CATDOLL : CATDOLL: How are triploid fish formed?

How do triploid fish form?

Biological principles of triploid fish generation

1. Formation of normal diploid fish

Except for a few species, most fish in nature exist in diploid form and reproduce by sexual fusion, requiring artificial induction to produce triploids. One of the characteristics of fish chromosomes is that they have great plasticity and are easy to double, allowing haploids to form diploids and then diploids to form polyploids. This is the theoretical basis for the artificial induction of triploids.

When the egg cell is in the metaphase of the second meiotic division, there are two sets of chromosomes in the cell. When the sperm with one set of chromosomes enters the egg, it stimulates the egg to continue to complete the second meiotic division. One of the two sets of chromosomes in the egg is expelled from the egg as the second polar body, and the set of chromosomes that remain in the egg combines with the set of chromosomes brought in by the sperm. The fertilized egg then becomes a normal diploid and begins to develop.

2. Methods for directly obtaining triploid fish

If the fertilized egg is treated with high temperature, low temperature, high pressure or chemical drugs when the sperm enters the egg and the second polar body has not yet been discharged, the second polar body cannot be discharged from the egg, forming an individual with three sets of chromosomes, which is called artificially induced triploid. This combination of chromosome sets is that two sets of chromosomes come from the mother and one set of chromosomes comes from the father. By destroying the spindle fibers so that the chromosomes cannot separate, or by preventing the formation of contractile rings and grooves on the cell surface to stop the cytoplasmic division, the second meiosis can be prevented, forming a triploid fertilized egg, and then developing into a triploid fish.

3. Methods for artificially inducing triploidy

There are two ways to obtain triploids artificially. One is the direct way, that is, to treat the fertilized eggs with physical or chemical methods to prevent the second polar body from being expelled; the other is the indirect way, that is, to first induce tetraploids, and then naturally mate with diploid individuals after the tetraploid individuals are sexually mature to obtain triploids. Theoretically, the second way has its advantages. Once the tetraploid individuals are obtained, there is no need for physical or chemical induction, which avoids the damage to the embryo during the induction process. If a stable tetraploid strain can be established, it will become a very easy thing to obtain triploids. However, most of the artificially induced triploids are currently obtained by the first way, because the method of preventing the second polar body from being expelled is relatively mature. The induction of tetraploids is to prevent the first cleavage, which is much more difficult than inducing triploids, and the success rate is very low. Only in a few fish such as rainbow squats and crucian carp, sexually mature tetraploids have been obtained.

How to cultivate triploid

1 Principle

The method of artificially inducing polyploidy is adopted. For example, the seeds or seedlings of diploid plants are treated with colchicine (a plant alkaloid) to hinder the formation of spindle fibers and primary walls in the metaphase of cell division, so that the replicated chromosome sets cannot be divided to the two poles, and a secondary wall is formed in the middle. As a result, cells with doubled chromosome sets are formed, and the chromosome sets of ordinary diploid watermelons are doubled to obtain tetraploid watermelon plants. Then hybridize with diploid watermelon plants (as the father) to obtain triploid seeds. The triploid plants developed from triploid seeds cannot form normal reproductive cells due to the disorder of homologous chromosomes during the reduction process. The mature pollen of the diploid plant is then used to stimulate the ovary of the triploid plant flower to form a triploid fruit. Because its ovules cannot develop into seeds, it is called a triploid plant.

2 Specific measures (taking seedless watermelon as an example)

(1) Soak the seeds of diploid common watermelon in 0.2-0.4% colchicine (C22H26O6N) liquid for 12-24 hours, or drip 0.2-0.4% colchicine liquid on the growth point of the stem tip of the seedlings at 6-7 pm every day for four consecutive days.

(2) After treatment, the seeds or seedlings should be washed with clean water. During the treatment and seedling acclimatization period, the plants should be placed under diffuse light to avoid direct sunlight, which may cause the decomposition and destruction of colchicine. At the same time, high temperatures should be avoided, because under high temperatures, the toxicity of colchicine to plants increases, which can easily cause seedling death. Therefore, good cultivation conditions and careful management should be provided before survival.

(3) Identification of polyploids: ① After treatment with colchicine solution, pay attention to observe polyploid plants. If polyploids are formed, the thickness of its budding fat increases significantly, and the tip of the young root swells; ② Inspect the body shape of the plant. Polyploids are characterized by larger body size. For example, the leaves are larger, the rhizomes become thicker, the flowers, fruits, and seeds are larger, the stem and leaf tissues are relatively rough, the color is dark green, and sometimes there is a wrinkle phenomenon; ③ The stomata on the leaf surface of tetraploids are larger, the number of stomata per unit area is relatively reduced, and the pollen grains are more than twice as large as those of diploids; ④ In order to ensure the accuracy of identification, it is necessary to directly check under a microscope whether the number of chromosomes has doubled (ordinary diploid watermelon 2N=22, tetraploid watermelon 4N=44).

(4) Triploid watermelon seed production: Use tetraploid watermelon as the female parent, fertilize with diploid pollen grains, and produce triploid watermelon seeds (existing in the watermelon developed from the tetraploid ovary). For small-scale seed production, bag the watermelons on time every afternoon according to the flowering habits of watermelons (to prevent free pollination). Artificial pollination is carried out the next morning, and markers are hung at the same time. For large-scale seed production, it is necessary to isolate and cultivate tetraploid female and diploid male plants in a certain ratio, and the female plants must be emasculated in time to allow the tetraploid plants to receive pollen from the diploid male plants to produce triploid seeds for use.

The above is the work of the first year. In the second year, the triploid seeds are interplanted with the diploid seeds, and the diploid pollen is naturally or artificially stimulated to stimulate the ovary of the triploid plant to develop into watermelon, that is, triploid seedless watermelon. In field production, triploid seeds can also be planted without interplanting with diploid seeds, but only triploid seeds can be planted. During the flowering period of the plant, low concentration of 2.4-D (2---4---dichlorophenoxyacetic acid) is sprayed on the flower buds to make the ovary develop into seedless watermelon fruit.

It should be noted that seeds develop from various parts of the ovule, and fruits develop from the ovary. Therefore, the watermelon obtained in the first year should be a tetraploid watermelon, and the seeds in it are triploid. To be precise, the embryo of the seed is triploid, and the seed coat is tetraploid (the seed coat is developed from the tetraploid integuments). The watermelon obtained in the second year is a triploid watermelon, which has no seeds, or to be precise, only the seed coat (developed from the triploid ovule) but no embryo (pollination without fertilization, can not form a fertilized egg, so there is no embryo).