CN111296286A - Moringa oleifera tetraploid induction and propagation method - Google Patents
Moringa oleifera tetraploid induction and propagation method Download PDFInfo
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Abstract
The invention discloses a method for inducing and propagating moringa oleifera tetraploids. According to the invention, the moringa leaves are used as explant materials, the moringa explants are treated by colchicine, conditions influencing regeneration and polyploid induction of the moringa explants by the colchicine are optimized, the tetraploid yield reaches 25%, the regeneration efficiency reaches 86.67%, and the rooting rate reaches 93.3%, so that the moringa explant has remarkable advantages; the quality of the moringa oleifera is improved, the high-quality moringa tetraploid variety is cultivated, the resource requirement of the moringa oleifera germplasm is met, and a technical method and a theoretical basis are provided for the quality improvement of the moringa oleifera through a polyploid breeding means.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for inducing and propagating moringa oleifera tetraploids.
Background
Moringa oleifera (Moringa oleifera Lam.) as a multifunctional tree species contains various minerals and vitamins in fruit pods, leaves and roots, and has certain health-care function as a vegetable with nutritional value. The moringa oleifera is rich in nutrition, meets the standard of high-quality feed, has high crude protein content and rich vitamin content in leaves, is used as a feed or an auxiliary material of the feed, can promote the growth of animals, improve the immunity and meat quality of the animals, and can save the feed cost. The moringa oleifera has high yield, simple cultivation, wide adaptability and strong stress resistance, can be used as woody feed to make up for the problem of serious shortage of protein feed, and is also a good mixed agriculture and forestry tree species. In recent years, along with the development of research and development of moringa resources, the moringa is also more and more valued.
After the plant chromosome is induced and doubled, due to the gene interaction effect and the gene dosage effect, the physiological balance of plant cells is broken, polyploids show 'giant', the adaptability and the stress resistance of the polyploids are enhanced, and the biomass of nutritive organs such as roots, stems, leaves and the like is also obviously increased. Therefore, if the moringa oleifera polyploid can be obtained through induction, the quality of the moringa oleifera can be improved, high-quality moringa oleifera polyploid varieties can be cultivated, and the requirement of moringa oleifera germplasm resources is met.
Disclosure of Invention
The invention aims to provide a method for inducing and propagating moringa oleifera tetraploids.
The invention relates to a method for inducing and propagating moringa oleifera tetraploids, which comprises the following steps:
a. pre-culturing leaf explants:
cutting sterile young leaves of moringa oleifera seedlings to serve as explants, after small wounds are scratched on the paraxial surfaces of the young leaves, placing the paraxial surfaces downwards to be inoculated on an induction culture medium for pre-culture for 3 days, wherein the induction culture medium comprises the following components: each liter contains 0.8mg of 6-BA, 0.2mg of KT, 0.05mg of NAA, 30g of cane sugar and 4.5g of agar, the balance is MS culture medium, and the pH value is 5.8-6.0;
b. and (3) colchicine induction culture:
transferring the pre-cultured explant to a colchicine induction culture medium for dark culture for 21d, wherein the culture temperature is 26 ℃; the colchicine induction culture medium comprises: each liter contains colchicine 100mg, 6-BA0.8 mg, KT 0.2mg, NAA0.05mg, sucrose 30g and agar 4.5g, the rest is MS culture medium, pH5.8-6.0;
c. moringa root tip chromosome counting:
cutting off adventitious bud strains subjected to colchicine induced culture to an MS solid culture medium for culture, and taking root tips for chromosome counting when the regenerated roots grow to about 2cm, and identifying the moringa oleifera tetraploid;
d. propagation culture of the moringa tetraploid:
cutting obtained moringa tetraploid stem nodes to the length of 1-2cm to be used as an explant, horizontally inoculating the explant in a moringa tetraploid adventitious bud induction culture medium, and inducing and regenerating; the induction culture medium for the adventitious bud of the moringa oleifera tetraploid comprises: each liter contains 6-BA1.2mg, NAA0.05mg, sucrose 30g and agar 4.5g, the rest is MS culture medium, pH5.8-6.0;
e. rooting culture of the moringa tetraploid:
cutting off adventitious buds obtained by regeneration of the moringa oleifera tetraploid, inserting the adventitious buds into a culture medium to a depth of about 0.5cm, inoculating the adventitious buds into an adventitious root induction culture medium of the moringa oleifera tetraploid for culture, and inducing the moringa oleifera tetraploid to root to form a moringa oleifera tetraploid plant; the induction culture medium for the adventitious roots of the moringa oleifera tetraploid is as follows: contains NAA 0.1mg, sucrose 30g and agar 4.5g per liter, and MS culture medium in balance, and has pH of 5.8-6.0.
Preferably, the culture conditions in steps a, c, d and e are as follows: the temperature is 26 ℃, the illumination intensity is 2000lx, and the illumination time is 12 h/d.
Preferably, the MS solid culture medium of step c contains 30g of sucrose and 4.5g of agar per liter, and the balance is MS culture medium, and the pH value is 5.8-6.0.
The invention relates to a method for inducing and propagating a horseradish tree (Moringa oleifera Lam.) tetraploid, which takes horseradish tree leaves as an explant material, treats the horseradish tree explant with colchicine, optimizes various conditions influencing the regeneration of the horseradish tree explant and the induction of polyploids by the colchicine, achieves 25% of tetraploid obtaining rate, 86.67% of regeneration efficiency and 93.3% of rooting rate, and has remarkable advantages. According to the invention, the moringa oleifera tetraploid is obtained by inducing moringa oleifera explant leaves with colchicine, so that the quality of the moringa oleifera is improved, a high-quality moringa oleifera tetraploid variety is cultivated, the resource requirement of moringa oleifera germplasm is met, and a technical method and a theoretical basis are provided for improving the quality of the moringa oleifera by a polyploid breeding means.
Drawings
FIG. 1 is a diploid (2n ═ 28) Moringa oleifera root tip chromosome;
fig. 2 is a tetraploid (4 n-56) moringa root tip chromosome.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1
The method of the present invention is specifically described below in connection with tetraploid induction and expansion of Moringa oleifera (Moringa oleifera Lam.) M-2 clone.
Preparation of main reagents:
kabaoyinhong dye liquor: preparing a stock solution A (3 g of basic fuchsin is dissolved in 70% ethanol aqueous solution with volume fraction and the volume is fixed to 100mL), a stock solution B (10 mL of the stock solution A is added into 5% carbolic acid aqueous solution and the volume is fixed to 90mL), and a stock solution C (55 mL of the stock solution B is added with 6mL of glacial acetic acid and 6mL of formalin), wherein the stock solution A and the stock solution C can be stored for a long time, and the stock solution B is used up within two weeks generally. And adding 80-90mL of acetic acid aqueous solution with volume fraction of 45% into the stock solution C10-20mL, and adding 1.8g of sorbitol to prepare a 10-20% Kabao fuchsin solution. The solution is preferably used after two weeks, and the immediate use of the solution results in poor staining.
Induction and propagation of moringa oleifera tetraploid
1. Pre-culturing leaf explants:
selecting sterile young leaves of Moringa oleifera seedling (i.e. leaves at the second development stage, single leaflet about 0.5 cm)3Left and right size) as an explant, on a clean bench, marking a tiny wound on a leaflet paraxial surface by using a sterile scalpel blade, and placing the paraxial surface downwards on an induction culture medium for culture under the culture conditions: the temperature is 26 ℃, the illumination intensity is 2000lx, the illumination time is 12h/d, the moringa oleifera leaf explants are pre-cultured for different days (0d, 3d, 6d and 9d), the influence of different pre-culture durations on regeneration of the moringa oleifera leaf explants and chromosome doubling of the regenerated seedlings is analyzed, and the results are respectively shown in tables 1 and 2. Induction medium used: each liter contains 6-BA0.8mg, KT 0.2mg, NAA0.05mg, sucrose 30g, agar 4.5g and the balance of MS culture medium, and the pH is adjusted to 5.8-6.0; is prepared by mixing the above materials, and sterilizing at 121 deg.C and 1.1Mpa for 15 min.
TABLE 1 Effect of Pre-incubation time on regeneration of Moringa leaf explants
The same letter after each column number in the table indicates no significant difference in Duncan multiplex experiments (P <0.05)
TABLE 2 Effect of Pre-culture time on chromosome doubling of regeneration seedlings of Moringa leaf explants
The same letter after each column number in the table indicates no significant difference in Duncan multiplex experiments (P <0.05)
2. And (3) colchicine induction culture:
transferring the pre-cultured moringa leaf explants to a colchicine induction culture medium (containing colchicine with the final concentration of 100 mg/L), carrying out dark culture treatment at 26 ℃ for different times (0d, 7d, 14d, 21d and 28d), transferring the explants to the induction culture medium for continuous culture after the treatment is finished, and analyzing the influence of different colchicine treatment durations on regeneration of the moringa leaf explants and chromosome doubling of regenerated seedlings, wherein the results are respectively shown in tables 3 and 4. Because colchicine is easy to decompose after high-temperature and high-pressure sterilization, the colchicine is sterilized by adopting a filtration mode. Sterilizing an induction culture medium solution (containing 0.8mg of 6-BA, 0.2mg of KT, 0.05mg of NAA0.05mg, 30g of sucrose, 4.5g of agar and the balance of MS culture medium, wherein the pH value is 5.8-6.0) at 121 ℃ and 1.1Mpa for 15min, and adding a colchicine aqueous solution mother solution which is filtered and sterilized by a 0.45 mu m bacterial filter when the temperature is reduced to 50-60 ℃ so that the final concentration of the colchicine in the culture medium is 100mg/L to obtain the colchicine induction culture medium.
TABLE Effect of colchicine treatment 3100 mg/L on regeneration of Moringa leaf explants
The same letter after each column number in the table indicates no significant difference in Duncan multiplex experiments (P <0.05)
TABLE 4100 mg/L colchicine treatment Effect on Moringa leaf explant regenerated plantlet chromosome doubling at different times
The same letter after each column number in the table indicates no significant difference in Duncan multiplex experiments (P <0.05)
Moringa root tip chromosome counting: when the adventitious bud generated by the leaf blade after colchicine induction culture grows to about 2cm, cutting the adventitious bud and inoculating the cut adventitious bud into an MS solid culture medium (containing 30g of sucrose and 4.5g of agar per liter, and the balance being the MS culture medium, the pH value is 5.8-6.0; the components are mixed uniformly and sterilized for 15min at the temperature of 121 ℃ and under the pressure of 1.1 Mpa) for culture, and the culture conditions are as follows: the temperature is 26 ℃, the illumination intensity is 2000lx, and the illumination time is 12 h/d. When the roots of the regenerated plants in the tissue culture bottle grow to about 2cm, the roots are taken and put into a centrifuge tube filled with purified water (the purified water is used for submerging materials) before and after 11 am, and then the centrifuge tube filled with the materials is quickly placed on ice for pretreatment for 6 hours. After the pretreatment is finished, the root tip is put into Carnoy's stationary liquid (V)95% ethanol：VGlacial acetic acid3: 1) and (5) fixing for 5-24h at room temperature. After the fixation is finished, taking out the root tip, putting the root tip into 1mol/L HCl solution, and carrying out acidolysis in water bath at 60 ℃ for 10-15 min. And (4) washing the root tip subjected to acidolysis under running water for 20min, and washing the root tip with hydrochloric acid, wherein the later chromosome coloring is influenced otherwise. The rinsed root tip is hypotonic for 5min in pure water. Then, the root tip is placed on a clean glass slide, the white part of the root tip is cut by a blade to be 0.5-1mm, and the white part is dripped with Kabaohuang to be dyed for 20 min. The coverslip was then covered and gently tapped to disperse the cells. Finally, the counts were observed under a microscope.
Propagation culture of the moringa tetraploid: cutting the obtained stem nodes of the moringa oleifera tetraploid to 1-2cm in length by using a sharp surgical blade in a super clean workbench to be used as an explant, horizontally inoculating the explant in moringa oleifera tetraploid adventitious bud induction culture media (each liter contains 6-BA1, NAA0.05mg, sucrose 30g and agar 4.5g with corresponding concentrations, and the balance is MS culture media with pH of 5.8-6.0) containing different 6-BA concentrations (0.4, 0.8, 1.2, 1.6mg/L) and NAA of 0.05mg/L, and uniformly mixing the components, sterilizing the mixture for 15min at the temperature of 121 ℃ and the pressure of 1.1Mpa, and culturing, inducing and regenerating the obtained product. The effect of different 6-BA concentrations on regeneration of moringa tetraploids was analyzed and the results are shown in Table 5. The culture conditions are as follows: the temperature is 26 ℃, the illumination intensity is 2000lx, the illumination time is 12h/d, and the culture is carried out for 30 days.
TABLE 56 Effect of BA concentration on regeneration of Moringa tetraploids
The same letter after each column number in the table indicates no significant difference in Duncan multiplex experiments (P <0.05)
Rooting culture of the moringa tetraploid: cutting adventitious buds obtained by regeneration of the moringa tetraploid from a parent tissue by using a sharp surgical blade in an ultraclean workbench, inserting the adventitious buds into a culture medium to the depth of about 0.5cm, inoculating the buds into culture media (containing NAA, sucrose and agar with corresponding concentrations of 30g and 4.5g per liter, and the balance being MS culture medium, and the pH value of the culture media is 5.8-6.0) containing NAA (0, 0.05, 0.1 and 0.15mg/L) with different concentrations, uniformly mixing the components, sterilizing the mixture at the temperature of 121 ℃ and under the pressure of 1.1MPa for 15min, and allowing the adventitious buds to root to induce the buds to form the moringa tetraploid plant. The effect of NAA concentration on rooting of moringa tetraploids is shown in table 6. The culture conditions are as follows: the temperature is 26 ℃, the illumination intensity is 2000lx, and the illumination time is 12 h/d.
TABLE 6 influence of NAA concentration on rooting of tetraploid Moringa oleifera
The same letter after each column number in the table indicates no significant difference in Duncan multiplex experiments (P <0.05)
Second, result analysis
The regeneration efficiency of the explant can be effectively improved by pre-culturing the explant before colchicine induction, the regeneration efficiency and the tetraploid inductivity of the explant are comprehensively considered, so that the pre-culturing effect is the best 3d, under the condition, the regeneration efficiency of the explant is 33.33%, and the tetraploid inductivity is 20%. In addition, the influence of colchicine treatment time on explant regeneration efficiency and moringa tetraploid inductivity is great, the best treatment is carried out for 21 days by 100mg/L colchicine, and the tetraploid yield reaches 25%.
Taking the obtained moringa oleifera tetraploid stem nodes as an explant material, wherein the optimal moringa oleifera tetraploid adventitious bud induction culture medium comprises the following components: each liter contains 1.2mg of 6-BA, 0.05mg of NAA, 30g of sucrose and 4.5g of agar, the balance is MS culture medium, and the pH value is 5.8-6.0; the regeneration efficiency reaches 86.67%, and each explant can obtain 2.87 adventitious buds on average; the optimal induction culture medium for adventitious roots of the moringa oleifera tetraploid is as follows: each liter contains NAA 0.1mg, sucrose 30g and agar 4.5g, the rest is MS culture medium, pH is 5.8-6.0, and rooting rate reaches 93.3%.
Claims (3)
1. A method for inducing and propagating moringa oleifera tetraploids is characterized by comprising the following steps:
a. pre-culturing leaf explants:
cutting sterile young leaves of moringa oleifera seedlings to serve as explants, after small wounds are scratched on the paraxial surfaces of the young leaves, placing the paraxial surfaces downwards to be inoculated on an induction culture medium for pre-culture for 3 days, wherein the induction culture medium comprises the following components: each liter contains 0.8mg of 6-BA, 0.2mg of KT, 0.05mg of NAA0.05mg, 30g of sucrose and 4.5g of agar, the balance is MS culture medium, and the pH value is 5.8-6.0;
b. and (3) colchicine induction culture:
transferring the pre-cultured explant to a colchicine induction culture medium for dark culture for 21d, wherein the culture temperature is 26 ℃; the colchicine induction culture medium comprises: each liter contains colchicine 100mg, 6-BA0.8 mg, KT 0.2mg, NAA0.05mg, sucrose 30g and agar 4.5g, the rest is MS culture medium, pH 5.8-6.0;
c. moringa root tip chromosome counting:
cutting off adventitious bud strains subjected to colchicine induced culture to an MS solid culture medium for culture, and taking root tips for chromosome counting when the regenerated roots grow to about 2cm, and identifying the moringa oleifera tetraploid;
d. propagation culture of the moringa tetraploid:
cutting obtained moringa tetraploid stem nodes to the length of 1-2cm to be used as an explant, horizontally inoculating the explant in a moringa tetraploid adventitious bud induction culture medium, and inducing and regenerating; the induction culture medium for the adventitious bud of the moringa oleifera tetraploid comprises: each liter contains 1.2mg of 6-BA, 0.05mg of NAA, 30g of sucrose and 4.5g of agar, the balance is MS culture medium, and the pH value is 5.8-6.0;
e. rooting culture of the moringa tetraploid:
cutting off adventitious buds obtained by regeneration of the moringa oleifera tetraploid, inserting the adventitious buds into a culture medium to a depth of about 0.5cm, inoculating the adventitious buds into an adventitious root induction culture medium of the moringa oleifera tetraploid for culture, and inducing the moringa oleifera tetraploid to root to form a moringa oleifera tetraploid plant; the induction culture medium for the adventitious roots of the moringa oleifera tetraploid is as follows: contains NAA 0.1mg, sucrose 30g and agar 4.5g per liter, and MS culture medium in balance, and has pH of 5.8-6.0.
2. The method according to claim 1, wherein the culture conditions in steps a, c, d, e are: the temperature is 26 ℃, the illumination intensity is 2000lx, and the illumination time is 12 h/d.
3. The method of claim 1, wherein the MS solid medium of step c comprises 30g of sucrose and 4.5g of agar per liter, and the balance is MS medium, pH 5.8-6.0.
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| US11800842B2 (en) * | 2021-12-14 | 2023-10-31 | Tropical Crops Genetic Resources Institute, Chinese Academy Of Tropical Agricultural Sciences | Method for obtaining adventitious tetraploid bud of Blumea balsamifera |
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