For every gardener, whether a professional or an amateur, it is important to know how to propagate your plantings. There are many ways, and one of the most interesting and effective is the method of micropropagation. What it is, how it works and all its main wisdom - in our material.
What is this?
Let's start with the most important thing. In the phrase "microclonal reproduction" the second word is clear to everyone, but the first - only to the elite. Let's clarify the situation. What is "microclonal"?
Speaking in "smart" scientific terms, this is a special subspecies of vegetative propagation using a technique called "in vitro" (in vitro), which makes it possible to obtain plants in a shorter time. We will understand more clearly and in more detail further, and for this we first recall what vegetative propagation is, and explain what the term "invitro" means.
In the scientific wilderness
From the course of the schoolIn biology, we know that plants can be propagated in two ways: seed (when we scatter seeds into the soil) and vegetative. Vegetative propagation is asexual, it occurs by separating some part from the parent plant. Budding, rooting of young shoots, transplanting bulbs - all this is vegetative propagation.
It would seem that with the help of seeds it is much easier to increase the number of plants - there is no such trouble. However, this method has quite a few downsides; in some cases, it is impossible to use seeds at all - and the vegetative method, the undeniable advantage of which over the first one is to preserve the totality of the genes of the parent plant, remains the only accessible and convenient one. But unfortunately, he also has shortcomings. For example, the lack of the desired efficiency (for example, in plants such as oak, pine, and so on), "older" tree species (which are more than 15 years old) are not able to propagate by cuttings, such procedures are quite laborious and energy-consuming, the resulting plants do not always correspond norm and sample (may be infected) - and so on.
And it is for these cases that micropropagation technology exists, which, like Chip and Dale, rushes to the rescue. As mentioned above, it is carried out using the "in vitro" technique, which is translated from Latin as "in vitro". Thus, this technique makes it possible to “clone” in a “test tube” a plant with genes exactly the same aslike the parent. This is due to the fact that the cell is able to give life to a new organism under the influence of external factors.
The technology of micropropagation undoubtedly has a number of advantages and disadvantages. We'll talk about them later.
Which is better than micropropagation
To many! And first of all, the absence of viruses and infections in bred plants (because special cells are used for this - they are called meristem cells, their feature lies in the incessant division and the presence of physiological activity throughout life). Also, in plants "extracted" in this way, there is a fairly high volume of reproduction, and the entire breeding process is much faster. With the help of micropropagation technology, it is possible to carry out this procedure for those plants for which it is extremely problematic to do this by conventional, "traditional" methods. Finally, in the "in vitro" technique, plants can be grown throughout the year, not limited to any one interval. So there are a lot of advantages to such a technique. And before delving into the essence of microclonal propagation of plants, let's touch on a little history of the emergence of this method. Who came up with this idea and how?
History of the method
The first successful experiment on orchids was made by a French scientist back in the fifties of the last century. At the same time, he did not initially engage in the "invitro" technique - it was developed before him, and quite successfully. However, it is JeanMorel - such is the name of the French experimenter - decided on a similar experiment and carried it out quite successfully. The works that tell about this technique appeared several decades before him - in the twenties of the last century.
A "test tube clone" of a woody plant - specifically aspen - was obtained in the sixties. It turned out to be more difficult to work with wood than with flowers and other types of plants, however, these difficulties were overcome after a certain period of time. Currently, more than 200 species of trees from more than forty families can be obtained by the "test-tube" method. The technology of micropropagation of plants justifies itself and bears fruit.
More about the method
As you might have guessed, there are many subtleties in the development and application of micropropagation of plants. So, for example, there are special stages for this technology, which are simply necessary to follow in order to obtain the desired result. It must be understood that neglecting the sequence of actions or some stage can bring absolutely not the result that the breeder expects. So, we will talk about the stages of this technique further.
Stages of micropropagation of plants
This technology involves four "steps" on the way to obtaining the coveted "clones". We will try to talk about them as unscientifically as possible, since the terms of biotechnology are still not the most understandable thing for a wide audience. AND,By the way, we will immediately explain one of these terms: explant - this is how scientists in this field call a new organism separated from the parent organism. That is, the very "guinea pig" that will be grown further.
So, let's move on to our "steps". The first step is the choice of the parent itself - or the donor. This issue should be approached with the utmost seriousness and responsibility, because in order to obtain a good, strong, he althy plant, we and the "original" must choose the same one. An apple, as you know, does not fall far from the tree.
At the same stage, it is necessary to isolate and sterilize the explants, and then organize such conditions so that the growth of these same explants in the "in vitro" technique occurs as comfortably as possible.
The second "step" couldn't be easier - it's reproduction itself. It is possible in a month and a half, when the mini-cuttings have already reached the size of peas and have the rudiments of all vegetative organs. It, in turn, is followed by the rooting of the shoots that were obtained in the previous stage. It is carried out when the plant has already formed a good root system.
The last step is to help plants adapt to "life" in the soil, growing them in a greenhouse, then transplanting them into the ground or selling them - so to speak, "departure to the big world". This stage, oddly enough, is the most time-consuming and costly, because very often, unfortunately, it happens that, once in the soil, the plant beginslose leaves, stop growing - and then it may die altogether. All this happens because test-tube plants lose a lot of water when transplanted into the ground. Therefore, it is necessary to prevent such a possibility during transplantation - for which it is recommended to spray the leaves with a 50% aqueous solution of glycerin or a mixture of paraffin. This must be done throughout the acclimatization period. In addition, in some cases, it is advisable to intentionally mycorrhize - that is, the artificial introduction of fungi into plant tissues that infect it. This is done so that the plant receives as many useful nutrients and organic substances as possible, and is also protected from various pathogens.
That's all the stages of micropropagation, in which, as we see, there is nothing globally complex or supernatural, however, we repeat once again, this whole event requires great responsibility and attention.
Influencing factors
The process of micropropagation, like any other, is influenced by certain factors. Let's list them, because "you need to know the enemy in person."
- Varietal, species and physiological characteristics of the parent plant - it must be he althy, grow intensively, if necessary, treated with temperature exposure.
- Age, structure and origin of the explant.
- Cultivation duration.
- Sterilization efficiency.
- Good breeding ground.
- Hormones, mineral s alts, carbohydrates, vitamins.
- Temperature andlighting.
What you need for micropropagation
There is one very important requirement for plants that will be propagated in the above way - in addition to the fact that they must be he althy. This is an indispensable preservation of genetic stability throughout all the above stages. This requirement is best met by apical meristems, as well as axillary buds of stem origin, which is why they are preferred to be used for the procedure of interest to us.
The above terms should be incomprehensible to the average layman. Below we will try to explain what kind of animals they are and what to serve them with.
Apical meristems
Above we have already mentioned the existence of special meristem cells - in other words, educational ones. These are cells that are constantly dividing, always in a state of physical activity - due to which the mass of the plant grows and a special tissue of this plant is formed. It is called the meristem. There are many types of meristems. In general, they can be divided into general and special. The concept of common meristems includes three groups, which, as it were, follow one from the other. The very first meristem in a plant is the meristem of the embryo, from which the apical meristem of interest to us originates.
The word "apical" comes from the Latin "apix" and it translates as "top". Thus, this is the apical tissue system located at the very tip of the embryo - and it is from it that the shoot is subsequently formed and its growth and development begins. So, speaking of the apical meristem as an object for microcloning, we must understand that we take the tip of the embryo for our needs.
Axillary buds are a little easier. Everyone knows what kidneys are. The axillary bud is the one that was born from the axil of the leaf. The leaf axil, in turn, is the angle between the leaf and its stem; from there a kidney or an escape will grow. This very part, that is, the future side shoot, is also taken for subsequent micropropagation.
Now that some light has been shed over the veil of mystery, we can finally move on to micropropagation methods.
Micro breeding methods
Microclonal propagation is still good, which basically implies the possibility of using several different techniques at once. We will try to cover each of them as simply as possible. There are four micropropagation methods in total.
First. Activation of already existing meristems in the plant
What does this mean? In a plant, even such a tiny micropiece, certain meristems are already laid. This is the top of the stem and its axillary buds. In order to microclone a plant, it is possible to "wake up" these hitherto dormant meristems "in vitro". This is achieved either by removing the apical meristem of the microsprout, or rather, its stem, and then cutting the shoot using the "in vitro" technique, or by introducing special substances into the nutrient medium of the plant that activate the growth and development of axillary shoots. Methodactivation of "sleeping" meristems is the main, most popular and effective, and it was developed back in the seventies of the last century. The first "guinea pig" in the application of micropropagation of plants of this type was the strawberry. It is important to note, however, that it is forbidden to propagate crops indefinitely in this way, since this is fraught with the loss of the ability to root, and in some cases, the death of the plant.
Second. The emergence of adventitious buds by the forces of the plant itself
Any isolated part of a plant has a truly magical ability, its own superpower. If during microclonal propagation the nutrient medium of the plant and all other living conditions are favorable and comfortable, then it can restore the missing parts. A kind of regeneration takes place - the tissues of the plant form adventitious, or adnexal buds - that is, those that appear, as it were, "from old reserves", and not from new tissues. Such buds are unusual in that they appear, as a rule, in those places where you would not expect them to appear - on the roots, for example. It is in this way that many flowers are often propagated, again - strawberries. This is the second most popular and effective method of micropropagation of plants.
Third. Somatic embryogenesis
With the second word, everything should be more or less clear. Let's touch on the first - what does somatic mean? This word in this vein is directly related to the cells of the same name. Such cells are called those that make up the body of multicellular organisms and do notparticipate in sexual reproduction. In short, these are all cells, with the exception of gametes. Somatic embryogenesis is carried out in a rather simple way: embryoids are formed from the above cells (that is, somatic) using the “in vitro” technique, which subsequently, when they organize suitable conditions for development with an optimal nutrient medium, turn into an independent whole plant. In this case, we can talk about such a concept as totipotency (the ability of any cell, due to division, to initiate any cell type of an organism). It is believed that eventually such embryos develop into a seedling. Somatic embryogenesis is also good because it is possible to obtain artificial seeds in this way. This method was first discovered in the middle of the last century in carrot cells.
An actively similar method of micropropagation of plants is used in the propagation of oil palm. The thing is that, since it has neither shoots nor lateral shoots, its vegetative propagation is impossible (or, in any case, very, very difficult), just as cuttings are impossible. Thus, the above methodology is the only one of all the most accessible and optimal when working with this plant.
Fourth. Working with callus tissue
Another term smoothly "floated" into the network of our narrative, and first of all, it is required to clarify its meaning. What is callus tissue? Everyone knows that on the wound, when it lives a little, a drying crust appears. And if you pull it off, the wound starts to bleed again. Toythe crust itself, in other words, “the healing tissue, is the callus tissue. The cells of this tissue, not only contribute to the healing of wounds, are also totipotent - that is, as already explained above, they allow a new plant to arise. And that is why adnexal buds (adventive - we have already introduced this term before) can also appear on such tissue.
This method of all the four above is perhaps the least popular. First of all, this is due to the fact that too frequent separation of callus tissue cells can lead to gene disorders and mutations of various levels. Since the preservation of the genotype is very important for micropropagation, and tissue culture must be maintained at the highest level. In addition, with the above violations, other shortcomings appear: short stature, susceptibility to disease, and so on. However, in some cases, reproduction is possible exclusively in a similar way - for example, for sugar beet, there is simply no other method.
Next, for example, we will say a few words about the cloning of specific plants, but first, we need to share information about the recovery of plants used as planting material. How can this be achieved?
Recovery
There are several ways to turn a plant from a diseased into a he althy one, and the first of them is to place the sprout in a special chamber, or box, where sterile conditions are maintained, and “stuffing” it with antibiotics. This method is good for everyone, except that it does not cope with all bacteria.and viruses to which plants can be exposed. In such cases, in order to disinfect plants, they are given thermotherapy - in other words, heat treatment in special isolated chambers, where the temperature is increased daily for several days in a row. Chemotherapy is another way to fight infections and bacteria for infected plants.
About potato cloning
Potato, by the way, is one of the few crops that can be propagated by the fourth of the above methods. But, of course, this is far from the only way - and often they also resort to the activation of "sleeping" apical and axillary meristems. The tubers obtained after cloning are exactly the same as the "original" ones - they differ only in a smaller size, these are the so-called microtubers. And besides, they will certainly be he althy and free of viruses.
In microclonal propagation of potatoes, it is grown in test tubes of two cuttings, the test tubes are placed under the light of fluorescent lamps with a power of six to eight thousand lux, the temperature is maintained at night within eighteen degrees, during the day - about twenty-five. In Russia, it is potatoes that are most actively grown using cloning.
About apple tree cloning: what you need to know
In the micropropagation of apple trees, the first method is widely used - propagation using axillary buds. There is a high ability of this culture to root and the survival rate of moreexplants.
They were placed in a liquid nutrient medium, which was constantly - daily - updated. The temperature for test-tube plants was also maintained at twenty-five degrees during the day, the experiment was carried out for three to four weeks.
Interesting facts
- This technique, as you can easily guess, got its name from the concept of “clone”, which appeared in 1903. From the Greek language, this word is translated as "offspring" or "cutting".
- The first place in our country where the first experimental attempts at microclonal propagation of plants were carried out was the Timiryazev Moscow Institute.
- Clonal micropropagation is an excellent method to control viruses and produce he althy, infection-free plants.
- The period that a plant goes through before flowering and fruiting is called juvenile - and in those organisms that are obtained by cloning, it is minimized.
- The United States of America, the Netherlands, Italy, Poland, Israel and India are considered to be the leading countries in the production of plants in the above way.
- Almost two and a half thousand species and varieties of plants can now be propagated using the "in vitro" technique.
- In the early stages, plants grown in vitro may differ in appearance, but as they grow, all differences disappear, and in the end the plants become similar, like twins.
- Explants from young plants are best rooted than frommature.
- One of the important conditions in microclonal propagation is the selection of the most favorable nutrient medium for the plant, and it can be both liquid and solid state.
- Cells of meristematic tissues usually do not contain viruses.
- The size of the explant is directly related to the possible presence of viruses in it. The smaller it is, the lower the risk of infections.
- Another name for micropropagation is meristem propagation.
This is the information about plant micropropagation, a topic as complex as it is interesting.
