Cultivation method and application of octaploid actinidia arguta
By soaking tetraploid jujube kiwi seeds in colchicine solution and following treatment, octaploid jujube kiwi fruit was successfully induced, solving the problem of failure to achieve tetraploid to octaploid conversion in the prior art. , realizes efficient multi-sports methods.
Patent Information
- Application Number
- CN202411662177.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-20
- Publication Date
- 2025-02-07
AI Technical Summary
The prior art has not yet studied how to process tetraploid jujube kiwi into octaploid jujube kiwi, and there is a lack of methods to achieve multi-sported jujube kiwi.
By soaking the tetraploid jujube kiwi seeds in colchicine solution for 20 to 28 hours, then rinsing, drying, and culture until germination, then sowing and cultivation, the octaploid jujube kiwi fruit was successfully induced.
Induction of soft jujube kiwi from tetraploid to octaploid is achieved, with the induction rate of octaploid as high as 2.75%, and the method is simple and suitable for widespread promotion.
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Figure CN119385060A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of polyploid breeding, and in particular to a method for cultivating octoploid Actinidia arguta and application thereof. Background Art
[0002] Actinidia arguta, also known as soft date fruit and kiwi fruit, is a perennial deciduous vine plant of the Actinidia family and the genus Actinidia. It is widely distributed, mainly in North China, Northwest my country, Northeast China and the Yangtze River Basin in China. The stock resources are particularly abundant in Heilongjiang, Jilin and Liaoning provinces. The average weight of a single fruit of Actinidia arguta is 8g, the peel is green and smooth, the flesh is green, juicy, delicate, sweet and sour, and it matures in about October every year. The fruit is rich in nutrients, with a VC content of up to 430mg / 100g, which is 100 times that of apples and 3 times that of Chinese kiwifruit. The soluble solids content of Actinidia arguta is about 15%, the total sugar content is 8.8%, and the organic acid content is 1.5%. Its fruit, roots, stems, leaves and other organs can be used as medicine, which has a certain effect on diseases such as hypertension and hyperlipidemia, and has extremely high nutritional and health value. At the same time, it has good economic value and good market prospects.
[0003] Polyploid breeding is a breeding method for improving the quality of Actinidia arguta, which can obtain Actinidia arguta with strong resistance and large fruit. New polyploid varieties are needed for people's growing material living standards. Research on chromosome doubling of Actinidia arguta can promote the industrialization of Actinidia arguta and the progress of polyploid breeding of Actinidia arguta. There is no research in the prior art on processing tetraploid Actinidia arguta to obtain octoploid Actinidia arguta.
[0004] Based on this, the present invention is proposed. Summary of the invention
[0005] The purpose of the present invention is to provide a cultivation method of octoploid Actinidia arguta and application thereof.
[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0007] The present invention provides a method for cultivating octoploid Actinidia arguta, comprising the following steps:
[0008] (1) Soaking tetraploid Actinidia arguta seeds in a colchicine solution for 20 to 28 hours to obtain treated seeds;
[0009] (2) washing the treated seeds, draining them, and culturing them until they germinate to obtain germinated seeds;
[0010] (3) sowing and cultivating the germinated seeds to obtain octoploid Actinidia arguta.
[0011] Preferably, in step (1), the concentration of the colchicine solution is 0.15-0.25 wt %.
[0012] Preferably, in step (1), the variety of the tetraploid Actinidia arguta seeds is "Longcheng No. 2".
[0013] Preferably, in step (2), the flushing is performed 4 to 5 times.
[0014] Preferably, in step (2), the culturing includes dark culturing and light culturing;
[0015] The temperature of the light culture is 20-30°C; the time of the light culture is 15-17h / d;
[0016] The temperature of the dark culture is 12-24° C.; the time of the dark culture is 7-9 h / d.
[0017] Preferably, in step (3), the sowing density is 1 to 3 seeds per hole; and the sowing depth is 0.5 to 1.5 cm.
[0018] Preferably, in step (3), the cultivation temperature is 20-26° C.; the cultivation air humidity is 50-70%.
[0019] The invention also provides application of the culture method in culturing octoploid Actinidia arguta.
[0020] Beneficial effects of the present invention:
[0021] The invention uses colchicine to induce tetraploid Actinidia arguta “Longcheng No. 2”, and can successfully induce octoploid Actinidia arguta. It is found that the concentration of colchicine solution and the treatment time are key factors affecting the successful induction of Actinidia arguta. The colchicine induction for 24 hours at a concentration of 0.2% is the best treatment, and the octoploid induction rate can reach 2.75%.
[0022] The cultivation method of the octoploid Actinidia arguta of the present invention is simple and suitable for wide promotion. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 is the germination rate of each group of Actinidia arguta, wherein ck is the blank control group, 0.05% 24h is the comparative example 4 group, 0.1% 24h is the comparative example 2 group, 0.2% 24h is the example 1 group, 0.05% 48h is the comparative example 5 group, 0.1% 48h is the comparative example 3 group, and 0.2% 48h is the comparative example 1 group;
[0024] Figure 2 This is the result of flow cytometry test of tetraploid Actinidia arguta;
[0025] Figure 3 This is the result of flow cytometry test of octoploid Actinidia arguta;
[0026] Figure 4 This is the result of flow cytometry detection of mixed ploid Actinidia arguta. DETAILED DESCRIPTION
[0027] The present invention provides a method for cultivating octoploid Actinidia arguta, comprising the following steps:
[0028] (1) Soaking tetraploid Actinidia arguta seeds in a colchicine solution for 20 to 28 hours to obtain treated seeds;
[0029] (2) washing the treated seeds, draining them, and culturing them until they germinate to obtain germinated seeds;
[0030] (3) sowing and cultivating the germinated seeds to obtain octoploid Actinidia arguta.
[0031] In the present invention, in step (1), the tetraploid aralia seeds need to be subjected to a dormancy-breaking treatment before being soaked in a colchicine solution, and the specific steps are as follows: soaking the dormant tetraploid aralia seeds in water at 40 to 60° C. for 50 to 70 minutes, taking out the seeds and placing them on gauze to control the moisture, and then soaking them in 400 to 600 mg / L gibberellin (GA3) for 20 to 28 hours to obtain the tetraploid aralia seeds;
[0032] The temperature of the water during the soaking is preferably 45-55°C, more preferably 50°C;
[0033] The dormant tetraploid Actinidia arguta seeds are preferably soaked in water for 55 to 65 minutes, more preferably 60 minutes;
[0034] The concentration of the gibberellin is preferably 450-550 mg / L, more preferably 500 mg / L;
[0035] The soaking time using gibberellin is preferably 22 to 26 hours, and more preferably 24 hours.
[0036] In the present invention, in step (1), the concentration of the colchicine solution is 0.15-0.25 wt %, preferably 0.18-0.22 wt %, and more preferably 0.20 wt %.
[0037] In the present invention, in step (1), the variety of the tetraploid Actinidia arguta seeds is "Longcheng No. 2".
[0038] In the present invention, in step (2), the number of flushing is 4 to 5 times, preferably 5 times.
[0039] In the present invention, in step (2), the culturing includes dark culturing and light culturing;
[0040] The temperature of the light culture is 20-30°C, preferably 23-27°C, and more preferably 25°C; the time of the light culture is 15-17h / d, preferably 16h / d;
[0041] The temperature of the dark culture is 12 to 24° C., preferably 16 to 20° C., and more preferably 18° C.; the time of the dark culture is 7 to 9 h / d, and preferably 8 h / d.
[0042] In the present invention, in step (3), the sowing density is 1 to 3 seeds / hole, preferably 2 seeds / hole; the sowing depth is 0.5 to 1.5 cm, preferably 1 cm.
[0043] In the present invention, in step (3), the cultivation temperature is 20-26°C, preferably 22-24°C, and more preferably 23°C; the cultivation air humidity is 50-70%, preferably 55-65%, and more preferably 60%.
[0044] The invention also provides application of the culture method in culturing octoploid Actinidia arguta.
[0045] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.
[0046] The variety of tetraploid soft kiwifruit used in the embodiments, comparative examples and experimental examples of the present invention is "Longcheng No. 2". The seeds of the tetraploid soft kiwifruit "Longcheng No. 2" in 2023 were collected, and the dormant tetraploid soft kiwifruit seeds "Longcheng No. 2" were obtained after cleaning and drying, and stored in a refrigerator at 4°C for standby use.
[0047] Example 1
[0048] A method for cultivating octoploid Actinidia arguta:
[0049] (1) Soaking dormant tetraploid Actinidia arguta seeds "Longcheng No. 2" in 50°C water for 60 min, taking out the seeds and placing them on gauze to control the moisture, and then soaking them in 500 mg / L gibberellin (GA3) for 24 h to obtain tetraploid Actinidia arguta seeds; soaking the tetraploid Actinidia arguta seeds in 0.2 wt% colchicine solution for 24 h to obtain treated seeds;
[0050] (2) The treated seeds were rinsed with tap water for 5 times, dried on gauze, and germinated in a culture dish covered with a double layer of moist filter paper. The culture dish was placed in an incubator and cultured at 25°C, 16 h light / 18°C, 8 h dark to obtain germinated seeds;
[0051] (3) Sowing the germinated seeds, 2 seeds per hole, with a sowing depth of 1 cm, and cultivating at a temperature of 23° C. and an air humidity of 60% to obtain octoploid Actinidia arguta.
[0052] Example 2
[0053] A method for cultivating octoploid Actinidia arguta:
[0054] (1) Soaking dormant tetraploid Actinidia arguta seeds "Longcheng No. 2" in 55°C water for 70 min, taking out the seeds and placing them on gauze to control the moisture, and then soaking them in 450 mg / L gibberellin (GA3) for 26 h to obtain tetraploid Actinidia arguta seeds; soaking the tetraploid Actinidia arguta seeds in 0.19 wt% colchicine solution for 26 h to obtain treated seeds;
[0055] (2) The treated seeds were rinsed with tap water for 4 times, dried on gauze, and germinated in a culture dish covered with a double layer of moist filter paper. The culture dish was placed in an incubator and cultured at 28°C, 15 h light / 20°C, 9 h dark to obtain germinated seeds;
[0056] (3) The germinated seeds were sown, 2 seeds were sown in each hole, the sowing depth was 1.3 cm, and the seeds were cultivated at a temperature of 22° C. and an air humidity of 70% to obtain octoploid Actinidia arguta.
[0057] Example 3
[0058] A method for cultivating octoploid Actinidia arguta:
[0059] (1) Soaking dormant tetraploid Actinidia arguta seeds "Longcheng No. 2" in 60°C water for 50 min, taking out the seeds and placing them on gauze to control the moisture, and then soaking them in 600 mg / L gibberellin (GA3) for 20 h to obtain tetraploid Actinidia arguta seeds; soaking the tetraploid Actinidia arguta seeds in 0.22 wt% colchicine solution for 22 h to obtain treated seeds;
[0060] (2) The treated seeds were rinsed with tap water for 5 times, dried on gauze, and germinated in a culture dish covered with a double layer of moist filter paper. The culture dish was placed in an incubator set at 22° C., 17 h light / 16° C., 7 h dark to obtain germinated seeds;
[0061] (3) sowing the germinated seeds, 3 seeds per hole, with a sowing depth of 0.8 cm, and cultivating at a temperature of 24° C. and an air humidity of 55% to obtain octoploid Actinidia arguta.
[0062] Comparative Example 1
[0063] The difference from Example 1 is that the tetraploid Actinidia arguta seeds are soaked in a 0.2wt% colchicine solution for 48 hours to obtain treated seeds; the other steps are the same as in Example 1.
[0064] Comparative Example 2
[0065] The difference from Example 1 is that the tetraploid Actinidia arguta seeds are soaked in a 0.1wt% colchicine solution for 24 hours to obtain treated seeds; the other steps are the same as those in Example 1.
[0066] Comparative Example 3
[0067] The difference from Example 1 is that the tetraploid Actinidia arguta seeds are soaked in a 0.1wt% colchicine solution for 48 hours to obtain treated seeds; the other steps are the same as those in Example 1.
[0068] Comparative Example 4
[0069] The difference from Example 1 is that the tetraploid Actinidia arguta seeds are soaked in a 0.05wt% colchicine solution for 24 hours to obtain treated seeds; the other steps are the same as those in Example 1.
[0070] Comparative Example 5
[0071] The difference from Example 1 is that the tetraploid Actinidia arguta seeds are soaked in a 0.05wt% colchicine solution for 48 hours to obtain treated seeds; the other steps are the same as in Example 1.
[0072] Experimental Example 1
[0073] Cultivation was performed using the methods described in Example 1 and Comparative Examples 1 to 5, which were recorded as Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, and Comparative Example 5. The tetraploid Actinidia arguta seeds were soaked in water instead of colchicine as a blank control group. Each group was treated with 150 tetraploid Actinidia arguta seeds, and the process was repeated 3 times.
[0074] After each group has been germinated [step (2) for each group] for 7 days, the germination rate of each group of seeds is counted and the statistics are continued until the 12th day. The test results are as follows: Figure 1 , as shown in Table 1;
[0075] The calculation formula of germination rate is as follows:
[0076] Germination rate (%) = number of seeds that germinate normally / number of seeds sown × 100%.
[0077] Table 1 Germination rate of seeds in each group on the 12th day
[0078] Group Germination rate (%) Example 1 72.67±2.40 Comparative Example 1 60.67±1.33 Comparative Example 2 86.67±2.91 Comparative Example 3 72.00±4.16 Comparative Example 4 74.00±5.29 Comparative Example 5 85.00±0.58 Blank control group 51.33±4.67
[0079] from Figure 1 As can be seen from Table 1, the germination rates of seeds were the highest after being soaked in 0.1% colchicine for 24 hours and 0.05% colchicine for 48 hours on the 12th day after treatment, which were 86.67% and 85% respectively.
[0080] Experimental Example 2
[0081] The germinated seeds in Example 1 and Comparative Examples 1 to 5 were sown in 72-hole plug trays, the substrate in the plug tray was peat soil, 2 seeds were sown in each plug tray, the sowing depth was 1 cm, the culture temperature was 23° C., the air humidity was 60%, and after 3 true leaves grew, the ploidy level was detected by flow cytometry and somatic cell chromosome counting methods, the number of octoploid plants was counted, and the octoploid induction rate was calculated. The results are as follows: Figures 2 to 4 As shown in Table 2;
[0082] Among them, octoploid induction rate % = number of octoploid plants / total number of plants × 100%;
[0083] Flow cytometric analysis was performed according to the method of Pinto et al. (Pinto G, Loureiro J, Lopes T, et al. Analysis of the genetic stability of Eucalyptus globulus Labill. somatic embryos by flow cytometry [J]. Theoretical and Applied Genetics, 2004, 109: 580-587.), with the following specific steps: 0.5 g of leaves were taken and soaked in a buffer solution (mixed with 45 mmol / L magnesium chloride, 30 mmol / L sodium citrate, 20 mmol / L 4-morpholinepropanesulfonic acid, and 0.1% Triton X-100, then adjusted to pH 7.0 and filtered under a filter membrane), and then finely chopped with a blade and filtered through a 30 μm nylon mesh to remove the broken leaves; 5 μg / mL of 4',6-diamidino-2-phenylindole (DAPI) was added to the filtrate to stain DNA for 5 minutes, and then the DNA was analyzed by flow cytometry (CyFlow Ploidy Analyzer, Sysmex Partec GmbH, Germany) to analyze the ploidy of the plants to be tested;
[0084] Table 2 Octoploid induction results of Actinidia arguta seeds
[0085] Group Octoploid induction rate (%) Example 1 2.75 Comparative Example 1 0 Comparative Example 2 0 Comparative Example 3 0 Comparative Example 4 0 Comparative Example 5 0
[0086] As can be seen from Table 2, the best induction effect was found when immersing the plants for 24 hours in a concentration of 0.2% colchicine, with an octoploid induction rate of 2.75%. No doubled plants were found in the other groups.
[0087] As can be seen from the above embodiments, the present invention provides a method for cultivating an octoploid Actinidia arguta and its application. The present invention uses colchicine to induce the tetraploid Actinidia arguta "Longcheng No. 2", and can successfully induce an octoploid Actinidia arguta.
[0088] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A method for cultivating octoploid Actinidia arguta, characterized in that: The steps include: (1) Soaking tetraploid Actinidia arguta seeds in a colchicine solution for 20 to 28 hours to obtain treated seeds; (2) washing the treated seeds, draining them, and culturing them until they germinate to obtain germinated seeds; (3) sowing and cultivating the germinated seeds to obtain octoploid Actinidia arguta.
2. The culture method according to claim 1, characterized in that In step (1), the concentration of the colchicine solution is 0.15-0.25 wt %.
3. The culture method according to claim 2, characterized in that In step (1), the variety of the tetraploid Actinidia arguta seeds is "Longcheng No. 2".
4. The culture method according to claim 3, characterized in that In step (2), the flushing is performed 4 to 5 times.
5. The culture method according to claim 4, characterized in that In step (2), the culturing includes dark culturing and light culturing; The temperature of the light culture is 20-30°C; the time of the light culture is 15-17h / d; The temperature of the dark culture is 12-24° C.; the time of the dark culture is 7-9 h / d.
6. The culture method according to claim 5, characterized in that: In step (3), the sowing density is 1 to 3 seeds per hole; the sowing depth is 0.5 to 1.5 cm.
7. The culture method according to claim 6, characterized in that: In step (3), the cultivation temperature is 20-26° C.; the cultivation air humidity is 50-70%.
8. Use of the cultivation method according to any one of claims 1 to 7 in cultivating octoploid Actinidia arguta.