A method for cultivating pentaploid Actinidia arguta

Abstract

The present invention relates to the field of agricultural breeding technology, and in particular to a method for cultivating pentaploid Actinidia chinensis. The present invention provides a method for cultivating pentaploid Actinidia chinensis, comprising the following steps: (1) selecting a high-yielding hexaploid Actinidia chinensis female variety 'Yisai' as the female parent and a tetraploid Actinidia chinensis male variety 'Weiki' with a large number of male flowers as the male parent; (2) collecting pollen from the male plant of the male parent and pollinating the pollen when the stigma of the female plant of the female parent is about to emerge. The present invention provides a method for creating high-yielding pentaploid Actinidia chinensis breeding materials, provides new breeding germplasm for the creation of Actinidia chinensis varieties, and invents a set of methods for stably cultivating pentaploid Actinidia chinensis, solving the problem of pentaploid shortage in the breeding germplasm selection process. This method is of great significance for changing the current breeding status of the Actinidia chinensis industry, which is characterized by a single variety and a lack of breeding technology.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural breeding, and in particular to a method for cultivating pentaploid Actinidia arguta. Background Art

[0002] Actinida arguta, a perennial woody vine in the genus Actinida, Actinidaceae, is a diverse genus with complex ploidy variation, including diploid, tetraploid, hexaploid, and octoploid series. Its small chromosomes and high ploidy variation significantly hinder genetic research and breeding using traditional introgression methods. In hybrid breeding practices, improper parental selection can lead to hybrid failure and infertility.

[0003] Since most soft-fleshed kiwifruits are dioecious, hybrid breeding using excellent soft-fleshed kiwifruit resources at home and abroad cannot select target traits based on fruit indicators, and can only judge their yield based on flowering characteristics. The selection of hybrid varieties is more difficult, so it is difficult to create varieties through hybridization methods.

[0004] Therefore, how to obtain hybrid offspring for variety creation through hybridization methods and solve the technical problem of the difficulty of hybridization of soft-fleshed kiwifruit is an urgent problem that technical personnel in this field need to solve. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for cultivating pentaploid Actinidia arguta.

[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 pentaploid Actinidia arguta, comprising the following steps:

[0008] (1) Select high-yielding hexaploid Actinidia arguta female varieties as female parents and tetraploid Actinidia arguta male varieties with large male flower numbers as male parents;

[0009] (2) Collect pollen from the male parent and pollinate the stigma of the female flower when it is about to emerge;

[0010] The hexaploid Actinidia arguta varieties include: Yisai;

[0011] The tetraploid Actinidia arguta varieties include: Wiki.

[0012] Preferably, the pollen collection method in step (2) is: collecting branches that are about to bloom in late May, cultivating them, and collecting pollen when the branches shed pollen.

[0013] Preferably, the length of the branches is 30 to 50 cm.

[0014] Preferably, the pollen viability during the pollination in step (2) is ≥50%.

[0015] Preferably, the pollination in step (2) is performed 2 to 3 times, with an interval of 20 to 28 hours between each pollination.

[0016] Preferably, the method further comprises: collecting seeds after they mature, storing the seeds in sand, and then sowing them to obtain pentaploid Actinidia arguta hybrid seedlings.

[0017] Preferably, the temperature of the sand storage is 3-5°C; and the time of the sand storage is 140-160 days.

[0018] Preferably, the water content of the sand during storage is 8-12%.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. In breeding practice, the sowing ratio of seeds after hybridization of two different ploidys (respectively denoted as M and N) is not equal to (M+N) / 2. Pentaploid Actinidia plants have not been found in nature or existing resources. The present invention has created a pentaploid Actinidia plant for the first time.

[0021] The present invention used a hybridization of the Japanese hexaploid female variety 'Yisai' and the German tetraploid male variety 'Weiki'. Both varieties produce abundant flowers and fruit, with a flowering period of over 10 days, making them ideal male and female parent materials. However, 'Yisai' self-pollinates, and the two varieties have different ploidy levels, making hybridization difficult.

[0022] The present invention, using a bagging isolation method, investigated the self-pollination fruit set rate of 'Yisai' in Dalian, finding a self-pollination fruit set rate of 28%. Self-pollination can hinder cross-pollination, leading to unsuccessful hybridization. The present invention, by pre-pollinating before the female flowers open (i.e., before the male flowers fully develop), overcomes the effects of self-pollination and effectively enables hybridization between two Actinidia arguta plants of different ploidy.

[0023] 2. The present invention provides a method for creating high-yield pentaploid soft-fleshed kiwifruit breeding materials, provides new breeding germplasm for the creation of soft-fleshed kiwifruit varieties, and invents a set of methods for stably cultivating pentaploid soft-fleshed kiwifruit, which solves the problem of pentaploid shortage in the breeding germplasm selection process. It is of great significance to changing the current breeding status of the soft-fleshed kiwifruit industry with a single variety and a lack of breeding technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0025] Figure 1 This is the fluorescence intensity diagram of the peak value of the Yisai sample in Experimental Example 2;

[0026] Figure 2 This is the fluorescence intensity graph of the peak value of the Wiki sample in Experimental Example 2;

[0027] Figure 3 This is the fluorescence microscopy observation image of pollen tubes from Experimental Example 2.

[0028] Figure 4 This is the fluorescence microscope observation image of the pollen tube of Isay in Experimental Example 2;

[0029] Figure 5 The peak fluorescence intensity diagram of the YSWJ-1 sample in Experimental Example 3;

[0030] Figure 6 The peak fluorescence intensity diagram of the YSWJ-2 sample in Experimental Example 3;

[0031] Figure 7 The peak fluorescence intensity diagram of the YSWJ-3 sample in Experimental Example 3;

[0032] Figure 8 The peak fluorescence intensity diagram of the YSWJ-4 sample in Experimental Example 3;

[0033] Figure 9 The peak fluorescence intensity diagram of the YSWJ-5 sample in Experimental Example 3;

[0034] Figure 10 This is the fluorescence intensity diagram of the peak value of the YSWJ-6 sample in Experimental Example 3. DETAILED DESCRIPTION

[0035] The present invention provides a method for cultivating pentaploid Actinidia arguta, comprising the following steps:

[0036] (1) Select high-yielding hexaploid Actinidia arguta female varieties as female parents and tetraploid Actinidia arguta male varieties with large male flower numbers as male parents;

[0037] (2) Collect pollen from the male parent and pollinate the stigma of the female flower when it is about to emerge;

[0038] The hexaploid Actinidia arguta varieties include: Yisai;

[0039] The tetraploid Actinidia arguta varieties include: Wiki.

[0040] In the present invention, the hexaploid Actinidia arguta variety is preferably Isai; the tetraploid Actinidia arguta variety is preferably Weiji.

[0041] In the present invention, the method for collecting pollen in step (2) is as follows: collecting branches that are about to bloom in late May, cultivating them, and collecting pollen when the branches shed pollen.

[0042] In the present invention, the length of the branches is 30 to 50 cm, preferably 34 to 46 cm, more preferably 38 to 42 cm, and even more preferably 40 cm.

[0043] In the present invention, the pollen viability during the pollination in step (2) is ≥50%; preferably 50-90%; more preferably 60-80%; and more preferably 70%.

[0044] In the present invention, the pollination is performed 2 to 3 times, each time with an interval of 20 to 28 hours; preferably, the pollination is performed 2 to 3 times, each time with an interval of 21 to 27 hours; more preferably, the pollination is performed 2 times, each time with an interval of 22 to 26 hours; and more preferably, the pollination is performed 2 times, each time with an interval of 24 hours.

[0045] The present invention further comprises: collecting seeds after they mature, storing the seeds in sand, and then sowing them to obtain pentaploid Actinidia arguta hybrid seedlings.

[0046] In the present invention, the temperature of the sand storage is 3-5°C, preferably 4°C.

[0047] In the present invention, the sand storage time is 140 to 160 days, preferably 144 to 156 days, more preferably 148 to 152 days, and even more preferably 150 days.

[0048] In the present invention, the water content of the sand during storage is 8-12%, preferably 9-11%, and more preferably 10%.

[0049] In the present invention, the characteristics of the Yisai variety are: fruiting period is mid-to-late October; the fruit is cylindrical or oval, small, single fruit weighs 4-9g, the peel and flesh are both green, high sweetness, and fragrant; the tree has medium vigor, compact growth, can self-pollinate, can bear fruit in the first year of planting, the maturity period is inconsistent, the minimum tolerance temperature is -28°C, the chromosome ploidy is 6X, and it can self-fertilize; the characteristics of the Weiji variety are a flowering branch rate of 97.8%, a polychasium inflorescence, 5-10 flowers, an average of 37.8 anthers, a flowering period of 12-15 days, and a chromosome ploidy of 4X.

[0050] The present invention adopts the agar method (refer to "Study on the Viability of Pollen of Actinidia arguta", Li Renhao, Sun Yang, Liu Zhenpan, et al., Liaoning Forestry Science and Technology, 2018(3)) to culture pollen overnight to achieve rapid determination of pollen viability.

[0051] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0052] Example 1

[0053] A method for cultivating pentaploid Actinidia arguta, comprising the following steps:

[0054] S1: Selection of female maternal plants: Select the hexaploid Actinidia arguta ‘Yisai’ and store it in a greenhouse. When the maternal plants bloom in mid-to-late May, seal them with sulfuric acid paper bags.

[0055] S2: Selection of male male plants: The tetraploid Actinidia arguta ‘Wei Ji’ was selected and stored in the resource garden;

[0056] S3: Collection of highly active pollen: Collect branches about to bloom around May 30 in spring. The branches are 30 cm long and inserted into a tissue culture bottle filled with 1 / 2 water. Place it in a tissue culture room at 25°C. When the pollen is released, use a collection bottle to quickly collect fresh pollen. Store at -80°C and it can be used for 2 years.

[0057] S4: Cross pollination: Before pollination, the pollen vitality must be measured to reach 50%. When the female flowers are about to emerge, all the stigmas of the female flowers are fully pollinated. Pollination is carried out twice in a row, with an interval of 20 hours between each pollination.

[0058] S5: Obtaining pentaploid Actinidia arguta breeding materials: Seeds were collected after maturity and stored in sand at 3°C for 140 days to improve the germination rate. The initial moisture content of the sand during storage was 8%. Hybrid seedlings were obtained by sowing. Ploidy was identified using 'Yisai' and 'Weiki' as controls. Pentaploid hybrid progeny were marked and preserved for subsequent research.

[0059] Example 2

[0060] A method for cultivating pentaploid Actinidia arguta, comprising the following steps:

[0061] S1: Selection of female maternal plants: Select the hexaploid Actinidia arguta ‘Yisai’ and store it in a greenhouse. When the maternal plants bloom in mid-to-late May, seal them with sulfuric acid paper bags.

[0062] S2: Selection of male male plants: The tetraploid Actinidia arguta ‘Wei Ji’ was selected and stored in the resource garden;

[0063] S3: Collection of highly active pollen: Collect branches about to bloom around May 30 in spring. The branches are 50 cm long and inserted into a tissue culture bottle filled with 1 / 2 water. Place it in a tissue culture room at 25°C. When the pollen is released, use a collection bottle to quickly collect fresh pollen. Store at -80°C and it can be used for 2 years.

[0064] S4: Cross pollination: Before pollination, the pollen vitality must be measured to reach 90%. When the female flowers are about to emerge, all the stigmas of the female flowers are fully pollinated. Pollination should be carried out three times in a row, with an interval of 28 hours between each pollination.

[0065] S5: Obtaining pentaploid Actinidia arguta breeding material: Seeds were collected after maturity and stored in sand at 5°C for 160 days to improve the germination rate. The initial moisture content of the sand during storage was 12%. Hybrid seedlings were obtained by sowing. Ploidy was identified using 'Yisai' and 'Weiki' as controls. Pentaploid hybrid progeny were marked and preserved for subsequent research.

[0066] Example 3

[0067] A method for cultivating pentaploid Actinidia arguta, comprising the following steps:

[0068] S1: Selection of female maternal plants: Select the hexaploid Actinidia arguta ‘Yisai’ and store it in a greenhouse. When the maternal plants bloom in mid-to-late May, seal them with sulfuric acid paper bags.

[0069] S2: Selection of male male plants: The tetraploid Actinidia arguta ‘Wei Ji’ was selected and stored in the resource garden;

[0070] S3: Collection of highly active pollen: Collect branches about to bloom around May 30 in spring. The branches should be 40 cm long and placed in a tissue culture bottle filled with 1 / 2 water. Place the bottle in a 25°C tissue culture room. When the pollen is released, use a collection bottle to quickly collect fresh pollen. Store at -80°C and it can be used for 2 years.

[0071] S4: Cross pollination: Before pollination, the pollen vitality must be measured to reach 70%. When the female flowers are about to emerge, all the stigmas of the female flowers are fully pollinated. Pollination should be carried out twice in a row, with an interval of 24 hours between each pollination.

[0072] S5: Obtaining pentaploid Actinidia arguta breeding material: Seeds were collected after maturity and stored in sand at 4°C for 150 days to improve the germination rate. The initial moisture content of the sand during storage was 10%. Hybrid seedlings were obtained by sowing. Ploidy was identified using 'Yisai' and 'Weiki' as controls. Pentaploid hybrid progeny were marked and preserved for subsequent research.

[0073] Example 4

[0074] The only difference from Example 3 is that Longcheng No. 2 is used as the female parent and Adam is used as the male parent.

[0075] Example 5

[0076] The only difference from Example 3 is that Longcheng No. 2 is used as the female parent and Wikipedia is used as the male parent.

[0077] Example 6

[0078] The only difference from Example 3 is that Yisai is used as the maternal parent and Adam is used as the paternal parent.

[0079] Example 7

[0080] The only difference from Example 3 is that Kukuwa is used as the maternal copy and Wiki is used as the paternal copy.

[0081] Example 8

[0082] The only difference from Example 3 is that Kukuwa is used as the maternal parent and Adam is used as the paternal parent.

[0083] Experimental Example 1

[0084] Pollination affinity was verified for the different ploidy resources of Examples 3 to 8, the number of pollinated female flowers, the number of seeds set, the seed set rate, and the ploidy of the hybrid offspring were determined. The results are shown in Table 1.

[0085] Table 1 Investigation on pollination fruit setting rate between male and female plants of different ploidy

[0086]

[0087] As shown in Table 1, the ploidy and success rates of hybrid offspring from different resources vary, and the success of hybridization is closely related to the choice of parents. The best hybridization results were achieved when Yisai was the female parent and Weiji was the male parent.

[0088] Experimental Example 2

[0089] 1. Based on the results obtained in Example 1, the peak fluorescence intensity of the best hybridization results, Yisai and Weiji, was further analyzed. Figure 1 and Figure 2 shown.

[0090] Results: The peak fluorescence intensity of the Yisai sample was 142.98, indicating a hexaploid; the peak fluorescence intensity of the Weiji sample was 99.84, indicating a quadruploid, and this was used to identify the pentaploid Actinidia arguta plants.

[0091] 2. The flowering period of Isai and Viki was investigated in 2021, and the results are shown in Table 2.

[0092] Table 2 Flowering period of Actinidia arguta resources

[0093]

[0094] Actinidia arguta flower buds are mixed, undergoing physiological differentiation in the fall and morphological differentiation after budding in the spring of the following year. Plants are typically dioecious, with female flowers optimally pollinated two days after opening. Both Isai and Weiji produce abundant flowers and a flowering period of over 10 days, making them ideal parental material.

[0095] 3. In step S4 of Example 3, the pollinated stigmas were taken 24 hours after pollination, fixed by FAA, and observed under a fluorescence microscope.

[0096] The results showed that when the FAA-fixed materials were observed under a fluorescence microscope, the pollen tubes appeared green. Using this method for pollination, it was found that a large number of pollen grains on the stigma of the male plant 'Wei Ji' had germinated 24 hours after pollination with affinity pollen, as shown in ( Figure 3 ), indicating that self-pollination was incompatible during this period.

[0097] Experimental Example 3

[0098] Six hybrid progenies were obtained by hybridizing Isai and Weiji in Example 3, namely YSWJ-1 to YSWJ-6, and peak fluorescence intensity tests were performed on them. The results are shown in Table 3.

[0099] Table 3 Ploidy identification of hybrid progeny

[0100]

[0101]

[0102] From the contents recorded in Table 3, it can be seen that all 6 hybrid offspring are 5-ploid, indicating that the hybridization of Yisai and Weiji is successful and that 5-ploid Actinidia arguta can be stably cultivated.

[0103] 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 principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for cultivating pentaploid Actinidia arguta, characterized in that: The steps include: (1) Select a hexaploid Actinidia arguta variety as the female parent and a tetraploid Actinidia arguta variety as the male parent; (2) Collect pollen from the male parent and pollinate the stigma of the female flower when it is about to emerge; The hexaploid Actinidia arguta variety is Yisai; The tetraploid Actinidia arguta variety is Wiki; The pollen collection method in step (2) is as follows: collecting branches that are about to bloom in late May, cultivating them, and collecting pollen when the branches shed pollen; The length of the branches is 30 to 50 cm; The pollen viability during the pollination in step (2) is ≥50%; The pollination in step (2) is performed 2 to 3 times, with an interval of 20 to 28 hours between each pollination.

2. The method for cultivating pentaploid Actinidia arguta according to claim 1, wherein: Also includes: The seeds are collected after they mature, stored in sand and then sown to obtain pentaploid Actinidia arguta hybrid seedlings.

3. The method for cultivating pentaploid Actinidia arguta according to claim 2, wherein: The temperature of the sand storage is 3-5°C; the time of the sand storage is 140-160 days.

4. The method for cultivating pentaploid Actinidia arguta according to claim 2 or 3, wherein: The water content of the sand during the sand storage is 8-12%.

Images