A method for creating polyploid germplasm of non-ex vitro buds of Idesia polycarpa

Abstract

The invention discloses a method for creating polyploid germplasm of non-explant buds of Idesia polycarpa, belonging to the field of plant breeding. By directly treating the buds on the Idesia polycarpa plant with a pendimethalin aqueous solution, polyploid shoot germplasm can be directly produced. By inducing polyploids from non-explant buds of Idesia polycarpa, polyploid germplasm can be obtained in the year of induction without further isolation and purification. The polyploid mutagenesis rate of Idesia polycarpa can reach 52.32%, and the damage to the tree body is relatively small. The shoots of the new ploidy germplasm can be completely lignified in the same year and can be used for grafting and propagation in the following year. When this technology is applied to the practice of polyploid breeding of Idesia polycarpa, it is easy to operate, has a high mutagenesis efficiency, can greatly shorten the breeding cycle, and improve the efficiency of polyploid breeding.

Description

Technical Field

[0001] The invention belongs to the field of plant breeding, and in particular relates to a method for creating non-in vitro bud polyploid germplasm of Castanopsis japonici. Background Art

[0002] Idesia polycarpa Maxim., also known as oil grape, water winter melon, red fruit tree, etc., is a deciduous tree of the family Idesia. Idesia polycarpa has multiple advantages such as strong environmental adaptability, wide range of suitable habitats, high ornamental value, good material, high fruit yield, high oil content, good oil quality, and long fruit-bearing period. It has the reputation of "oil depot on tree" and is a woody oil plant with great development prospects. At present, Idesia planting is mostly based on wild or semi-wild mother trees for seed breeding, with messy germplasm and serious alternate years, resulting in unstable yield and poor comprehensive benefits. Therefore, strengthening the innovation of Idesia germplasm resources and cultivating high-quality and high-yield new varieties have become key issues to be urgently solved in the development and utilization of Idesia industry. Germplasm resources are the material basis of breeding, and creating key germplasm resources is the key to cultivating breakthrough varieties. Therefore, studying innovative methods of germplasm resources is of great significance to better utilize Idesia resources and cultivate breakthrough new Idesia varieties.

[0003] The main methods of plant germplasm resource innovation and new variety breeding include seedling breeding, bud mutation breeding, hybrid breeding, mutation breeding, haploid breeding, polyploid breeding, genetic engineering breeding, etc. Polyploid breeding is an important means of innovation of Castanopsis chinensis germplasm resources and new variety breeding. Polyploidy refers to individuals with three or more chromosome sets in somatic cells. The doubling of chromosome sets often brings morphological and physiological changes, such as enlarged organs, increased metabolite content, and increased stress resistance in polyploid plants. The creation of polyploid resources is the core of Castanopsis chinensis polyploid breeding. At present, most plant polyploidy induction is basically the callus induction of seeds, leaves, stem tips, axillary buds, root segments and their organs or tissues under tissue culture conditions to produce polyploids. Although this method can quickly obtain polyploids, these polyploids are mostly chimeras, and the separation and purification cycle is long and the technical difficulty is high; at the same time, there are problems such as severe vitrification, difficulty in seedling formation, and a long period from successful mutagenesis to flowering and fruiting in the field.

[0004] At present, chemical induction is the main method for artificially inducing polyploidy, among which colchicine is the most widely used. However, it is harmful to both human body and test materials, and high concentration will inhibit plant growth and even cause death. Can less toxic chemical reagents be used to replace colchicine? Some studies have reported that dinitroaniline herbicides are less toxic to human body and test materials, and can effectively induce plants at lower concentrations. However, whether this conclusion is feasible for Castanopsis sylvestris remains to be verified. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for creating polyploid germplasm of non-explant buds of Idesia polycarpa, so as to shorten the polyploid breeding cycle.

[0006] The technical solution of the present invention is: a method for creating polyploid germplasm of non-explant buds of Idesia polycarpa, which directly induces the buds on the Idesia polycarpa plant with a pendimethalin aqueous solution to directly produce polyploid shoot germplasm.

[0007] Further, the volume concentration of the pendimethalin aqueous solution is 2-8%, and it contains 0.5% by volume of dimethyl sulfoxide.

[0008] Further, the time of the induction treatment is 24-48 hours.

[0009] Further, the period of the induction treatment is: at the bud sprouting stage of Idesia polycarpa, when the appearance shows that the buds of Idesia polycarpa are significantly swollen to slightly cracked scales, and a small amount of green part can be seen.

[0010] Further, the method of the induction treatment is: wrap the selected buds of Idesia polycarpa separately with absorbent cotton, cover them with plastic film, inject the prepared pendimethalin aqueous solution onto the absorbent cotton wrapped around the buds with a syringe until the absorbent cotton absorbs the induction agent to a saturated state, supplement the liquid medicine 2-3 times a day, and remove the absorbent cotton in time after the induction treatment is completed.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] By inducing polyploid production in non-explant buds of Idesia polycarpa, polyploid germplasm can be obtained in the same year of mutagenesis without further separation and purification. The polyploid mutagenesis rate of Idesia polycarpa can reach 52.32%, and the damage to the tree body is relatively small. The shoots of the new ploidy germplasm can bloom and be completely lignified in the same year, and can be used for grafting and propagation in the spring of the second year. This technology is applied to the polyploid breeding practice of Idesia polycarpa, which is easy to operate and has a high mutagenesis efficiency, can greatly shorten the breeding cycle, and improve the polyploid breeding efficiency. Specific Embodiments

[0013] The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all obtained from commercial channels unless otherwise specified.

[0014] The Idesia polycarpa variety used is the female plant of "Haitong No. 1", which is planted in the Idesia polycarpa cutting orchard of the Yangma Experimental Base of the Chengdu Academy of Agriculture and Forestry Sciences. It was planted in March 2022, and the grafted seedlings of "Haitong No. 1" Idesia polycarpa grew well. The cotton soaking method was used to induce polyploidy in Idesia polycarpa according to the following method.

[0015] The experiment was designed in a randomized block design with 10 plants in each block and 3 replicates. This experiment used the concentration of chemical induction agents (pendimethalin, colchicine) and impregnation duration as factors for a complete experimental design, with a total of 14 treatments (see Table 1). The following steps were carried out for the induction operation of Idesia polycarpa Maxim polyploid:

[0016] Table 1 Experimental design table for polyploid induction of non-in vitro buds of Idesia polycarpa Maxim

[0017]

[0018]

[0019] (1) Induction materials: Select the buds of healthy and disease-free, vigorously growing female plants of Idesia polycarpa Maxim as the induction treatment materials.

[0020] (2) Induction time: During the budding period of Idesia polycarpa Maxim, the appearance shows that the buds of Idesia polycarpa Maxim are significantly swollen to slightly cracked scales, and a small amount of green part can be seen (from March 5th to 10th).

[0021] (3) Selection and preparation of induction agents: Select pendimethalin (330 g / L, emulsifiable concentrate) and colchicine (AR, >99%) as chemical mutagenesis agents respectively, and use dimethyl sulfoxide (AR, ≥99.9%) as a penetration enhancer. The concentrations of the pendimethalin solution are 2%, 4%, and 8% (v / v) respectively, and the concentration of dimethyl sulfoxide is 0.5%. This concentration means adding 2 mL, 4 mL, and 8 mL of pendimethalin emulsifiable concentrate to an appropriate amount of water respectively, then adding 0.5 mL of dimethyl sulfoxide, and making up the volume to 100 mL and mixing well. The concentrations of the colchicine solution are 0.1%, 0.2%, and 0.4% (w / v) respectively, and the concentration of dimethyl sulfoxide is 0.5%. This concentration means dissolving 0.1 g, 0.2 g, and 0.4 g of colchicine in an appropriate amount of water respectively, then adding 0.5 mL of dimethyl sulfoxide, and making up the volume to 100 mL and mixing well. The induction agents are prepared and used immediately. Use distilled water as the control treatment, with dimethyl sulfoxide as the penetration enhancer. This concentration means adding 0.5 mL of dimethyl sulfoxide to an appropriate amount of water and making up the volume to 100 mL and mixing well.

[0022] (4) Impregnating the buds with the induction agent: Wrap the selected buds of Idesia polycarpa Maxim separately with absorbent cotton, with a cotton layer thickness of 3 ± 0.5 mm. After wrapping with plastic wrap, tie it with a rubber band to prevent loosening and falling off. Use a disposable medical syringe to inject the prepared induction agent onto the absorbent cotton wrapped around the buds until the absorbent cotton absorbs the induction agent to a saturated state. Supplement the liquid medicine 2 - 3 times a day to keep the buds of Idesia polycarpa Maxim in an impregnated state for 24 h or 48 h (the duration used for each treatment is shown in Table 1). After the treatment, remove the tied rubber band, plastic wrap, and absorbent cotton in time.

[0023] (5) Ploidy detection: When the length of the germinated branches of the buds to be processed reaches 20 - 30 cm, collect the young leaves of each treatment, and use flow cytometry to detect the chromosome ploidy of Idesia polycarpa Maxim.

[0024] (6) Implementation effect

[0025] The results are shown in Table 2. Using the cotton soaking method, with pendimethalin or colchicine as the induction agent, polyploids of Idesia polycarpa Maxim can be induced at both 24 h and 48 h. Using water as the control treatment does not double the Idesia polycarpa Maxim. From the perspective of obtaining polyploids of Idesia polycarpa Maxim, the induction effect of pendimethalin as the inducer is significantly stronger than that of colchicine. Except for the T1 treatment, the polyploid mutagenesis rate with pendimethalin as the mutagen is significantly higher than that of colchicine (p < 0.05). Especially for the T4 treatment (4.0% pendimethalin, 0.5% dimethyl sulfoxide, treated for 48 hours), the polyploid mutagenesis rate of Idesia polycarpa Maxim can reach 52.32%, and the effect is significant. Further analysis shows that the reason for this result is the huge difference in the survival rate of the induced materials between the two. Colchicine has a certain toxicity to the tested materials, and too high a concentration or too long application time will inhibit plant growth and even cause its death. As can be seen from Table 2, the survival rate of the buds of Idesia polycarpa Maxim with pendimethalin as the mutagen is significantly higher than that of colchicine (p < 0.05), and the survival rate of the buds of Idesia polycarpa Maxim in the treatment with a lower concentration of pendimethalin (T1, T2, T3, T4) is above 98%.

[0026] Table 2 Induction effect of polyploid induction of Idesia polycarpa Maxim by cotton soaking method

[0027] Serial number Processing code Survival rate % Polyploid mutagenesis rate % 1 CK1 100±0a 0±0h 2 CK2 100±0a 0±0h 3 T1 100±0a 19.44±1.36f 4 T2 100±0a 26.37±1.16d 5 T3 100±0a 31.14±2.86c 6 T4 98.83±0.98b 52.32±4.98a 7 T5 95.66±1.04c 43.94±3.22b 8 T6 94.1±1.15c 41.63±3.46b 9 T7 86.94±2.36d 11.12±1.31g 10 T8 79.19±1.97e 12.62±0.92g 11 T9 77.83±3.45ef 20.25±2.18ef 12 T10 66.94±1.94g 26.37±1.82d 13 T11 74.54±2.80f 23.33±2.16e 14 T12 58.04±1.14h 11.29±1.11g

[0028] Note: Different lowercase letters for the same index indicate significant differences (p < 0.05).

Claims

1. A method for creating non-in vitro bud polyploid germplasm of Castanopsis affine, characterized in that: The buds on the Castanopsis japonici plants are directly induced by a pendimethalin aqueous solution, thereby directly producing polyploid branch germplasm. The pendimethalin aqueous solution has a volume concentration of 4-8% and contains dimethyl sulfoxide with a volume concentration of 0.5%. The induction treatment time is 24-48 hours.

2. The method according to claim 1, characterized in that: The induction treatment period is: during the budding period of the Castanopsis schrenkiana buds, when the Castanopsis schrenkiana buds are obviously enlarged to the point where the scales are slightly cracked and a small amount of green parts are visible.

3. The method according to any one of claims 1 to 2, characterized in that: The induction treatment method is as follows: the selected Castanopsis japonici buds are individually wrapped with cotton wool and covered with a plastic film, a prepared pendimethalin aqueous solution is injected onto the cotton wool wrapped with the buds using a syringe until the cotton wool absorbs the induction agent to a saturated state, the solution is replenished 2-3 times a day, and the cotton wool is removed in time after the induction treatment is completed.

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