Method for inducing generation of pear polyploidy through colchicine
By inducing young pear tree tissues with colchicine and combining it with specific culture media and hormone ratios, the problem of chromosome doubling in pear tree breeding was solved, achieving efficient induction and survival of polyploid pear trees and providing a new pathway for genetic variation.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JIANGSU ACAD OF AGRI SCI
- Filing Date
- 2024-01-31
- Publication Date
- 2026-04-24
AI Technical Summary
Conventional hybridization in pear breeding is unlikely to produce groundbreaking varieties. Polyploid breeding can provide new genetic variations and superior germplasm, but existing technologies are insufficient to efficiently induce chromosome doubling in pear trees.
Colchicine was used to induce polyploidy in young pear tissues. By combining different concentrations and durations of culture media and hormone ratios, polyploid chromosomes were screened using flow cytometry and sectioning. Pear tissues were cultured under specific conditions to induce polyploidy.
It improved the success rate and survival rate of pear polyploid production, met the needs of creating pear polyploid materials, and provided a higher possibility of genetic variation.
Abstract
Description
Technical Field
[0001] This invention relates to the field of pear breeding technology, specifically to a method for inducing polyploid production in pears using colchicine. Background Technology
[0002] Pear trees are widely distributed in my country, ranking as the country's third largest fruit tree. Most pear trees are diploid, making it difficult to produce groundbreaking varieties through conventional hybridization. Polyploid breeding technology, a recent hot topic in fruit tree breeding, involves controlling the number of chromosomes in fruit trees based on artificial mutagenesis and natural variation to create triploid and tetraploid breeding materials, serving as a crucial means for germplasm innovation. Polyploid breeding differs from conventional breeding in that the resulting fruit trees have different chromosome numbers and gene expression patterns, potentially leading to more novel genetic variations. Compared to conventional varieties, polyploid varieties exhibit larger fruits or vegetative organs, improved nutritional content and quality, and enhanced environmental adaptability. Polyploid pear breeding is a vital pathway for the innovation of superior pear germplasm and the selection of groundbreaking varieties, providing a new technological approach for high-yield and high-quality pear breeding. Summary of the Invention
[0003] Therefore, the present invention provides a method for inducing polyploidy in pears by colchicine, in order to solve the above-mentioned problems in the prior art.
[0004] To achieve the above objectives, the present invention provides the following technical solution: According to a first aspect of the present invention, a method for inducing polyploidy in pears by colchicine includes the following steps: Step 1: Plant Acquisition. The pear variety selected is the early-maturing sand pear 'Sucui No. 1'. In April or May, tender tissues are cut from mature pear trees, and the cut tissues are disinfected. Step two, plant culture: Before culturing the excised tissue, the tissue is sterilized using mercuric chloride for 3-6 minutes. The sterilized tissue is then placed on a sterile culture medium for further culture. Step 3: Induction of differentiation. Take tissues that have been sterilely cultured for 5-10 days and have survived, rinse them with distilled water, and then place them in a solution containing colchicine for further culture. Step 4, plant culture: The colchicine-induced tissues were placed on MS medium and cultured at a temperature of 25±2℃ and a pH of 5.8.
[0005] Furthermore, the tissues of the pear tree include leaves, stem tips, or terminal buds. When cutting leaves, take tender leaf tissue in April or May. When cutting stem tips, take the stem tip 1 cm from the end of the newly growing stem. When cutting terminal buds, take the tender terminal buds.
[0006] Furthermore, when the pear tree tissue was in the form of leaves, it was cultured using a solution containing 0.15% of the solution for 25-35 hours. The culture medium used after culture was MS + 0.1 mg / L 6-BA + 2.5 mg / L IIA + 0.15 mg / L 2,4-D + 0.1 mg / L ZT + 30 g / L sugar + 7 g / L agar powder + 1 g / L activated carbon.
[0007] Furthermore, when the pear tree tissue was at the shoot tip, it was cultured using a solution containing 0.18% of the solution for 25-35 hours. The culture medium used after culture was MS + 0.2 mg / L 6-BA + 2 mg / L IIA + 0.15 mg / L 2,4-D + 0.25 mg / L ZT + 30 g / L sugar + 7 g / L agar powder + 1 g / L activated carbon.
[0008] Furthermore, when the pear tree tissue is the terminal bud, the pear tree tissue is cultured using a solution containing 0.2% for 25-35 hours. The culture medium used after culture is MS + 0.15 mg / L 6-BA + 1 mg / L LIAAA + 0.15 mg / L 2,4-D + 0.3 mg / L ZT + 30 g / L sugar + 7 g / L agar powder + 1 g / L activated carbon.
[0009] Furthermore, flow cytometry and / or sectioning were used to analyze pear tissue cultured at different colchicine concentrations to screen for the concentration at which polyploid chromosomes could be obtained from pear tissue.
[0010] Furthermore, the concentration of colchicine was 0.15% when the pear tree tissue was in the leaf form, 0.18% when the pear tree tissue was in the stem tip form, and 0.2% when the pear tree tissue was in the terminal bud form. The pear tree tissue was then soaked at a temperature of 12°C.
[0011] Furthermore, pear tree tissues were cultured on MS medium for 30 days under a light intensity of 1500-3000 lux and a daily light duration of 8-12 hours. After the culture was completed, the rooted plants were transferred to soil for further cultivation.
[0012] Furthermore, during the pear tissue culture on MS medium, each 10-day period was divided into stages. In the first 10 days, the temperature was set at 18°C, the light intensity was 1500 lux, and the light duration was 8 hours. In the second 10 days, the light intensity was 2000 lux and the light duration was 10 hours. In the third 10 days, the light intensity was 3000 lux and the light duration was 12 hours.
[0013] The present invention has the following advantages: In implementing the technical solution of this application, different concentrations of colchicine are used to induce different pear tree tissues to obtain pear tree tissues with successfully doubled chromosomes. Furthermore, by selecting and limiting the culture medium and the content of various hormones during the cultivation process, the production of polyploids in pear tree tissues is improved, thus satisfying people's purpose of creating pear tree polyploid materials. Detailed Implementation
[0014] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example
[0015] The following method was used to induce differentiation in young leaf tissues during April and May: A method for inducing polyploidy in pears using colchicine includes the following steps: Step 1: Plant Acquisition. The pear variety selected is the early-maturing sand pear 'Sucui No. 1'. In April or May, tender tissues are cut from mature pear trees, and the cut tissues are disinfected. Step two, plant culture: Before culturing the excised tissue, the tissue is sterilized using mercuric chloride for 3-6 minutes. The sterilized tissue is then placed on a sterile culture medium for further culture. Step 3: Induction of differentiation. Take tissues that have been sterilely cultured for 5-10 days and have survived, rinse them with distilled water, and then place them in a solution containing colchicine for further culture. Step 4, plant culture: The induced differentiation tissues are placed on MS medium and cultured at a temperature of 25±2℃ and a pH of 5.8.
[0016] Experimental Group 1: Pear tree tissues were cultured using a solution containing 0.15% colchicine for 25 hours. The culture medium used after culture was MS + 0.1 mg / L 6-BA + 2.5 mg / L IIAAA + 0.15 mg / L 2,4-D + 0.1 mg / L ZT + 30 g / L sugar + 7 g / L agar powder + 1 g / L activated carbon.
[0017] The second experimental group: pear tree tissues were cultured using a solution containing 0.15% colchicine for 30 hours. The culture medium used after culture was MS + 0.1 mg / L 6-BA + 2.5 mg / L IIAAA + 0.15 mg / L 2,4-D + 0.1 mg / L ZT + 30 g / L sugar + 7 g / L agar powder + 1 g / L activated carbon.
[0018] The third experimental group: pear tree tissues were cultured using a solution containing 0.15% colchicine for 35 hours. The culture medium used after culture was MS + 0.1 mg / L 6-BA + 2.5 mg / L IIAAA + 0.15 mg / L 2,4-D + 0.1 mg / L ZT + 30 g / L sugar + 7 g / L agar powder + 1 g / L activated carbon.
[0019] Experimental Group 4: Pear tree tissues were cultured using a solution containing 0.1% colchicine for 30 hours. The culture medium used after culture was MS medium containing 0.1 mg / L 6-BA, 2.5 mg / L IIAAA, 0.15 mg / L 2,4-D, 0.1 mg / L ZT, 30 g / L sugar, 7 g / L agar powder, and 1 g / L activated carbon. Experimental Group 5: Pear tree tissues were cultured using a solution containing 0.2% colchicine for 30 hours. The culture medium used after culture was MS + 0.1 mg / L 6-BA + 2.5 mg / L IIAAA + 0.15 mg / L 2,4-D + 0.1 mg / L ZT + 30 g / L sugar + 7.7 g / L agar powder + 1 g / L activated carbon.
[0020] The pear tree tissues cultured at different colchicine concentrations were analyzed by flow cytometry and / or sectioning to screen for the concentration at which pear tree tissues yielded a higher number of chromosome polyploids.
[0021] Pear tree tissues were cultured on MS medium for 30 days under a light intensity of 1500-3000 lux and a daily light duration of 8-12 hours. After the culture was completed, the rooted plants were transferred to soil for further cultivation.
[0022] During the pear tissue culture on MS medium, each stage was divided into 10-day periods. In the first 10 days, the temperature was set at 18℃, the light intensity was 1500 lux, and the light duration was 8 hours. In the second 10 days, the light intensity was 2000 lux and the light duration was 10 hours. In the third 10 days, the light intensity was 3000 lux and the light duration was 12 hours.
[0023] The probability of induced polyploidy and the number of pear trees that successfully differentiated from the five experimental groups were statistically analyzed: In the first experimental group, the probability of successfully inducing polyploids was between 18% and 27%, and the proportion of surviving plants that successfully grew into pear trees was between 63% and 77%. In the second experimental group, the probability of successfully inducing polyploids was between 36% and 47%, and the number of surviving plants that successfully grew into pear trees was between 81% and 88%. The probability of successfully inducing polyploids in the third experimental group was between 22% and 32%, and the number of surviving plants that successfully grew into pear trees was between 54% and 62%. The fourth experimental group: the probability of successfully inducing polyploids was between 8% and 14%, and the number of surviving plants that successfully grew into pear trees was between 22% and 36%. The fifth experimental group showed that the probability of successfully inducing polyploids was between 11% and 21%, and the number of surviving plants that successfully grew into pear trees was between 11% and 29%. Based on the above five sets of experimental data, it can be concluded that culturing pear leaves with a solution containing 0.15% colchicine for 25-35 hours yields the highest probability of successfully inducing polyploidy in pear leaf tissue. Example
[0024] When inducing shoot tip differentiation in pear trees, take a 1 cm section of the shoot tip from the end of a newly grown stem and use the following method: A method for inducing polyploidy in pears using colchicine includes the following steps: Step 1: Plant Acquisition. The pear variety selected is the early-maturing sand pear 'Sucui No. 1'. In April or May, tender tissues are cut from mature pear trees, and the cut tissues are disinfected. Step two, plant culture: Before culturing the excised tissue, the tissue is sterilized using mercuric chloride for 3-6 minutes. The sterilized tissue is then placed on a sterile culture medium for further culture. Step 3: Induction of differentiation. Take tissues that have been sterilely cultured for 5-10 days and have survived, rinse them with distilled water, and then place them in a solution containing colchicine for further culture. Step 4, plant culture: The induced differentiation tissues are placed on MS medium and cultured at a temperature of 25±2℃ and a pH of 5.8.
[0025] The first group of experiments: pear tree tissues were cultured using a solution containing 0.18% colchicine for 25 hours. The culture medium used after culture was MS + 0.2 mg / L 6-BA + 2 mg / L IIAAA + 0.15 mg / L 2,4-D + 0.25 mg / L ZT + 30 g / L sugar + 7.7 g / L agar powder + 1 g / L activated carbon.
[0026] The second group of experiments: pear tree tissues were cultured using a solution containing 0.18% colchicine for 30 hours. The culture medium used after culture was MS + 0.2 mg / L 6-BA + 2 mg / L IIAAA + 0.15 mg / L 2,4-D + 0.25 mg / L ZT + 30 g / L sugar + 7 g / L agar powder + 1 g / L activated carbon.
[0027] The third group of experiments: pear tree tissues were cultured using a solution containing 0.18% colchicine for 35 hours. The culture medium used after culture was MS + 0.2 mg / L 6-BA + 2 mg / L IIAAA + 0.15 mg / L 2,4-D + 0.25 mg / L ZT + 30 g / L sugar + 7 g / L agar powder + 1 g / L activated carbon.
[0028] The fourth experiment: pear tree tissue was cultured using a solution containing 0.15% colchicine for 30 hours. The culture medium used after culture was MS + 0.2 mg / L 6-BA + 2 mg / L IIAAA + 0.15 mg / L 2,4-D + 0.25 mg / L ZT + 30 g / L sugar + 7 g / L agar powder + 1 g / L activated carbon.
[0029] The fifth experiment: pear tree tissue was cultured using a solution containing 0.2% colchicine for 30 hours. The culture medium used after culture was MS + 0.2 mg / L 6-BA + 2 mg / L IAA + 0.15 mg / L 2,4-D + 0.25 mg / L ZT + 30 g / L sugar + 7.7 g / L agar powder + 1 g / L activated carbon.
[0030] Chromosome doubling in pear tissue was determined using flow cytometry and / or sectioning, and the pear tissue was immersed in colchicine solution at 12°C.
[0031] Pear tree tissues were cultured on MS medium for 30 days under a light intensity of 1500-3000 lux and a daily light duration of 8-12 hours. After the culture was completed, the rooted plants were transferred to soil for further cultivation.
[0032] During the pear tissue culture on MS medium, each stage was divided into 10-day periods. In the first 10 days, the temperature was set at 18℃, the light intensity was 1500 lux, and the light duration was 8 hours. In the second 10 days, the light intensity was 2000 lux and the light duration was 10 hours. In the third 10 days, the light intensity was 3000 lux and the light duration was 12 hours.
[0033] The survival rate of plants and the number of pear trees that successfully differentiated from the five experimental groups were statistically analyzed: In the first experimental group, the probability of successfully inducing polyploids was between 19% and 31%, and the proportion of surviving plants that successfully grew into pear trees was between 55% and 72%. In the second experimental group, the probability of successfully inducing polyploids was between 33% and 51%, and the number of surviving plants that successfully grew into pear trees was between 74% and 83%. The probability of successfully inducing polyploids in the third experimental group was between 26% and 41%, and the number of surviving plants that successfully grew into pear trees was between 54% and 68%. The fourth experimental group: the probability of successfully inducing polyploids was between 11% and 18%, and the number of surviving plants that successfully grew into pear trees was between 21% and 35%. The fifth experimental group showed that the probability of successfully inducing polyploids was between 11% and 21%, and the number of surviving plants that successfully grew into pear trees was between 9% and 25%. Based on the above five sets of experimental data, it can be concluded that the highest probability of successfully inducing polyploids in pear shoot tip tissue is achieved by culturing pear shoot tips with a solution containing 0.18% colchicine for 25-35 hours. Example
[0034] When inducing differentiation of the terminal buds of pear trees, the newly grown tender terminal buds are treated using the following method: A method for inducing polyploidy in pears using colchicine includes the following steps: Step 1: Plant Acquisition. The pear variety selected is the early-maturing sand pear cultivar 'Sucui No. 1'. In April or May, tender tissues are cut from mature pear trees, and the cut tissues are then processed. Step two, plant culture: Before culturing the excised tissue, the tissue is sterilized using mercuric chloride for 3-6 minutes. The sterilized tissue is then placed on a sterile culture medium for further culture. Step 3: Induction of differentiation. Take tissues that have been sterilely cultured for 5-10 days and have survived, rinse them with distilled water, and then place them in a solution containing colchicine for further culture. Step 4, plant culture: The induced differentiation tissues are placed on MS medium and cultured at a temperature of 25±2℃ and a pH of 5.8.
[0035] The first group of experiments: pear tree tissues were cultured using a solution containing 0.2% colchicine for 25 hours. The culture medium used after culture was MS + 0.15 mg / L 6-BA + 1 mg / L IIAAA + 0.15 mg / L 2,4-D + 0.3 mg / L ZT + 30 g / L sugar + 7 g / L agar powder + 1 g / L activated carbon.
[0036] The second group of experiments: pear tree tissues were cultured using a solution containing 0.2% colchicine for 30 hours. The culture medium used after culture was MS + 0.15 mg / L 6-BA + 1 mg / L IIAAA + 0.15 mg / L 2,4-D + 0.3 mg / L ZT + 30 g / L sugar + 7 g / L agar powder + 1 g / L activated carbon.
[0037] The third group of experiments: pear tree tissues were cultured using a medium containing 0.2% colchicine for 35 hours. The culture medium used after culture was MS + 0.15 mg / L 6-BA + 1 mg / L LIAAA + 0.15 mg / L 2,4-D + 0.3 mg / L ZT + 30 g / L sugar + 7.7 g / L agar powder + 1 g / L activated carbon.
[0038] The fourth experiment: pear tree tissues were cultured using a medium containing 0.15% colchicine for 30 hours. The culture medium used after culture was MS + 0.15 mg / L 6-BA + 1 mg / L IIAAA + 0.15 mg / L 2,4-D + 0.3 mg / L ZT + 30 g / L sugar + 7 g / L agar powder + 1 g / L activated carbon.
[0039] The fifth experiment: pear tree tissues were cultured using a medium containing 0.25% colchicine for 30 hours. The culture medium used after culture was MS + 0.15 mg / L 6-BA + 1 mg / L IIA + 0.15 mg / L 2,4-D + 0.3 mg / L ZT + 30 g / L sugar + 7 g / L agar powder + 1 g / L activated carbon.
[0040] Chromosome doubling in pear tissue was determined using flow cytometry and / or sectioning, and the pear tissue was immersed in colchicine solution at 12°C.
[0041] Pear tree tissues were cultured on MS medium for 30 days under a light intensity of 1500-3000 lux and a daily light duration of 8-12 hours. After the culture was completed, the rooted plants were transferred to soil for further cultivation.
[0042] During the pear tissue culture on MS medium, each stage was divided into 10-day periods. In the first 10 days, the temperature was set at 18℃, the light intensity was 1500 lux, and the light duration was 8 hours. In the second 10 days, the light intensity was 2000 lux and the light duration was 10 hours. In the third 10 days, the light intensity was 3000 lux and the light duration was 12 hours.
[0043] The survival rate of plants and the number of pear trees that successfully differentiated from the five experimental groups were statistically analyzed: In the first experimental group, the probability of successfully inducing polyploids was between 33% and 41%, and the proportion of surviving plants that successfully grew into pear trees was between 63% and 77%. In the second experimental group, the probability of successfully inducing polyploids was between 28% and 45%, and the number of surviving plants that successfully grew into pear trees was between 81% and 88%. The third experimental group: the probability of successfully inducing polyploids was between 36% and 65%, and the number of surviving plants that successfully grew into pear trees was between 54% and 62%. The fourth experimental group showed that the probability of successfully inducing polyploids was between 11% and 18%, and the number of surviving plants that successfully grew into pear trees was between 15% and 27%. The fifth experimental group: the probability of successfully inducing polyploids was between 15% and 23%, and the number of surviving plants that successfully grew into pear trees was between 7% and 20%. Based on the above five sets of experimental data, it can be concluded that the highest probability of successfully inducing polyploids in pear tree terminal buds is achieved by culturing pear tree terminal buds in a medium containing 0.20% colchicine for 25-35 hours.
Claims
1. A method for inducing polyploidy in pears by colchicine, characterized in that, Includes the following steps, Step 1: Plant Acquisition. The pear variety selected is the early-maturing sand pear new variety 'Sucui No. 1'. In April or May, tender tissues are cut from mature pear trees, and the cut tissues are disinfected. Step two, plant culture: Before culturing the excised tissue, the tissue is sterilized using mercuric chloride for 3-6 minutes. The sterilized tissue is then placed on a sterile culture medium for further culture. Step 3: Induction of differentiation. Take tissues that have been sterilely cultured for 5-10 days and have survived, rinse them with distilled water, and then place them in a solution containing colchicine for further culture. Step 4, plant culture: The colchicine-induced tissues were placed on MS medium and cultured at a temperature of 25±2℃ and a pH of 5.
8.
2. The method for inducing polyploidy in pears by colchicine according to claim 1, characterized in that, The tissues of a pear tree include leaves, stem tips, or terminal buds. When cutting leaves, take tender leaf tissue in April or May. When cutting stem tips, take the stem tip 1 cm from the end of the newly growing stem. When cutting terminal buds, take the tender terminal buds.
3. A method for inducing polyploidy in pears by colchicine according to claim 2, characterized in that, When the pear tree tissue is in the form of leaves, it is cultured using a solution containing 0.15% colchicine for 25-35 hours. The culture medium used after culture consists of MS + 0.1 mg / L 6-BA + 2.5 mg / L L1AA + 0.15 mg / L 2,4-D + 0.1 mg / L ZT + 30 g / L sugar + 7 g / L agar powder + 1 g / L activated carbon.
4. The method for inducing polyploidy in pears by colchicine according to claim 2, characterized in that, When the pear tree tissue is at the shoot tip, it is cultured using a solution containing 0.18% colchicine for 25-35 hours. The culture medium used after culture consists of MS + 0.2 mg / L 6-BA + 2 mg / L IIA + 0.15 mg / L 2,4-D + 0.25 mg / L ZT + 30 g / L sugar + 7 g / L agar powder + 1 g / L activated carbon.
5. The method for inducing polyploidy in pears by colchicine according to claim 2, characterized in that, When the pear tree tissue is the terminal bud, it is cultured with a solution containing 0.2% colchicine for 25-35 hours. The culture medium used after culture is MS + 0.15 mg / L 6-BA + 1 mg / L LIAAA + 0.15 mg / L 2,4-D + 0.3 mg / L ZT + 30 g / L sugar + 7 g / L agar powder + 1 g / L activated carbon.
6. The method for inducing polyploidy in pears by colchicine according to claim 2, characterized in that, The pear tree tissues cultured at different colchicine concentrations were analyzed by flow cytometry and / or sectioning to screen for the concentration at which pear tree tissues yielded a higher number of chromosome polyploids.
7. A method for inducing polyploidy in pears by colchicine according to claim 6, characterized in that, The concentration of colchicine was 0.15% when the pear tree tissue was in the leaf stage, 0.18% when the pear tree tissue was in the stem tip stage, and 0.2% when the pear tree tissue was in the terminal bud stage. The pear tree tissue was soaked at a temperature of 12°C.
8. A method for inducing polyploidy in pears by colchicine according to claim 3, 4, or 5, characterized in that, Pear tree tissues were cultured on MS medium for 30 days under a light intensity of 1500-3000 lux and a daily light duration of 8-12 hours. After the culture was completed, the rooted plants were transferred to soil for further cultivation.
9. A method for inducing polyploidy in pears by colchicine according to claim 8, characterized in that, During the pear tissue culture on MS medium, each 10-day period was divided into stages. In the first 10 days, the temperature was set at 18℃, the light intensity was 1500 lux, and the light duration was 8 hours. In the second 10 days, the light intensity was 2000 lux and the light duration was 10 hours. In the third 10 days, the light intensity was 3000 lux and the light duration was 12 hours.