A distant hybridization method of pine based on stockroot-scion interaction

Abstract

The present invention relates to the technical field of pine hybridization. The present invention provides a method for distant hybridization of the genus Pinus based on stock-scion interaction, comprising the following steps: (1) selecting stock and scion of different tree species for grafting to obtain a grafted plant; (2) cultivating the grafted plant, removing the stamens and bagging before the female flowers of the grafted plant turn white; (3) after the female flowers of the grafted plant turn white, artificial pollination is performed using pollen of a pine tree of the same variety as the stock. The present invention uses pine trees of different factions for grafting and hybridization, and utilizes the horizontal transfer of small molecules between the stock and scion to achieve the possibility of interspecific hybridization, thereby overcoming the problem of highly incompatible hybridization between different subgenera of pine trees.

Description

Technical Field

[0001] The invention relates to the technical field of pine hybridization, and in particular to a distant hybridization method of pine based on stock and scion interaction. Background Art

[0002] The genus Pinus is divided into two subgenera: monovascular pine and bivascular pine. Interspecific hybridization within the monovascular pine subgenus is highly compatible. Interspecific hybridization within the bivascular pine subgenus is different. It is easier to hybridize within a subgenus, but it is not easy to succeed between subgenuses. The Pinus tabulaeformis subgenus includes Masson pine, Yunnan pine, South Asian pine, Laya pine, and Daming pine; the New World bivascular pine subgenus includes Longleaf pine, Torch pine, Caribbean pine, and Wetland pine. Hybridization between the two subgenera is highly incompatible. Therefore, how to break through the hybridization restrictions has become an urgent problem to be solved. Summary of the invention

[0003] The purpose of the present invention is to provide a distant hybridization method of pine based on stock and scion interaction, so as to improve the fertility of interspecific hybridization of pine.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a method for distant hybridization of pine based on stockroot-scion interaction, comprising the following steps:

[0006] (1) selecting rootstocks and scions of different tree species for grafting to obtain grafted plants;

[0007] (2) cultivating the grafted plants, removing the stamens and bagging the grafted plants before the female flowers of the grafted plants turn white;

[0008] (3) After the female flowers of the grafted plant turn white, artificial pollination is performed using pollen from a pine tree of the same variety as the rootstock.

[0009] Preferably, the rootstock and the scion in step (1) both belong to the subgenus Pinus;

[0010] The rootstock and scion are Pinus tabulaeformis or New World double-vascular bundle pine;

[0011] The Pinus tabulaeformis pine trees include Pinus massoniana, Pinus yunnanensis, Pinus asiatica, Pinus rapa and Pinus damingensis.

[0012] The New World two-dimensional bundle pine trees include longitudinal pine, loblolly pine, Caribbean pine, and wetland pine.

[0013] Preferably, the rootstock in step (1) is 5 to 10 months old and has a ground diameter of 0.6 to 1.5 cm.

[0014] Preferably, the scion in step (1) is a branch that sprouts in the current year, and the diameter of the scion is 0.4 to 0.8 cm.

[0015] Preferably, the grafting time in step (1) is from November to February of the following year or from April to May, and the grafting method is cleft grafting, cut grafting, pith-cambrium grafting or peeling grafting.

[0016] Preferably, the height of the grafting in step (1) is 8 to 20 cm from the ground.

[0017] Preferably, a sulfuric acid paper bag is used when removing the bag in step (2).

[0018] Preferably, the time for emasculation in step (2) is from mid-December to early March.

[0019] Preferably, the artificial pollination in step (3) is carried out from late December to late March of the following year.

[0020] The present invention provides a method for distant hybridization of pine based on stockroot-scion interaction, comprising the following steps:

[0021] (1) Selecting rootstocks and scions of different tree species for grafting to obtain grafted plants; (2) Cultivating grafted plants and castrating the males before the female flowers of the grafted plants turn white; (3) After the female flowers of the grafted plants turn white, artificial pollination is performed using pollen from pine trees of the same variety as the rootstock. The present invention aims to improve stem shape, increase growth, increase resin production, improve resin quality, improve ecological adaptability, advance the age of flowering, and enhance stress resistance as important goals of distant hybridization of pine species. Pines from different factions are used for grafting and hybridization, and the horizontal transfer of small molecules from the rootstock and scion is used to achieve the possibility of interspecific hybridization, thus overcoming the problem of highly incompatible hybridization between different subgenera of pine. The horizontal transfer of chloroplast and mitochondrial genes in the grafting region of different rootstocks is utilized, and the intercellular transfer of genomes is achieved by selecting different pine species for grafting, breaking the boundaries of species kinship and achieving the creation of new germplasm from distant hybridization. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The effect of hybridization of different tree species;

[0023] Figure 2 For pine trees after detasseling and bagging;

[0024] Figure 3 Male flowers of Masson pine used for hybridization;

[0025] Figure 4 Male flowers of slash pine used for hybridization;

[0026] Figure 5 The process of drying pollen;

[0027] Figure 6 The process of collecting pollen;

[0028] Figure 7 The process of cold storage of pollen at -10℃;

[0029] Figure 8 For emasculated and bagged female flowers;

[0030] Fig. 9 The female flowers are removed from the bag and successfully pollinated;

[0031] Fig.10 For the effect of grafting. DETAILED DESCRIPTION

[0032] The present invention provides a method for distant hybridization of pine based on stockroot-scion interaction, comprising the following steps:

[0033] (1) selecting rootstocks and scions of different tree species for grafting to obtain grafted plants;

[0034] (2) cultivating the grafted plants, removing the stamens and bagging the grafted plants before the female flowers of the grafted plants turn white;

[0035] (3) After the female flowers of the grafted plant turn white, artificial pollination is performed using pollen from a pine tree of the same variety as the rootstock.

[0036] In the present invention, the rootstock and the scion in step (1) preferably both belong to the subgenus Pinus.

[0037] In the present invention, the rootstock and scion in step (1) are preferably Pinus tabulaeformis or New World double-vascular pine.

[0038] The Pinus tabulaeformis pine trees preferably include Pinus massoniana, Pinus yunnanensis, Pinus asiatica, Pinus rapa and Pinus damingensis;

[0039] The New World double-dimensional bundled pine trees preferably include slash pine, loblolly pine, Caribbean pine, and wetland pine.

[0040] In the present invention, the rootstock is selected from pine seedlings with good growth, no diseases and insect pests and the seedling age is more than 5 months, and the ground diameter is preferably 0.6-1.5 cm, and there is no strict requirement for the seedling height.

[0041] The cultivation time is different for different tree species. Among them, the diameter growth of wetland pine, wetland pine and loblolly pine is large in the seedling stage, and 5 to 7 months is suitable. However, the height growth of Masson pine, Caribbean pine, Yunnan pine, Raya pine and Daming pine is large in the seedling stage, and 7 to 10 months is suitable.

[0042] In the present invention, the age of the rootstock in step (1) is preferably 5 to 10 months, more preferably 7 to 9 months, and the ground diameter of the rootstock is preferably 0.6 to 1.5 cm, more preferably 1 to 1.1 cm.

[0043] In the present invention, high-quality, robust, pest-free semi-lignified branches with a shoot diameter of 0.4 to 0.8 cm are selected as scion sticks, which are numbered and labeled as they are collected, wrapped with a wet towel and placed in a ventilated and cool place for use.

[0044] In the present invention, the scion in step (1) is preferably a branch that shoots out in the current year, and the diameter of the scion is preferably 0.4 to 0.8 cm, and more preferably 0.6 cm.

[0045] In the present invention, grafting can be carried out in two ways. One is to centrally graft and transplant the seedlings to the mountain in a nursery. Different pine seedlings with ground diameters that meet the grafting requirements are transplanted into 14cm×20cm nutrient cups, with a substrate ratio of 60% loess soil + 20% coconut bran + 15% peat soil + 5% substrate. Grafting is carried out after the seedlings have recovered and stabilized 20 to 30 days after transplantation, and post-grafting care is carried out in a centralized nursery. After the growth is stable, the seedlings are transplanted to the mountain. The second is to directly graft on the mountain. In the mountain grafting, qualified rootstocks are planted according to the planting map, and grafting is carried out after 25 days of recovery. In this method, post-grafting care is carried out on the mountain.

[0046] In the present invention, the grafting time in step (1) is preferably from November to February of the following year or from April to May, and further preferably from December to January of the following year or from late April to early May. The grafting method is preferably cleft grafting, cut grafting, pith cambium docking or peeling docking, among which pith cambium docking is the best. Select a rootstock that is 8 to 20 cm above the ground and relatively straight, and use a grafting knife to cut a 5 to 8 cm incision. Select a fringe strip that is similar in size to the rootstock and about 10 cm in length, and cut it downward 2 cm from the top of the fringe strip so that the length of the blade is equivalent to the length of the cut of the rootstock (5 to 8 cm). Place the fringe strip close to the rootstock, with the blade facing the blade, and aligning the cambium on at least one side. Use a grafting film to fully seal and tie it from bottom to top. During the tying process, the fringe strip is constantly pressed by hand to ensure that the rootstock and the fringe strip fit tightly.

[0047] In the present invention, the height of the grafting in step (1) is preferably 8 to 20 cm from the ground, and more preferably 12 to 16 cm from the ground.

[0048] In the present invention, when removing the bag in step (2), it is preferred to use a sulfuric acid paper bag, the sulfuric acid paper bag is light-transmitting and waterproof, length × width: 12-15cm × 35-50cm, and different specifications are selected depending on the length of the spring shoots of different pine trees.

[0049] In the present invention, the time for emasculation in step (2) is preferably from mid-December to early March.

[0050] In the present invention, the artificial pollination in step (3) is carried out from December to late March of the following year.

[0051] In the present invention, the time for collecting pollen and artificial pollination of Caribbean pine is from December to early January.

[0052] In the present invention, the time for collecting pollen and artificial pollination of Pinus elliottii and Pinus elliottii is from mid-January to early February.

[0053] In the present invention, the time for collecting pollen and artificial pollination of Masson pine is from mid-February to early March.

[0054] In the present invention, the time for collecting pollen and artificial pollination of Pinus rapa and Pinus rapa is from late February to early March.

[0055] In the present invention, the time for pollen collection and artificial pollination of loblolly pine, Yunnan pine and Daming pine is from mid-March to late March.

[0056] In the present invention, since the flowering periods of different pine trees do not coincide, in order to successfully complete hybrid pollination, the pollen is collected one year before artificial pollination, the moisture content of the collected pollen is reduced to 5-8%, and the pollen is sealed and stored at -10°C. The pollen is thawed and recovered and the germination rate is tested one month before pollination, and the pollen with a germination rate greater than 50% can be used.

[0057] In the present invention, the interface cannot touch water within 5 days after the grafting, otherwise the survival rate is seriously affected. After about 30 days, the fringe is seen to sprout and begin to emerge. A small opening is cut near the sprouting bud with a sharp blade to help the bud to elongate. The incision is as close to the bud point as possible and the smaller the opening, the better, which is conducive to avoiding rainwater from entering the interface in the grafting film and causing waterlogging and rotting. In order to avoid excessive growth of the stock, the growth of the stock is divided into 2-5 times for topping and cultivating the growth of the scion. About 2 months after the grafting, the scion extracts new buds, and the interface heals well to completely untie the grafting film, and the stock is broken within 7 days. When the scion grows well and grows vigorously, the stock above the grafting interface is completely cut off, and the grafted plant can survive stably after 1 month.

[0058] 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.

[0059] Example 1

[0060] A method for distant hybridization of pine based on stockroot-scion interaction comprises the following steps:

[0061] (1) A Masson pine with a seedling age of 10 months and a ground diameter of 0.6 cm was selected as a rootstock, and a slash pine branch with a diameter of 0.4 cm that was taken out in the current year was selected as a scion. The grafting was performed 8 cm above the ground using a cleft grafting method to obtain a grafted plant. The interface could not touch water within 5 days after grafting. After 31 days, the scion was seen to sprout and begin to emerge. A small incision was made near the sprouting bud with a sharp blade to help the bud to elongate. The incision was as close to the bud point as possible and the smaller the opening, the better, which was conducive to preventing rainwater from entering the interface in the grafting film and causing waterlogging and rotting; in order to prevent the rootstock from growing too vigorously and inhibiting the growth of the scion, the scion was topped and cultivated for growth 5 times depending on the growth of the rootstock; 2 months after grafting, the scion sprouted new buds, and the interface healed well, the grafting film was completely untied, the rootstock was cut off within 7 days, and when the scion grew well and grew vigorously, all the rootstocks above the grafting interface were cut off, and the grafted plant could survive stably after 1 month;

[0062] (2) Cultivate the surviving grafted plants, remove the stamens and put them in sulfuric acid paper bags on February 3;

[0063] (3) After the female flowers of the grafted plant turn white, they are artificially pollinated with pollen from Pinus massoniana to create a hybrid of Pinus elliottii × Pinus massoniana.

[0064] Example 2

[0065] A method for distant hybridization of pine based on stockroot-scion interaction comprises the following steps:

[0066] (1) Select a South Asian pine with a seedling age of 7 months and a ground diameter of 1 cm as a rootstock, select a loblolly pine branch with a diameter of 0.6 cm that year as a scion, adopt the peeling and docking method, and graft at 15 cm from the ground to obtain a grafted plant. The interface cannot touch water within 5 days after grafting. After 27 days, the fringe is seen to sprout and begin to emerge. Use a sharp blade to cut a small opening near the sprouting bud to help the bud elongate. The incision is as close to the bud point as possible and the smaller the opening, the better, which is conducive to preventing rainwater from entering the interface in the grafting film and causing waterlogging and rotting; in order to avoid the excessive growth of the rootstock to inhibit the growth of the fringe, the scion is topped and cultivated for growth 5 times depending on the growth of the rootstock; 2 months after grafting, the scion shoots out new buds, the interface is well healed, the grafting film is completely untied, the rootstock is cut off within 7 days, and when the scion grows well and grows vigorously, the rootstock above the grafting interface is completely cut off, and the grafted plant can survive stably after 1 month;

[0067] (2) Cultivate the surviving grafted plants, remove the stamens and put them in sulfuric acid paper bags on March 11;

[0068] (3) After the female flowers of the grafted plant turn white, artificial pollination is carried out using pollen from Pinus taeda to create a hybrid of Pinus taeda and Pinus taeda.

[0069] Example 3

[0070] A method for distant hybridization of pine based on stockroot-scion interaction comprises the following steps:

[0071] (1) A Caribbean pine with a seedling age of 5 months and a ground diameter of 1.5 cm is selected as a rootstock, and a Masson pine branch with a diameter of 0.8 cm that is produced in the current year is selected as a scion. The medullary cambium docking method is used to graft at 20 cm above the ground to obtain a grafted plant. The interface cannot be exposed to water within 5 days after grafting. After 35 days, the scion is seen to sprout and begin to emerge. A small incision is made near the sprouting bud with a sharp blade to help the bud elongate. The incision is as close to the bud point as possible and the smaller the opening, the better, which is conducive to preventing rainwater from entering the interface in the grafting film and causing waterlogging and rotting; in order to prevent the rootstock from growing too vigorously and inhibiting the growth of the scion, the scion is topped and cultivated for growth 5 times depending on the growth of the rootstock; 2 months after grafting, the scion sprouts new buds, the interface is well healed, the grafting film is completely untied, the rootstock is cut off within 7 days, and when the scion grows well and grows vigorously, the rootstock above the grafting interface is completely cut off. After 1 month, the grafted plant can survive stably;

[0072] (2) Cultivate the surviving grafted plants, remove the stamens and put them in sulfuric acid paper bags on February 25;

[0073] (3) After the female flowers of the grafted plant turned white, they were artificially pollinated using pollen from Caribbean pine to create a hybrid of Masson pine and Caribbean pine.

[0074] Test example

[0075] The rootstock and scion of the test group were selected according to the combination in Table 1, and the treatment was carried out according to the following steps, and the success rate was calculated.

[0076] 1. Rootstock cultivation

[0077] The rootstock should be well-growing, disease-free, and more than 5-month-old pine seedlings with a ground diameter of 1.5 cm. The cultivation time is different for different tree species. The diameter growth of wetland pine, wetland pine, and loblolly pine is large in the seedling stage, so 6 months of age should be selected. The height growth of Masson pine, Caribbean pine, Yunnan pine, Raya pine, Daming pine, etc. is large in the seedling stage, so 10 months of age should be selected.

[0078] 2. Grafting

[0079] Different pine seedlings with ground diameters that meet the grafting requirements are transplanted into 14cm×20cm nutrient cups. The substrate ratio is 60% loess soil + 20% coconut bran + 15% peat soil + 5% substrate. Grafting is carried out 30 days after transplantation. Post-grafting care is carried out in a centralized nursery and transplanted to the mountain after stable growth.

[0080] Select high-quality, strong, disease-free, and semi-lignified branches with a diameter of 0.6 cm in the current year as scion sticks. Number and label the scion sticks as they are collected, wrap them with a wet towel, and place them in a ventilated and cool place for use. The best time for grafting is November 20; the best grafting method is to use the pith cambium docking. Select a 15 cm above the ground and relatively straight surface of the stock and use a grafting knife to cut an 8 cm incision. Select a scion stick that is similar in size to the stock and 10 cm in length, and cut it down 2 cm from the top of the scion stick so that the length of the blade is equivalent to the length of the cut of the stock (8 cm). Put the scion stick close to the stock, with the blade facing the blade, and align the cambium on at least one side. Use the grafting film to seal and tie it from bottom to top. During the tying process, press the scion stick with your hands to ensure that the stock and the scion stick fit tightly.

[0081] 3. Post-reception management

[0082] The interface cannot be exposed to water within 5 days after grafting, otherwise the survival rate will be seriously affected. After 3 scions sprout and begin to emerge, use a sharp blade to cut a small hole near the sprouting bud to help the buds to elongate. The incision should be as close to the bud point as possible and open. Depending on the growth of the rootstock, top the scion for 5 times to cultivate the growth of the scion. Two months after grafting, the scion will sprout new buds. If the interface heals well, the grafting film will be completely untied, and the rootstock will be cut off within 7 days. When the scion grows well and grows vigorously, all the rootstocks above the grafting interface will be cut off. After 1 month, the grafted plant can survive stably and bloom and bear fruit in 6 years.

[0083] 4. Artificial pollination

[0084] The grafted plants were hybridized between Pinus massoniana and Pinus longituba (female parent and male parent). On February 21, before the female flowers turned white, the plants were de-tasseled and covered with sulfuric acid paper bags. The frozen Pinus longituba pollen was thawed and tested for germination rate. Pollen with a germination rate greater than 50% was considered qualified. When the female flowers turned white, artificial pollination was performed.

[0085] Control example: The rootstock and scion of the mother plant were grafted with the same pine variety, and the specific grafting process was the same as that of other groups.

[0086] During hybridization, artificial hybridization was performed according to the combination of Tables 2 and 4, and the same steps were followed in the test group, and the success rate was counted. Since the control group lacked a stock-scion interaction bridge (the stock in the stock-scion interaction maternal parent was the same as the paternal parent, and the stock small molecules were transferred to the scion, becoming a link to improve the success rate of distant hybridization), the highest success rate of the hybrid combination of 15 control examples was only 16.7%, and the success rate of 4 combinations was 0 (Tables 2 and 4), which was significantly lower than the success rate of the stock-scion interaction embodiment (Tables 1 and 3).

[0087] Table 1 Statistics on the combination and success rate of hybridization between stock and scion

[0088]

[0089] Table 2 Statistics of hybridization combinations and success rates among the anvil factions (Table 1 for comparison)

[0090]

[0091] Table 3 Statistics of intra-faction hybridization combinations and success rates of stockstock-scion interaction

[0092]

[0093] Table 4: Statistics of hybridization test combinations and success rates within this anvil group (Table 3 for comparison)

[0094]

[0095]

[0096] Conclusion: The present invention utilizes the horizontal transfer of small molecules between stock and scion to realize the possibility of interspecific hybridization and overcome the problem of highly incompatible hybridization between different subgenera of pine.

[0097] As can be seen from the above embodiments, the present invention provides a method for distant hybridization of pine based on stock-scion interaction, comprising the following steps: (1) selecting stock and scion of different tree species for grafting to obtain a grafted plant; (2) cultivating the grafted plant, and emasculating the grafted plant before the female flowers of the grafted plant turn white; (3) after the female flowers of the grafted plant turn white, artificial pollination is performed using pollen of a pine tree of the same variety as the stock. The present invention uses pine trees of different factions for grafting and hybridization, and utilizes the horizontal transfer of stock-scion small molecules to achieve the possibility of interspecific hybridization, thereby overcoming the problem of highly incompatible hybridization between different subgenera of pine trees.

[0098] 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 distant hybridization of pine based on stock and scion interaction, characterized in that: The steps include: (1) selecting rootstocks and scions of different tree species for grafting to obtain grafted plants; (2) cultivating the grafted plants, removing the stamens and bagging the grafted plants before the female flowers of the grafted plants turn white; (3) After the female flowers of the grafted plant turn white, artificial pollination is performed using pollen from a pine tree of the same variety as the rootstock; The rootstock and the scion in step (1) both belong to the subgenus Pinus; The rootstock and scion are Pinus tabulaeformis or New World double-vascular bundle pine; The Pinus tabulaeformis pine is selected from Pinus massoniana, Pinus yunnanensis, Pinus asiatica, Pinus rapa and Pinus damingensis; The New World double-dimensional bundle pine is selected from the group consisting of slash pine, loblolly pine, Caribbean pine and wetland pine.

2. The method according to claim 1, characterized in that The rootstock in step (1) has a seedling age of 5 to 10 months and a ground diameter of 0.6 to 1.5 cm.

3. The method according to claim 2, characterized in that The scion in step (1) is a branch that has sprouted in the current year, and the diameter of the scion is 0.4 to 0.8 cm.

4. The method according to claim 3, characterized in that The grafting time in step (1) is from November to February of the following year or from April to May, and the grafting method is cleft grafting, cut grafting, pith cambium grafting or peeling grafting.

5. The method according to claim 4, characterized in that The height of the grafting in step (1) is 8 to 20 cm from the ground.

6. The method according to claim 5, characterized in that The bagging process in step (2) is carried out using a sulfuric acid paper bag.

7. The method according to any one of claims 1 to 6, characterized in that: The time for emasculation in step (2) is from mid-December to mid-March.

8. The method according to claim 7, characterized in that The artificial pollination in step (3) is carried out from late December to late March of the following year.

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