that contains a hormone called cytokinin. That cytokinin stimulates branching of that shoot just as if you had pinched it back to induce lateral branches to form. So basically, it turns into a very small bush or shrub-like structure, which we subdivide, place on fresh medium, repeat the process, and increase as much as we need to.
You can see here that if we have a five-fold multiplication rate every six weeks that will go through nine generations about a year's time and come up with about 1.9 million plants. There is no other asexual propagation technique that has the potential for that extremely high rate of increase.
Now, let's take a look at how it's actually done. I need to grab a Petri dish up here. This has been pre-sterilized. And this is a culture box that has been initiated already. And what we're doing is going through one of those rounds of multiplication.
Use the forceps here. We have to surface sterilize them in this red hot bact incinerator for about 10 seconds. Allow them to cool for just a moment. And then reach into the culture vessel, use them to pick out one of those clumps very gently.
Can't touch it with our fingers to avoid contamination. Lay it onto the surface of a sterile Petri dish. Sterilize the scalpel and use that to cut away the callus and basically divide that clump into a number of individual shoots.
There are five, so that's a five or six-fold multiplication rate. Then we can take the individual shoots and transfer them to a fresh nutrient media. Once again, containing the sugars, mineral nutrients, and the hormone cytokinin.
Take any one of the shoots and place it into the culture vessel. We can put about nine shoots into a vessel like this. And we can get the requisite number of shoots in there. Close it up.
Seal it off so it maintains aseptic conditions, and place it on a lighted growth shelf to grow on for another four to six weeks before we subculture it again.
And so once these microshoots have multiplied through several successive generations of subcultures, we have many, many shoots, as many as we need. Perhaps 1.9 million, if we need that many.
But these are shoots without roots. And so what we have to do is induce adventitious root formation on those shoots. We do that by either placing them on a fresh nutrient media that contains the rooting hormone auxin.
Or we can take these microshoots out of culture and place them up in the greenhouse under a special fog tent, and they will basically root just like a cutting that we discussed earlier, except on a very, very small scale. And we'll wind up with rooted microshoots that can be grown on into full sized plants.
The next micropropagation technique I'd like to talk about is somatic embryogenesis. This is regeneration through induction of asexual embryos. We talked about how it occurs naturally on the margins of leaves and in the case of clonal seeds called apomixis.
In this case, we're using a sterile micropropagation system. And this happens to be a cluster of callus and somatic embryos of American ginseng, panax quinquefolia. We're very much interested in this plant in our research program.
As it turns out, American ginseng is normally propagated from seed, but it is extremely difficult to propagate clonally. It has a single shoot that comes up from the ground. Only one determinant shoot with three or four leaves on it and a small flower cluster.
It will not root from cuttings. You can't propagate it effectively by division. Grafting is not an option. And so the only approach that has been even marginally successful is micropropagation.
What we do is we take a ginseng root. You can see in the photograph here it's a thick, fleshy root, highly valued as a medicinal herb, as a matter of fact. We take that root and surface sterilize it, cut it apart and take a small chunk of tissue out of the inner portion of that root and place it on the appropriate nutrient medium containing a combination of plant hormones auxins and cytokinins.
And these induce the cells from the root to begin to divide in an unorganized fashion and give rise to a callus, similar to the callus that we talked about in the case of graft union formation. And then by changing the nutrient media to a different combination of hormones, that callus can be induced to form somatic embryos.
And these are basically very similar in structure to normal sexual embryos, but they are occurring under these plant tissue culture micropropagation conditions. The thing is they do not have a regular seed code like a typical sexual seed does, so they have to be handled very, very gingerly. There's a procedure for getting them to germinate in culture, and then finally acclimatizing them so we can get them out of culture.
So let's go back to the culture room now where we grow these plants on under artificial lights. Micropropagation, then, is an important tool of the plant propagator in modern horticulture. It hasn't displaced the other forms of propagation that we've discussed like some folks thought it would 30 or 40 years ago.
But it is a very important tool used commercially to propagate such crops as rhododendrons, mountain laurels, and many of the house plants that you have in your home.
Now let's turn to the issue of the role of micropropagation in modern biotechnology. In addition to being used as a propagation tool per se, micropropagation is an important tool for plant breeders, those molecular plant breeders involved in genetic transformation or the production of genetically modified organisms.
Now, genetic transformation of plants cells is a different issue in some sense. It has to do with transferring genes from one organism, such as a bacterium, into another organism such as a plant. And that's not micropropagation per se.
But where micropropagation comes in is that once you have transformed those cells, then they need to be regenerated into whole plants. And that's either done through somatic embryogenesis or adventitious organogenesis.
Now, I'd like to just briefly talk about an issue that is confusing in some people's minds. We hear a lot of talk about human cloning and so forth, and we're also concerned about issues related to the genetically modified plants or genetic engineering.
There's a tendency for some people to confuse the two. In fact, genetic engineering utilizes micropropagation. It utilizes cloning. But the two aren't the same thing.
As you can see from this article in The New York Times, there is a tendency for even reporters at The New York Times to confuse the two. This is really an article about genetically modified organisms, but they talk in this particular highlighted sentence as if cloning were the same thing.
And so you see regardless of what you might think of human cloning or cloning of animals, the cloning of plants is a very important part of modern agriculture. In fact, a part of agriculture ever since the invention of agriculture 12,000 years ago.
Well, I hope I've piqued your interest in plant cloning. I'm sure you have questions. And if you do, I welcome you to ask them in the discussion board. I'd be happy to respond to them.
If you'd like more information about plant cloning, there are many books that I could recommend. And also I teach an online course in grafting. It's called "The How, When, and Why of Grafting." You can get more information at the URL that you can see here. Thank you.
In this Room I share with you my fascination with plant reproductive biology and its application to horticulture and related disciplines.
I begin by dispelling the widely held oversimplification that "plants grow from seeds" - indeed many of them do, but quite a few have evolved the capacity for asexual (clonal) reproduction. Even before the origins of agriculture, about 12,000 years ago, mankind has been observing wild plants performing feats of asexual reproduction.
From this increasingly sophisticated understanding of the natural history of cloning, early agriculturists domesticated a number of fruit, nut and other food crops and eventually a host of ornamentals as well. The Room includes hands-on demonstrations of clonal propagation by layering, cuttings, grafting and micropropagation.
This video is part 6 of 7 in the Natural and Human History of Plant Cloning series.
