---- Shellfish polyploid breeding technical methods classification and operation Shellfish induced artificial triploid method can be divided into physical, chemical and biological methods 3 aspects:
(1) The chemical method is mainly to use some chemical substances (ie, chemical inducers) that can inhibit the splitting of cells, so as to interfere with the normal cell division process and achieve the intended purpose. The main inducers are:
Cytochatin B (abbreviated as CB): A metabolite of fungi. CB treatment can induce a series of cytological effects, such as changes in the cytoskeleton, multinucleation, destruction of microfilaments [7]. It is generally believed that CB specifically destroys microfilaments, ultimately causing the disintegration of the contractile loop consisting of microfilaments of cell division, inhibiting cytoplasmic division, preventing the release of polar bodies, thereby generating polyploidy. CB is a carcinogen and has poor water solubility. It is generally dissolved in 1% dimethyl sulfoxide (DMSO). After treatment, fertilized eggs are washed with seawater containing 1 DMSO to remove residual CB.
6-dimethylaminopurine (6-DMAP): An analog of puromycin that inhibits the phosphorylation of proteins, acting on specific enzymes, destroying the polymerization centers of microtubules, and making microtubules Can not form, thereby inhibiting the formation and release of polar body [8]. 6-DMAP is non-carcinogenic and has water activity.
Caffeine: Its effect is to increase intracellular Ca2+. Since microtubules are sensitive to Ca2+ concentration, when the Ca2+ concentration is extremely low or higher than 10-3, it will cause depolymerization of the microtubule dimer and prevent cell division, resulting in polyploidy [9].
Colchicine: It can inhibit the assembly of microtubules and depolymerize existing microtubules, thus preventing the formation of spindles or destroying the formed spindles, doubling the chromosomes of the cells without separation [10]. The inducer is widely used in plants and has been successfully applied in shellfish and other animals.
In addition, chemical agents such as polyethylene glycol (PEG) [11] and calcium cyanide (CL) [12] have a certain role in inhibiting cell division and doubling chromosomes.
(2) The physical method applies physical factors to the cell division cycle to influence and interfere with the normal division of cells to achieve chromosome doubling.
Common physical methods include temperature shock (including high and low temperature shocks) and hydrostatic shock.
Temperature shock; its mechanism of action is that changes in temperature cause intracellular enzyme configuration changes, is not conducive to the enzymatic reaction, leading to cell division, the formation of spindles required ATP supply path is blocked, so that has completed chromosome doubled cells Can't split. The temperature used varies from one species to another.
Hydrostatic pressure: The pressure generated by a special hydrostatic apparatus is applied to the treatment object. Its mechanism of action is to inhibit the formation of microfilaments and microtubules in the spindle and prevent the movement of chromosomes, thereby inhibiting cell division and forming polyploidy.
In addition, electrical pulses can also induce polyploidy [13].
(3) Biological methods In addition to chemical methods and physical methods, polyploids (ie, biological methods) can also be cultivated using shellfish distant hybridization. However, due to the complexity of seed selection, this method has not been applied in shellfishes, and it has been used in fish.
The tetraploid and diploid mating of Pacific oysters successfully produced triploids [14].
The tetraploid (T) and diploid (D) hybrids produce shellfish triploids, triploid rate can be as high as 100%, the method is simple, easy to operate, to avoid the physical and chemical treatment of the embryonic development, can improve the embryo The hatching rate and the survival rate of larvae are suitable for productive breeding.
In addition, heterotetraploids can be generated by crossing tetraploids with heterodiploids. At present, allotriploid progenies have been successfully bred by hybridization between the tetraploid of O. pleurotus and the diploid of O. sinensis [15].
(1) The chemical method is mainly to use some chemical substances (ie, chemical inducers) that can inhibit the splitting of cells, so as to interfere with the normal cell division process and achieve the intended purpose. The main inducers are:
Cytochatin B (abbreviated as CB): A metabolite of fungi. CB treatment can induce a series of cytological effects, such as changes in the cytoskeleton, multinucleation, destruction of microfilaments [7]. It is generally believed that CB specifically destroys microfilaments, ultimately causing the disintegration of the contractile loop consisting of microfilaments of cell division, inhibiting cytoplasmic division, preventing the release of polar bodies, thereby generating polyploidy. CB is a carcinogen and has poor water solubility. It is generally dissolved in 1% dimethyl sulfoxide (DMSO). After treatment, fertilized eggs are washed with seawater containing 1 DMSO to remove residual CB.
6-dimethylaminopurine (6-DMAP): An analog of puromycin that inhibits the phosphorylation of proteins, acting on specific enzymes, destroying the polymerization centers of microtubules, and making microtubules Can not form, thereby inhibiting the formation and release of polar body [8]. 6-DMAP is non-carcinogenic and has water activity.
Caffeine: Its effect is to increase intracellular Ca2+. Since microtubules are sensitive to Ca2+ concentration, when the Ca2+ concentration is extremely low or higher than 10-3, it will cause depolymerization of the microtubule dimer and prevent cell division, resulting in polyploidy [9].
Colchicine: It can inhibit the assembly of microtubules and depolymerize existing microtubules, thus preventing the formation of spindles or destroying the formed spindles, doubling the chromosomes of the cells without separation [10]. The inducer is widely used in plants and has been successfully applied in shellfish and other animals.
In addition, chemical agents such as polyethylene glycol (PEG) [11] and calcium cyanide (CL) [12] have a certain role in inhibiting cell division and doubling chromosomes.
(2) The physical method applies physical factors to the cell division cycle to influence and interfere with the normal division of cells to achieve chromosome doubling.
Common physical methods include temperature shock (including high and low temperature shocks) and hydrostatic shock.
Temperature shock; its mechanism of action is that changes in temperature cause intracellular enzyme configuration changes, is not conducive to the enzymatic reaction, leading to cell division, the formation of spindles required ATP supply path is blocked, so that has completed chromosome doubled cells Can't split. The temperature used varies from one species to another.
Hydrostatic pressure: The pressure generated by a special hydrostatic apparatus is applied to the treatment object. Its mechanism of action is to inhibit the formation of microfilaments and microtubules in the spindle and prevent the movement of chromosomes, thereby inhibiting cell division and forming polyploidy.
In addition, electrical pulses can also induce polyploidy [13].
(3) Biological methods In addition to chemical methods and physical methods, polyploids (ie, biological methods) can also be cultivated using shellfish distant hybridization. However, due to the complexity of seed selection, this method has not been applied in shellfishes, and it has been used in fish.
The tetraploid and diploid mating of Pacific oysters successfully produced triploids [14].
The tetraploid (T) and diploid (D) hybrids produce shellfish triploids, triploid rate can be as high as 100%, the method is simple, easy to operate, to avoid the physical and chemical treatment of the embryonic development, can improve the embryo The hatching rate and the survival rate of larvae are suitable for productive breeding.
In addition, heterotetraploids can be generated by crossing tetraploids with heterodiploids. At present, allotriploid progenies have been successfully bred by hybridization between the tetraploid of O. pleurotus and the diploid of O. sinensis [15].