Efficient hybridization method for tetraploid apples and diploid apples

Abstract

The invention discloses an efficient hybridization method of tetraploid apples and diploid apples, which comprises the following steps: selecting diploid with persistent sepal on calyx as a female parent, and performing male and female parent hybridization without requirements on persistence of tetraploid male parent calyx or not, otherwise, selecting tetraploid with persistent sepal on calyx as the female parent, and performing male and female parent hybridization on the tetraploid with persistent sepal on calyx or the tetraploid with persistent sepal on calyx or the tetraploid with persistent sepal on calyx as the female parent. And carrying out reverse cross when no requirement is provided for the persistence of the diploid male parent fruit calyx. Fruit calyx is checked in the mature period, the hybrid seeds with persistent sepals are female parent selfing or non-target hybrid seeds, and the hybrid seeds without sepals are real target hybrid seeds. Moreover, the triploid yield is identified by a flow cytometry to confirm the effectiveness of the hybridization method, the method is simple and easy to implement, the screening success rate is high, and the screening process is simplified.

Description

Technical Field

[0001] The invention belongs to the technical field of hybrid breeding, and particularly relates to a high-efficiency hybridization method for tetraploid apples and diploid apples. Background Art

[0002] Hybridization has always been the most critical and widely used method in apple variety improvement. Traditional hybridization has played a vital role in apple breeding, producing numerous classic varieties. However, with the passage of time and the development of the industry, the limitations of traditional hybridization have become increasingly apparent. For one thing, most apple varieties developed in China over the past few decades are hybrids, seedlings, or bud selections of varieties like "Jinshuai," "Yuanshuai," and "Guoguang." This "inbreeding" phenomenon has resulted in a narrowing of the varieties' genetic base. The direct consequence of this narrow genetic base is a significant increase in genetic vulnerability, significantly reducing the varieties' adaptability and resistance to adverse factors such as environmental changes and pest and disease infestations. When faced with extreme climatic conditions or new pests and diseases, orchards of these varieties often suffer severe losses. Furthermore, the biological characteristics of apples also add numerous challenges to hybridization. Apples are highly heterozygous horticultural plants with a long growth cycle. The lengthy breeding cycle typically takes 20-30 years, requiring enormous human, material, and financial resources, and ultimately, the success of the breeding is uncertain. During the hybrid seedling cultivation stage, the evaluation and screening of hybrid seedlings and hybrid fruit are extremely laborious and arduous due to the long juvenile stage (the time from seed sowing to the first flowering and fruiting) of apple hybrids and their large size. Hybrid breeding methods are complex and any technical error in any step can lead to failure of the entire breeding effort. Furthermore, in traditional apple hybrid breeding, hybridization between tetraploids and diploids is associated with pollination incompatibility, low fruit set (typically <10%), and a high proportion of pseudo-hybrids. Summary of the Invention

[0003] In view of the problems existing in the prior art, the object of the present invention is to provide an efficient hybridization method for tetraploid apples and diploid apples, which simplifies the screening process by observing the persistence of the calyx as a morphological marker of the hybrid fruit.

[0004] In order to achieve the above object, the technical solution of the present invention is as follows:

[0005] A high-efficiency hybridization method for tetraploid apples and diploid apples, comprising:

[0006] (1) Parent selection: Select diploid and tetraploid parents whose flowers are in the "lantern stage" (petals not unfolded but anthers mature). The female parent is required to have persistent calyx, while the male parent is not required to have persistent calyx. (2) Anther collection and female parent emasculation.

[0007] (3) Pollination: Pollination should be carried out on a sunny day between 9 and 11 am, with a temperature of 18 to 25°C and an air humidity of 50% to 70%.

[0008] (4) Inspection after bagging: Remove the bag and observe after 5-7 days. If the style turns yellow and shrinks, pollination is unsuccessful; if the ovary is swollen and bright in color, pollination is successful.

[0009] (5) Screening of target hybrids: Check the calyx during the fruit ripening period. Those with persistent sepals are self-pollinated female parents or non-target hybrids, while those without sepals are true target hybrids.

[0010] Preferably, the collected anthers are placed in a dark environment at 25° C. and 30% humidity for 24 to 48 hours, and after the anthers naturally crack, impurities are removed and pure pollen is collected.

[0011] The vitality of the collected pure pollen was tested by 1% TTC staining, and pollen with vitality greater than 80% was selected for use.

[0012] Preferably, the diploid or tetraploid plant used as the mother parent is required to have persistent calyx, strong plant growth, no diseases or insect pests, and flowers in the lantern stage.

[0013] Dip a brush or a pencil with an eraser into the pollen of the male parent and lightly apply it to the stigma of the female parent until the pollen is evenly attached to the stigma visible to the naked eye; immediately put it in a sulfuric acid paper bag after pollination and fix the bag opening with wire to prevent it from falling off in the wind.

[0014] Management during the growing season: Field management throughout the entire growth cycle is the same as field fruit tree management.

[0015] The target hybrid described in the present invention refers to the offspring plants or varieties with a specific combination of excellent traits that are expected to be obtained through hybrid breeding technology.

[0016] Advantages of the present invention:

[0017] The present invention selects female parents with persistent sepals in their calyx, with no requirement for the male parent. Hybridization is then performed on these parents, and the calyxes are examined at fruit maturity. Those with persistent sepals are either self-pollinated female parents or non-target hybrids, while those without sepals are true target hybrids. Furthermore, the present invention verifies the effectiveness of the hybridization method by using flow cytometry to identify triploid yields. This method utilizes persistent calyx as a marker for hybrid fruit morphology, is simple and easy to implement, has a high screening success rate, and simplifies the screening process. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a comparison of the sepals of hybrid fruits that remain (left) and fall off (right);

[0019] Figure 2 This is a front view of the hybrid fruit sepals falling off. DETAILED DESCRIPTION

[0020] The present invention will be further described below with reference to specific examples, and the advantages and features of the present invention will become more apparent as the description proceeds. However, the specific experimental methods involved in the following examples, unless otherwise specified, are all conventional methods or are performed under the conditions recommended by the manufacturer's instructions.

[0021] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The experimental methods in the following examples are all conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used can be purchased from the market.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0023] Example A high-efficiency hybridization method for tetraploid apples and diploid apples

[0024] 1. Male pollen collection and pretreatment

[0025] Collection time: Choose a male parent whose flowers are in the "lantern stage" (petals have not unfolded but anthers are mature).

[0026] Pollen extraction: Place the anthers in a dark environment at 25°C and 30% humidity for 24 to 48 hours. After the anthers naturally crack, remove impurities and collect pure pollen.

[0027] Vitality detection: 1% TTC (triphenyltetrazolium chloride) staining method was used, and pollen with vitality greater than 80% was selected for use.

[0028] 2. Female parent selection and emasculation

[0029] Mother plant requirements: Choose a mother plant with persistent sepals on the calyx. The plant should be healthy and pest-free, with flowers in the lantern stage.

[0030] Emasculation: Remove the petals, sepals and all stamens by hand or with tweezers to avoid self-pollination.

[0031] 3 Pollination methods

[0032] Pollination time: 9-11 am on sunny days, temperature 18-25℃, air humidity 50%-70%.

[0033] Pollination technique: Dip a brush or a pencil with an eraser into the pollen of the male parent and lightly apply it to the stigma of the female parent until the pollen is evenly attached to the stigma visible to the naked eye.

[0034] Isolation protection: After pollination, immediately put the pollen into a sulfuric acid paper bag and fix the bag opening with wire to prevent it from falling off due to wind.

[0035] 4 Fruiting Season Management

[0036] Inspection after bagging: Remove the bag and observe after 5 to 7 days. If the style turns yellow and shrinks, pollination is unsuccessful; if the ovary is swollen and bright in color, pollination is successful.

[0037] Management during the growing season: Field management throughout the entire growth cycle is the same as field fruit tree management.

[0038] 5. Identification and harvesting of hybrid fruits

[0039] Pseudohybrid screening: Check the calyx during fruit maturity. Those with persistent sepals are self-pollinated female parents or non-target hybrids, while those without sepals are target true hybrids (diploid × tetraploid hybrids, Figure 2 ).

[0040] Seed processing: After washing the pulp, rinse the seeds clean and dry them for later use.

[0041] 6 The tetraploid 'Zi Yan' (4×) was used as the male parent and the diploid 'Lu Li' (2×) was used as the female parent ( Figure 1 ), the fruit setting rate after hybridization using this method reached 90%, and the pseudo-hybrid rate (non-target hybrid rate) was <5%.

[0042] In addition, hybrids were performed using different diploid and tetraploid parents, and the ploidy of the hybrids was determined by flow cytometry. The yield of triploid (3x) offspring in each hybrid combination was almost always above 60%, indicating that diploid and tetraploid hybridization is effective and that this method can be used to simply and effectively identify the target true hybrids.

[0043] Table 1 Triploid yields of various hybrid combinations

[0044]

[0045] The embodiments described above are only preferred embodiments of the present invention and are only used to explain the present invention, not to limit the scope of implementation of the present invention. For those skilled in the art, it is of course possible to easily make other implementation methods by replacing or changing the technical content disclosed in this specification. Therefore, all changes and improvements made on the principles of the present invention should be included in the scope of the patent application of the present invention.

Claims

1. A method for efficiently hybridizing tetraploid apples with diploid apples, characterized in that: The method comprises: (1) Selection of parents: Select male parents with flowers in the "lantern stage" and female parents with persistent sepals in the calyx; (2) anther collection and female plant emasculation; (3) Pollination: Pollination should be carried out on a sunny day between 9 and 11 am, with a temperature of 18 to 25°C and an air humidity of 50% to 70%. (4) Inspection after bagging: Remove the bag and observe after 5-7 days. If the style turns yellow and shrinks, pollination is unsuccessful; if the ovary is swollen and bright in color, pollination is successful. (5) Screening of target hybrids: Check the calyx during the fruit ripening period. Those with persistent sepals are self-pollinated female parents or non-target hybrids, while those without sepals are true target hybrids.

2. The efficient hybridization method of tetraploid apple and diploid apple according to claim 1, characterized in that: The collected anthers were placed in a dark environment at 25°C and 30% humidity for 24 to 48 hours. After the anthers opened naturally, impurities were removed and pure pollen was collected.

3. The efficient hybridization method of tetraploid apple and diploid apple according to claim 2, characterized in that: The vitality of the collected pure pollen was tested by 1% TTC staining, and pollen with vitality greater than 80% was selected for use.

4. The method for efficient hybridization of tetraploid apple and diploid apple according to claim 1, characterized in that: The female plant is emasculated by removing the petals, sepals and all stamens with tweezers or by hand.

5. The efficient hybridization method of tetraploid apple and diploid apple according to claim 1, characterized in that: After pollination, put on a sulfuric acid paper bag immediately and fix the bag opening with wire.

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