Method for cross breeding of cedrela plants by using dwarf seed orchard

Abstract

The invention discloses a method for cross breeding of cedrela plants by using a dwarf seed orchard, which comprises the following steps: collecting germplasm resources of cedrela tree species, and selecting orchard building parents from the collected germplasm resources; using branches of the garden building parent as scions to be grafted to the rootstock, building a grafting clone, and cultivating the grafting clone into a parent tree body; establishing a dwarf seed orchard by using the parent tree bodies, and shaping and managing the parent tree bodies; carrying out artificial supplementary pollination on the flowering parent tree bodies so as to hybridize the parent tree bodies; managing the parent tree body after pollination until the parent tree body fruit. According to the method, a bearing mother branch at the top of a parent material with excellent characters is grafted to a stock, the stock is planted in a seed garden after survival, the parent tree body is managed, so that the parent tree body is dwarfed, the crown is developed to be heart-shaped, and the hybrid seed can be obtained more easily through artificial supplementary pollination.

Description

Technical Field

[0001] This invention relates to the field of forest tree breeding technology. Specifically, it is a method for hybrid breeding of Toona sinensis plants using dwarf seed orchards. Background Technology

[0002] In forest tree breeding, many difficulties arise when performing hybridization, pollination, seed collection, and management on tall tree species. Dwarfing techniques, which significantly reduce tree height, make these breeding operations much easier to implement. Dwarfing techniques typically involve using plant growth regulators, selecting genotypes with dwarfing characteristics, and implementing specific cultivation and management measures.

[0003] In dwarf seed orchards, tree growth is effectively controlled, the canopy becomes more compact, and the branches are denser. This environment greatly improves seed yield and quality per unit area.

[0004] Hybrid breeding is a commonly used method in the genetic improvement of crops and forest trees, mainly including two types of hybridization: intraspecific hybridization and interspecific hybridization. Intraspecific hybridization refers to hybridization between different varieties or lines within the same species, with the primary purpose of integrating beneficial genes from different varieties to improve adaptability and yield. Interspecific hybridization, on the other hand, involves hybridization between different species, typically aiming to transfer specific desirable traits from one species to another, or to create new varieties possessing the advantages of both parents.

[0005] In forest tree breeding, both intraspecific or intragenus hybridization and interspecific or intergenus hybridization play crucial roles. However, due to the long growth cycle, complex genetic background, and hybridization incompatibility of forest trees, the application of hybridization breeding in forest trees is relatively limited.

[0006] As an important and precious tree species in my country, *Toona sinensis* possesses extremely high value in terms of timber, food, medicine, and ecology. However, traditional breeding methods for *Toona sinensis* are limited by the narrow genetic resources within the species, making it difficult to achieve breakthrough varieties. In recent years, although interspecific and intraspecific hybridization have broadened the genetic base to some extent, the breeding process remains slow due to problems such as large differences in the growth vigor of hybrid parents and low hybridization efficiency. Therefore, exploring an efficient hybridization breeding method for *Toona sinensis* is of paramount importance for accelerating the breeding of new varieties. Summary of the Invention

[0007] Therefore, the technical problem to be solved by this invention is to provide a method for hybrid breeding of Toona sinensis plants using a dwarfing seed orchard. This method involves grafting the fruiting branches at the top of parental materials with superior traits onto rootstocks. After survival, these branches are planted in a seed orchard to obtain parental trees. By strengthening the management of these parental trees, they are dwarfed, with heart-shaped crowns, further facilitating the acquisition of hybrids through artificial pollination.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0009] A method for hybridization breeding of Toona sinensis plants using dwarf seed orchards includes the following steps:

[0010] (1) Collect germplasm resources of Toona sinensis species and select parent trees for orchard establishment from the collected germplasm resources;

[0011] (2) Use the branches of the parent trees in the orchard as scions to graft onto the rootstock to construct a grafted clone, and cultivate the grafted clone into the parent tree;

[0012] (3) Use parent trees to build dwarf seed orchards, and shape and manage the parent trees;

[0013] (4) Artificial pollination is performed on flowering parent trees to enable hybridization of parent trees;

[0014] (5) Post-pollination management of the parent trees until the parent trees bear fruit;

[0015] (6) Harvest the mature fruits on the parent trees and extract the seeds from the fruits;

[0016] In step (3), when shaping and managing the parent trees in the dwarf seed orchard, pruning is used to control the height and crown width of the parent trees. The branches of the parent trees used for grafting are the fruiting mother branches at the top of the parent trees.

[0017] The above method, step (1) includes the following steps:

[0018] (1-1) Based on the target traits of the breeding, individuals with at least one target trait, vigorous growth, straight trunk, full fruit and fruit yield greater than the average fruit yield of the collected germplasm resources were selected from the collected Toona sinensis species to obtain candidate parents; the target traits include fast growth rate, high basic wood density, high heartwood ratio, disease and pest resistance and stress resistance;

[0019] (1-2) The genetic distance between individual plants of candidate parents was calculated using GenAIEx software. Based on the genetic distance calculation results and the traits of each candidate parent, candidate parents with a genetic distance between individual plants greater than the average of all genetic distances between individual plants and with complementary traits were selected for retention. Complementary traits mean that the candidate parents have different target traits. The retained candidate parents have a large genetic distance between individual plants and have complementary traits, which is beneficial for the hybrid offspring to have multiple good traits at the same time and is less likely to cause degradation problems due to inbreeding.

[0020] (1-3) Plant the candidate parents retained in (1-2) in the same plot and perform genetic testing on the candidate parents. Select the parent for orchard establishment from the candidate parents based on the genetic testing results. The indicators measured during genetic testing are the growth rate, adaptability, fruit yield and whether there are pests and diseases of the candidate parents. The criteria for selecting the parent for orchard establishment are: growth rate ranking in the top 90%, adaptability, fruit yield ranking in the top 90% and no pests and diseases.

[0021] (1-4) Determine the combining ability between the parent plants for orchard establishment, and then record the source, morphological characteristics, genetic testing results, combining ability testing results and genetic background of the parent plants for orchard establishment in order to establish a parent plant archive.

[0022] In the above method, in steps (1-3), when determining whether a candidate parent is adaptable, the growth rate and cold resistance of the candidate parent and the control plant are compared. If the growth rate of the candidate parent is greater than or equal to that of the control plant and no frost damage occurs, then the candidate parent is adaptable. The control plant is a plant from the same lineage as the candidate parent, and the control plant is planted in the place of origin of the candidate parent.

[0023] In steps (1-4), when determining the combining ability between the parent plants in the orchard, two parent plants containing at least one pair of contrasting traits are used as hybridization parents and form hybridization combinations. For each hybridization combination, the number of superior traits contained in the parent plants in the F1 generation of the hybridization combination is counted.

[0024] When the two parents in a hybridization possess quantitative traits, let the trait measurement result of the F1 generation be Y. F1 If the phenotypic measurement result of one hybrid parent is a, and the phenotypic measurement result of another hybrid parent is b, then as long as one of the F1 plants has a Y chromosome... F1 > (a+b) / 2, which means that the F1 generation exhibits the superior traits contained in the parents.

[0025] The above method, step (2) includes the following steps:

[0026] (2-1) During the winter dormancy period of Toona sinensis, healthy, disease-free, and plump one-year-old branches at the top of the parent plants for orchard establishment were collected as scions and stored under 4℃ cold storage conditions.

[0027] (2-2) When the sap begins to flow in spring, graft the scion onto the rootstock and wrap the contact area between the rootstock and the scion tightly with plastic film; the rootstock is a Chinese toon seedling with a ground diameter of 0.8-1.2 cm and free from diseases and pests; seedlings grow quickly and are highly adaptable, while the rootstock grown from cuttings is often not as good as the seedlings, which is not conducive to the vigorous growth of grafted clones;

[0028] (2-3) When the scion sprouts and produces new shoots, and the new shoots grow to 10-15cm, remove the plastic film wrapping and set up supports next to the new shoots to fix and protect them.

[0029] (2-4) Carry out fertilizer and water management and pest and disease control, and cultivate for 10 to 12 months until the grafted clonal line grows into the parent tree.

[0030] The above method, step (3) includes the following steps:

[0031] (3-1) Select plots with a slope of less than 30°, a soil organic matter content of more than 2.5%, and a soil bulk density of 1.0 to 1.3 g / cm³ as land for dwarfing seed orchards; in the dwarfing seed orchard land, designate one planting area for each parent tree species, with a spacing of more than or equal to 200m between each planting area;

[0032] (3-2) Before spring budding or after autumn leaf fall, transplant the parent trees to the planting area of ​​the dwarf seed orchard; within a planting area, the spacing between parent trees and the row spacing are both 6m, and one evergreen tree is planted between any two adjacent parent trees; planting one evergreen tree between any two adjacent parent trees facilitates interblock spacing and makes it easier to find the required parent trees during hybridization pollination;

[0033] (3-3) Regularly prune and shape the transplanted parent trees so that the height of the parent trees reaches and is maintained at 2.5-3.5m, the crown width reaches and is maintained at 3.0-3.5m, and the crown of the parent trees spreads out into a heart shape; carry out fertilizer and water management and pest and disease control for the parent trees.

[0034] In the above method, in step (3-3), the parent trees are pruned annually during the growing and dormant periods of the Chinese toon tree, wherein:

[0035] When pruning during the growing season, use methods such as heading back, shortening, and letting go.

[0036] When pruning during the dormant period, use thinning methods;

[0037] When managing fertilizer and water for parent trees, apply compound fertilizer to the parent trees in April each year. The mass ratio of nitrogen, phosphorus and potassium in the compound fertilizer is 15:15:15, and the amount of fertilizer applied to each parent tree is 2 kg.

[0038] The above method, step (4) includes the following steps:

[0039] (4-1) Select parent trees as male and female parents according to the breeding objectives. During the full bloom period of the male parent, collect mature pollen and screen, dry and store the collected pollen in sequence.

[0040] (4-2) Before the anthers of the female parent split open, the female parent is emasculated, and the pollen collected in step (4-1) is used to perform artificial pollination on the female parent; after artificial pollination, the female parent is bagged.

[0041] In the above method, in step (4-1), the pollen collection method is as follows: cut off the inflorescence, with a 3-5cm one-year-old branch at the base of the cut inflorescence; use a paintbrush or brush to brush the pollen from the anthers;

[0042] The pollen screening method is as follows: use a sieve with an aperture of 0.25 mm or less to screen the pollen; the material that passes through the sieve is the pollen.

[0043] Use a desiccator to dry the selected pollen; when storing the pollen, place it in a desiccator and store it at 4°C.

[0044] In the above method, in step (4-2), during artificial pollination, a pollination solution is used for liquid spray pollination. This pollination solution contains: 2–5 g / L pollen, 5 wt%–10 wt% sucrose, 0.005%–0.02% Tween 20 (volume fraction), 50–100 mg / L boric acid, 200–500 mg / L calcium chloride, and 1–5 mg / L of Toona sinensis leaf water extract; the pH of the pollination solution is 5.5–6.5. The sucrose in the pollination solution provides energy to the pollen and maintains the osmotic balance of the solution; Tween helps disperse pollen in water; boric acid promotes pollen tube germination; and calcium ions promote pollen germination and pollen tube elongation. The Toona sinensis leaf water extract contains various natural plant polyphenols and polysaccharides, which have antioxidant effects and provide nutrients to the pollen, prolonging its survival time; the slightly acidic environment is conducive to pollen germination. The amount of pollen used is mainly determined by its particle size and distribution in water. When the amount of pollen is within this range, it can be well dispersed in a solution containing 0.005% to 0.02% Tween 20, which is not easy to clog the spraying equipment and can fully cover the stigma. At the same time, when the amount of Tween 20 added is within this range, it will not affect the contact and binding between the pollen and the style, and will not affect pollen germination.

[0045] The pollination solution described above contains: 3 g / L pollen, 7 wt% sucrose, 0.015% Tween 20 (by volume), 80 mg / L boric acid, 400 mg / L calcium chloride, and 3 mg / L water extract of Toona sinensis leaves; the pH of the pollination solution is 6.

[0046] The technical solution of the present invention achieves the following beneficial technical effects:

[0047] 1. Significantly shortened breeding cycle, simplified and efficient operation: This invention uses scientific methods to select parent trees for orchard establishment, and uses branches of these parent trees as scions to construct dwarf seed orchards through grafting. Grafting significantly advances the flowering period of the parent trees by 5-7 years, generally requiring only 3-5 years to flower and bear fruit, significantly shortening the long cycle of traditional breeding. Furthermore, in the dwarf seed orchards constructed in this invention, the parent trees are dwarfed with heart-shaped crowns, making the entire breeding process simpler and easier, greatly improving the efficiency of artificial pollination, and further enhancing the success rate and efficiency of hybridization breeding.

[0048] 2. Dwarfing seed orchard technology not only reduces the difficulty of parent tree management but also lowers the overall cost of breeding. Through reasonable pruning and tree management, it not only controls tree height and crown width, forming a reasonable crown structure, but also reduces pests and diseases, allowing parent trees to grow healthily, ensuring the stable inheritance of excellent target traits in the parents, effectively reducing losses caused by pests and diseases, and thus improving economic benefits.

[0049] 3. The method provided in this invention ensures that the selected parents have excellent genetic background and hybridization potential through rigorous parent selection and genetic evaluation. When selecting parents for orchard establishment, this invention chooses parents with complementary traits; furthermore, it records and evaluates the combining ability of the parents for orchard establishment. During hybridization, parents with high combining ability are selected, which is beneficial for offspring to concentrate on expressing the excellent target traits contained in the parents, such as high yield, high quality, disease resistance, insect resistance, and stress resistance. This provides a solid foundation and effective approach for the genetic improvement and breeding of new varieties of Toona sinensis species. The establishment of dwarf seed orchards provides an ideal experimental platform for intraspecific and interspecific hybridization breeding, making breeding work more efficient and convenient, and promoting varietal innovation and genetic improvement of Toona sinensis species.

[0050] 4. The present invention also provides a method for artificially assisted pollination of Toona sinensis using pollination solution. The pollination solution can maintain the high vitality of pollen, promote pollen germination and pollen tube growth, and enable the sperm cells carried in the pollen to be delivered to the ovule more quickly and thoroughly, thereby greatly improving the fertilization rate; at the same time, it provides sufficient nutrition for the early development of seeds, making the seeds fuller. Attached Figure Description

[0051] Figure 1 The technical flowchart of this invention. Detailed Implementation

[0052] Example 1

[0053] I. Selecting Parental Trees for Garden Construction

[0054] Based on the target traits of the breeding, robust, disease- and pest-free individuals of the genus *Toona* (which can be *Toona sinensis*, *Toona ciliata*, or *Toona sinensis*) possessing the target traits are selected as candidate parents for orchard establishment. Target traits include: rapid growth rate, high basic wood density, high heartwood percentage, strong adaptability, resistance to diseases and pests, and stress resistance. The candidate parents are comprehensively evaluated through afforestation observation, genetic assessment, and molecular biology methods. Individuals with good genetic background and hybridization potential are selected from the candidate parents as the parent stock for orchard establishment. The relevant technical procedures are as follows: Figure 1 As shown. Specific methods include:

[0055] (1) Determine breeding objectives

[0056] Before starting the selection of candidate parents, the breeding objectives should be clearly defined. Generally speaking, the breeding objectives of Toona sinensis include fast growth rate, high basic wood density, high heartwood ratio, strong adaptability, and resistance to adverse conditions and pests. Technicians can determine the breeding objectives themselves according to specific needs.

[0057] (2) Collecting and evaluating germplasm resources

[0058] High-quality germplasm resources were collected from the natural distribution areas of Toona sinensis species, existing germplasm resource banks, and genetic testing forests. These germplasm resources included wild species, local varieties, improved varieties, or hybrid offspring with potential superior traits. A preliminary evaluation of the collected germplasm resources was conducted, including observation of their performance in terms of target traits, growth status, morphological characteristics (straight trunk), fruit (seed) yield, and fruit quality (fruits should be plump and not shriveled). Individuals exhibiting excellent performance were selected as candidate parents.

[0059] (3) Conduct genetic diversity analysis

[0060] Genetic diversity analysis of candidate parents was conducted using molecular marker techniques (such as SSR or SNP) to understand their genetic differences and relationships, providing a basis for subsequent hybridization. Furthermore, based on the results of the genetic diversity analysis, the inter-plant genetic distance between candidate parents was calculated using GenAIEx software, retaining candidate parents with significant differences in genetic background (inter-plant genetic distance greater than the average of all inter-plant genetic distances). When selecting candidate parents to retain, those with complementary traits should be kept to ensure that offspring inherit the superior target traits from both parents.

[0061] The term "complementary traits" refers to candidate parents having different target traits. For example, if candidate parent A has a high heartwood percentage but a slow growth rate, and candidate parent B has a low heartwood percentage but a fast growth rate, then candidate parents A and B are considered to have complementary traits. Conversely, if candidate parents C, D, and E all have high heartwood percentages and slow growth rates, then candidate parents C, D, and E are considered not to have complementary traits, and only one of them needs to be retained when choosing which candidate parent to keep.

[0062] (4) Genetic testing and comprehensive evaluation

[0063] Candidate parents were planted under identical environmental conditions (within the same plot) to conduct genetic testing. Replication and control sets were used in the genetic testing (each candidate parent was compared with plants from the same family grown in its place of origin) to assess the candidate parents' growth rate, adaptability, fruit yield, and susceptibility to pests and diseases.

[0064] Adaptability refers to the ability of candidate parent plants to adapt to different cultivation environments. When assessing adaptability, the growth rate of the candidate parent plant is compared with that of the control plant of the same family grown in the place of origin. The comparison is made to see if the growth rate of the candidate parent plant is slower and whether it is more cold-resistant. If the growth rate of the candidate parent plant is faster or equal and it can survive the winter normally, it is considered to be adaptable. If the candidate parent plant grows slower or suffers frost damage, it is considered to be unadaptable.

[0065] Based on the results of genetic testing, a comprehensive evaluation of candidate parental materials was conducted. Candidate parents ranking in the top 90% in growth rate, adaptability, and fruit yield, and free from pests and diseases, were selected as the initial orchard parents. For the selected initial orchard parents, their combining ability needed to be determined. Specifically, two initial orchard parents containing at least one pair of contrasting traits were used as hybridization parents to form hybrid combinations. For each hybrid combination, the number of superior traits exhibited by the hybrid parents in the F1 generation was counted.

[0066] A superior trait refers to the trait that better aligns with the breeding objective among a pair of contrasting traits. For example, powdery mildew-resistant individuals and powdery mildew-non-resistant individuals are used as parents to form a hybrid combination. Powdery mildew resistance and non-resistance are a pair of contrasting traits, and powdery mildew resistance is considered a superior trait. If powdery mildew-resistant individuals appear in the F1 generation, it is considered that the F1 generation exhibits a superior trait contained in one of the parents. The more types of superior traits exhibited by the F1 generation from the parents, the higher the combining ability between the two parents is considered to be.

[0067] In addition, many of the target traits in forest tree breeding are quantitative traits. When assessing whether the F1 generation has exhibited the superior traits contained in the parents, the inventors chose to compare the actual measured values ​​of the F1 generation traits with the mean values ​​of the corresponding measured values ​​of the parents to assess whether the F1 generation has exhibited the superior traits contained in the parents.

[0068] For example, there are two hybrid parents, P and P. i and P j P i The fruit yield was 5 kg / plant, which is considered a low-yield individual. (P) j The fruit yield of the first generation is 8 kg / plant, which is considered a high-yielding individual. Therefore, the average fruit yield of the two parents is (5+8)÷2=6.5 kg / plant. Let the fruit yield measurement result of the F1 generation be Y. F1 As long as there is one Y-type F1 plant F1 If the ratio is >6.5, then the F1 generation can be considered to have exhibited the superior trait of high fruit yield found in the parents.

[0069] (5) Establish parent files for the establishment of the orchard

[0070] Detailed files should be established for the selected parent lines used in the orchard, including information such as their origin, morphological characteristics, genetic testing results, combining ability testing results, and genetic background. This will help in tracking and managing the breeding process during subsequent hybridization.

[0071] II. Cultivating grafted asexual lines of parent plants for orchard establishment

[0072] Grafting techniques are used to graft branches of selected parent plants onto suitable rootstocks to obtain grafted clonal seedlings. Grafted clonal lines can maintain the superior traits of the parent plants and achieve rapid propagation. Grafted clonal seedlings should be cultivated and managed under suitable environmental conditions to promote their growth and development. The cultivation methods for grafted clonal lines include:

[0073] (1) Preparations before grafting

[0074] Rootstock selection: Select healthy Chinese toon seedlings with a ground diameter of 0.8-1.2cm and free from pests and diseases as rootstock.

[0075] Scion collection: Select healthy, disease-free, and plump one-year-old top branches (fruiting mother branches) with full buds from superior parent plants as scions. Scions are generally collected during the winter dormancy period of Toona sinensis. After collection, they should be stored at 4℃ to prevent dehydration and sprouting.

[0076] Preparation of grafting tools: Prepare the necessary grafting tools and materials such as knives, plastic film, and binding tape, and ensure that they are clean and sterile.

[0077] (2) Grafting operation

[0078] Grafting time: Grafting of the Toona sinensis genus is generally carried out in spring when the sap begins to flow, at which time the survival rate of grafting is relatively high.

[0079] Grafting methods: Commonly used grafting methods include cleft grafting, whip grafting, and side grafting. The appropriate grafting method should be selected based on factors such as the thickness of the rootstock and scion, and the grafting location. For example, cleft grafting can be used when the rootstock is thicker, while whip grafting can be used when the scion is thinner.

[0080] Key points for grafting: Ensure the cambium layers of the rootstock and scion are aligned and tightly wrapped with plastic film to prevent moisture loss and pathogen invasion. Leave the buds on the scion exposed during wrapping to facilitate subsequent sprouting and growth.

[0081] (3) Management after grafting

[0082] Check the survival rate: After grafting has been completed for a period of time (usually 2-3 weeks), check whether the scions have survived. Surviving scions should be kept fresh and plump, and scions that have not survived should be regrafted in a timely manner.

[0083] Unwrapping: When the scion sprouts and produces new shoots, and these shoots grow to a certain length (generally 10-15cm), the plastic film should be removed promptly to avoid affecting the normal growth of the scion. Care should be taken when removing the wrapping to avoid damaging the newly sprouted buds.

[0084] Setting up supports: Since the new shoots grow rapidly and are relatively fragile after grafting, supports (such as bamboo poles, branches, etc.) should be set up next to the new shoots for fixation and protection to prevent wind breakage or mechanical damage.

[0085] Fertilizer and water management: After grafting, clonal seedlings should be given enhanced fertilizer and water management to promote their rapid growth and development. Fertilization should be mainly nitrogen fertilizer, supplemented with appropriate amounts of phosphorus and potassium fertilizer; watering should follow the principle of "watering when dry" to avoid excessive drought or waterlogging.

[0086] Pest and disease control: Grafted clonal seedlings are more susceptible to pests and diseases due to their weaker growth. Therefore, pest and disease control should be strengthened by regularly spraying pesticides or taking other effective measures for prevention and control. At the same time, it is important to observe and record the occurrence of pests and diseases so that control measures can be adjusted promptly.

[0087] After grafting, cultivate for 10 to 12 months until the grafted clone grows into the parent tree, at which point it can be transplanted.

[0088] III. Construction of Dwarf Seed Orchards

[0089] At the selected location, parent trees are transplanted and customized according to a reasonable density and configuration to establish a dwarf seed orchard. Through appropriate pruning, the height and crown width of the parent trees in the dwarf seed orchard are controlled to create a compact crown structure, facilitating subsequent hybridization and seed collection. Simultaneously, soil management and fertilizer / water regulation are strengthened to provide favorable environmental conditions for the growth and development of the parent trees.

[0090] (1) Site selection and planning of dwarf seed orchard

[0091] Site selection: Choose a plot of land with a slope of less than 30°, a soil organic matter content of more than 2.5%, and a soil bulk density of 1.0 to 1.3 g / cm³ for the dwarf seed orchard. Avoid low-lying areas, saline-alkali land, or plots severely affected by pests and diseases.

[0092] Planning: Based on the area and shape of the dwarf seed orchard, rationally plan the planting areas for different parent tree species. Each parent tree species shall be designated as one planting area, and the distance between each planting area shall be greater than or equal to 200m.

[0093] (2) Planting and configuration

[0094] Planting time: Transplanting should be carried out in spring before budding or in autumn after leaf fall, and the parent trees should be transplanted to the planting area of ​​the dwarf seed orchard.

[0095] Planting density and configuration: Plant along horizontal strips (i.e., according to the horizontal strip planting method), with a plant spacing of 6m × 6m. Each planting area is divided into several sub-areas, with one clonal tree planted in each sub-area, and one evergreen tree such as Phoebe zhennan or Schima superba planted between sub-areas. That is, within a planting area, the plant spacing and row spacing of the parent trees are both 6mm, and one evergreen tree is planted between any two adjacent parent trees.

[0096] Planting method: Dig a suitable planting hole, place the clonal seedling in the hole, ensure that the roots are spread out, then fill the hole with soil and tamp it down, and water it thoroughly.

[0097] IV. Parent Tree Management

[0098] Regular pruning and shaping of parent trees in the dwarfing seed orchard are essential to maintain their aesthetic appeal and structural integrity. Pruning helps regulate branch growth and distribution, promoting ventilation, light penetration, and photosynthesis within the canopy. Simultaneously, strengthened pest and disease control and tree protection are crucial to ensure the healthy growth of parent trees and the successful inheritance of their superior traits. Specific management measures are as follows:

[0099] (1) Tree shaping and pruning

[0100] Shaping Goals: To maintain the good structure and shape of the parent trees, the shaping goal should be to form an open, well-ventilated, light-transmitting, layered, and structurally sound crown. The crown should ideally be heart-shaped to facilitate subsequent pollination and fruit harvesting.

[0101] Pruning timing: Pruning is mainly carried out during the growing season and dormancy period. Pruning during the growing season is mainly to adjust the tree's vigor and promote flower bud differentiation; pruning during the dormancy period is mainly for shaping and thinning out overly dense, diseased, and weak branches.

[0102] Pruning methods: Pruning is carried out according to the growth position and vigor of the branches, using methods such as shortening, heading back, thinning, and letting go. Shortening is mainly used to cultivate main branches and lateral branches; heading back is used to rejuvenate branches; thinning is used to remove overly dense, crossing, overlapping, and diseased or weak branches; letting go is used to promote branch growth and the formation of flower buds.

[0103] Therefore, when pruning during the growing season, the methods of shortening, heading back, and letting go are used; when pruning during the dormant season, the method of thinning is used.

[0104] The goal of pruning is to achieve and maintain the height of the parent tree at 2.5–3.5m and the crown width at 3.0–3.5m.

[0105] (2) Support and fixation of parent trees

[0106] Support material selection: Choose sturdy and durable materials that will not damage the tree as supports, such as bamboo poles, wooden sticks, and wire.

[0107] Support and fixation methods: Appropriate support and fixation methods should be adopted according to the growth condition of the parent tree and the parts that need support. For example, for a tilted main trunk or a large lateral branch, supports can be used to fix it; for small branches inside the canopy, thin wire can be used to tie and fix them.

[0108] Regular inspection and adjustment: Regularly inspect the support and fixation, and adjust and reinforce them in time if loosening or displacement is found.

[0109] (3) Fertilizer management

[0110] Apply general-purpose compound fertilizer (nitrogen, phosphorus and potassium in a mass ratio of 15:15:15) every April, at a rate of 2 kg / tree, to meet the normal growth and development of the tree.

[0111] (4) Pest and disease control

[0112] Prevention is key: Strengthen fertilizer and water management to enhance tree vigor and improve its resistance to diseases and pests. At the same time, maintain cleanliness in the seed orchard and promptly remove fallen leaves, diseased fruit, and other pathogens.

[0113] Biological control: Protecting and utilizing natural enemies such as insects and microorganisms to control pests and diseases. Examples include releasing natural enemy insects and using biological pesticides.

[0114] Chemical control: When pests and diseases are severe, low-toxicity, low-residue chemical pesticides can be used for control. However, attention should be paid to the concentration, timing, and safety interval of application to avoid adverse effects on the environment and trees.

[0115] (5) Other management measures

[0116] Weeding and loosening the soil: Regularly remove weeds from the seed garden and loosen the soil to maintain soil permeability and promote root growth.

[0117] Drainage: In case of waterlogging in summer, ensure proper drainage to prevent root rot caused by water accumulation.

[0118] V. Pollen Collection and Storage

[0119] Based on the breeding objectives, parent trees were selected as the male and female parents (i.e., the parental pairing scheme was determined). During the peak flowering period of the male parent, mature pollen was collected for artificial cross-pollination of the female parent. When collecting pollen, flowers with plump buds and abundant pollen should be selected. After collection, the pollen should be screened, dried, and properly stored to maintain its viability and ensure pollination effectiveness. Storage should be conducted in a low-temperature, dry, and dark environment.

[0120] (1) Pollen collection

[0121] Collection time: Select an appropriate time to collect pollen, usually in May when the flowers are open and the anthers are mature.

[0122] Collection method: Cut off the entire inflorescence with pruning shears, leaving 3-5 cm of one-year-old branch at the base of the inflorescence. Place the inflorescence on tracing paper and gently brush the pollen off the anthers with a clean, dry tool (such as a fine brush or small brush).

[0123] (2) Pollen treatment and storage

[0124] Processing method: Collected pollen should be processed as soon as possible to remove impurities and immature anthers. Fine sieves or gauze can be used for filtration to obtain pure pollen. The sieve aperture should be less than or equal to 0.25 mm; the material that passes through the sieve is pollen.

[0125] Storage conditions: Place the treated pollen in a desiccator and then place the desiccator in a refrigerator at 4°C.

[0126] VI. Artificial pollination

[0127] Based on the breeding objectives and parental pairing plan, artificial pollination was used for hybridization. During pollination, stored pollen should be evenly applied to the stigma of the female parent to ensure sufficient contact and effective pollination. After pollination, the pollen should be marked and recorded for subsequent seed collection and progeny identification. The specific methods are as follows:

[0128] (1) Preparation before pollination

[0129] Pollination tool preparation: Prepare fine tools such as a fine brush, small brush, and cotton swabs for precise pollination. Ensure these tools are clean and sterile to prevent cross-contamination.

[0130] Pollination timing: Closely observe the opening of the flowers and choose the best time for pollination. Generally, it is more effective to emasculate the female parent and pollinate it before the flowers are fully open and the anthers have dehisced.

[0131] Parental labeling: Selected parents are clearly labeled to avoid confusion.

[0132] (2) Pollination operation

[0133] Pollen acquisition: Take an appropriate amount of pollen from the stored paternal pollen, test it and ensure its viability (using conventional staining methods).

[0134] Pollination method: Use a fine brush, small brush, or cotton swab to gently apply pollen to the stigma of the pistil. To ensure successful pollination, repeat the application several times to ensure the pollen is evenly distributed on the stigma.

[0135] Post-pollination marking and recording: After pollination, clearly mark the pollinated flowers and record key data such as pollination date and parent information.

[0136] (3) Bagging and supplementary pollination

[0137] Bagging: After pollination, the inflorescence is covered with a sulfuric acid paper hybridization bag.

[0138] Supplemental pollination: On the second day after pollination, promptly supplemental pollination should be carried out on all pollinated flowers.

[0139] VII. Post-pollination management

[0140] After pollination, it is essential to strengthen the management and protection of the mother tree to promote fruit development and ripening. Through reasonable fertilization and irrigation, and pest and disease control measures, the quality and yield of the fruit can be improved. Simultaneously, the mother tree should be regularly observed and inspected, and the growth status and phenotypic characteristics of the fruit should be recorded.

[0141] (1) Protection of flowers and fruits

[0142] After pollination, the flowers and early fruits are very fragile. Use supports such as bamboo poles to protect the inflorescences and prevent them from falling off due to wind, rain, or other natural factors. Strengthen pest and disease control efforts, regularly check the health of the flowers and fruits, and take appropriate control measures immediately if any signs of pests or diseases are found to prevent their spread and ensure successful pollination.

[0143] (2) Fertilizer management

[0144] Apply appropriate amounts of phosphorus and potassium fertilizers after pollination until the fruit matures.

[0145] (3) Pruning

[0146] After pollination, the tree should be pruned appropriately to remove overly dense, crossing, and overlapping branches, thereby improving ventilation and light penetration in the canopy, which is beneficial to the growth and development of the fruit.

[0147] VIII. Fruit Harvesting and Processing

[0148] Harvest and process the fruit promptly when it is ripe. After collection, process the fruit immediately to maintain seed viability and quality. Store the fruit in a dry, well-ventilated, and dark environment.

[0149] Harvesting time and method: Select the appropriate harvesting time and method according to the maturity and intended use of the fruit. Generally speaking, fruits used for hybridization breeding can be harvested when the fruit color changes from green to brown.

[0150] Seed treatment and storage: After harvesting, the fruits are treated to extract the seeds. The fruits are placed in a dry, well-ventilated place to dry, and the seeds are collected after they fall off naturally. After cleaning, the seeds are packaged and labeled, and stored in a dry, cool, and well-ventilated warehouse for later use.

[0151] Through the implementation and management of the above steps, this invention can effectively carry out intraspecific and interspecific hybridization breeding of the Toona genus, providing strong support for the innovation and utilization of Toona germplasm resources.

[0152] Example 2

[0153] In this embodiment, based on Embodiment 1, the method of artificial pollination is changed, and the pollination liquid is sprayed to artificially pollinate the Chinese toon.

[0154] In this embodiment, the pollination solution was prepared by dispersing sucrose, Tween 20 (volume fraction 0.015%), boric acid (80 mg / L), calcium chloride (400 mg / L), and Chinese toon leaf water extract in sterile water, according to the formula: 1 L of pollination solution contains 7 wt% sucrose, 0.015% Tween 20 (volume fraction), 80 mg / L boric acid, 400 mg / L calcium chloride, and 3 mg / L Chinese toon leaf water extract. After stirring evenly, 3 g of dried pollen was added, the pH was adjusted to 6, and then the solution was stirred evenly with a magnetic force.

[0155] The preparation method of the water extract of Toona sinensis leaves is as follows: Select healthy tender Toona sinensis leaves, wash them, and add the leaves to the water at a ratio of 4 L of water to 1 kg of leaves. Heat the water to a boil and continue boiling for 30 minutes, then let it cool naturally to room temperature. Repeat the process of heating to a boil, boiling for 30 minutes, and then letting it cool naturally to room temperature. After solid-liquid separation, centrifuge at 3000 rpm for 15 minutes, and then evaporate the water at a temperature of 60°C or lower until dry.

[0156] When performing artificial pollination, use a handheld atomizer or spray bottle to spray the pollination solution onto the style of the female pistil, ensuring that each style is fully in contact with the pollination solution.

[0157] The pollination success rate of the artificial pollination method in this embodiment is approximately 92.3%, while the success rate of artificial pollination in Example 1 is approximately 70%. Meanwhile, the weight of 100 Toona sinensis seeds obtained in this embodiment is approximately 1.1g, significantly higher than the weight of 100 Toona sinensis seeds obtained in Example 1 (approximately 0.8g), indicating that the Toona sinensis seeds obtained in this embodiment are more plump.

[0158] In other embodiments, the contents of each component in the pollination solution are adjusted within the following ranges: 2–5 g / L pollen, 5 wt%–10 wt% sucrose, 0.005%–0.02% Tween 20 (volume fraction), 50–100 mg / L boric acid, 200–500 mg / L calcium chloride, and 1–5 mg / L Chinese toon leaf water extract.

[0159] In this embodiment, sucrose provides energy to the pollen and maintains the osmotic balance of the pollination solution. Tween 20 improves pollen dispersibility and prevents aggregation without interfering with the contact and binding between pollen and style. Boric acid and calcium ions promote pollen tube germination and elongation, respectively. Water extract of Toona sinensis leaves provides certain nutrients to the pollen and helps prevent pollen oxidation, thus prolonging pollen survival. A slightly acidic environment is conducive to pollen germination. Furthermore, the amount of pollen added in this embodiment is appropriate, effectively covering the style without easily clogging the nozzle.

[0160] Pollination solution helps maintain high pollen viability, promotes pollen germination and pollen tube growth, and allows sperm cells carried in the pollen to reach the ovules more quickly and thoroughly, thus significantly increasing the fertilization rate. Increased fertilization rate means more ovules develop normally. Simultaneously, pollination solution provides ample nutrition for early seed development, improving seed plumpness. In other words, spraying pollination solution not only improves pollen viability and promotes successful pollination, but also ensures that developing seeds receive more nutrients and become plumper.

[0161] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of the claims of this patent application.

Claims

1. A method for hybrid breeding of Toona sinensis plants using dwarf seed orchards, characterized in that, Includes the following steps: (1) Collect germplasm resources of Toona sinensis species and select parent trees for orchard establishment from the collected germplasm resources; (2) Use the branches of the parent trees in the orchard as scions to graft onto the rootstock to construct a grafted clone, and cultivate the grafted clone into the parent tree; (3) Use parent trees to build dwarf seed orchards, and shape and manage the parent trees; (4) Artificial pollination is performed on flowering parent trees to enable hybridization of parent trees; (5) Post-pollination management of the parent trees until the parent trees bear fruit; (6) Harvest the mature fruits on the parent trees and extract the seeds from the fruits; In step (3), when shaping and managing the parent trees in the dwarf seed orchard, pruning is used to control the height and crown width of the parent trees.

2. The method according to claim 1, characterized in that, Step (1) includes the following steps: (1-1) Based on the target traits of the breeding, individuals with at least one target trait, vigorous growth, straight trunk, full fruit and fruit yield greater than the average fruit yield of the collected Toona sinensis species were selected from the collected germplasm resources to obtain candidate parents. (1-2) Use GenAIEx software to calculate the genetic distance between individual plants among candidate parents. Based on the calculation results of the genetic distance between individual plants and the traits of each candidate parent, select candidate parents whose genetic distance between individual plants is greater than the average of the genetic distance between all individual plants and who have complementary traits for retention. (1-3) Plant the candidate parents retained in (1-2) in the same plot and perform genetic testing on the candidate parents. Select the parent for orchard establishment from the candidate parents based on the genetic testing results. The indicators measured during genetic testing are the growth rate, adaptability, fruit yield and whether there are pests and diseases of the candidate parents. The criteria for selecting the parent for orchard establishment are: growth rate ranking in the top 90%, adaptability, fruit yield ranking in the top 90% and no pests and diseases. (1-4) Determine the combining ability between the parent plants for orchard establishment, and then record the source, morphological characteristics, genetic testing results, combining ability testing results and genetic background of the parent plants for orchard establishment in order to establish a parent plant archive.

3. The method according to claim 2, characterized in that, In steps (1-3), when determining whether a candidate parent is adaptable, the growth rate and cold resistance of the candidate parent and the control plant are compared. If the growth rate of the candidate parent is greater than or equal to that of the control plant and no frost damage occurs, then the candidate parent is adaptable. The control plant is a plant from the same lineage as the candidate parent and is planted in the place of origin of the candidate parent. In steps (1-4), when determining the combining ability between the parent plants in the orchard, two parent plants containing at least one pair of contrasting traits are used as hybridization parents and form hybridization combinations. For each hybridization combination, the number of superior traits contained in the parent plants in the F1 generation of the hybridization combination is counted.

4. The method according to claim 1, characterized in that, Step (2) includes the following steps: (2-1) During the winter dormancy period of Toona sinensis, healthy, disease-free, and plump one-year-old branches at the top of the parent plants for orchard establishment were collected as scions and stored under 4℃ cold storage conditions. (2-2) When the sap begins to flow in spring, graft the scion onto the rootstock and wrap the contact area between the rootstock and the scion tightly with plastic film; the rootstock is a Chinese toon seedling with a ground diameter of 0.8-1.2 cm and free from diseases and pests; (2-3) When the scion sprouts and produces new shoots, and the new shoots grow to 10-15cm, remove the plastic film wrapping and set up supports next to the new shoots to fix and protect them. (2-4) Carry out fertilizer and water management and pest and disease control, and cultivate for 10 to 12 months until the grafted clonal line grows into the parent tree.

5. The method according to claim 1, characterized in that, Step (3) includes the following steps: (3-1) Select plots with a slope of less than 30°, a soil organic matter content of more than 2.5%, and a soil bulk density of 1.0 to 1.3 g / cm³ as land for dwarfing seed orchards; in the dwarfing seed orchard land, designate one planting area for each parent tree species, with a spacing of more than or equal to 200m between each planting area; (3-2) Before spring budding or after autumn leaf fall, transplant the parent trees to the planting area of ​​the dwarf seed garden; within a planting area, the spacing between parent trees and the row spacing are both 6m, and one evergreen tree is planted between any two adjacent parent trees. (3-3) Regularly prune and shape the transplanted parent trees so that the height of the parent trees reaches and is maintained at 2.5-3.5m, the crown width reaches and is maintained at 3.0-3.5m, and the crown of the parent trees spreads out into a heart shape; carry out fertilizer and water management and pest and disease control for the parent trees.

6. The method according to claim 5, characterized in that, In step (3-3), the parent trees are pruned annually during the growing season and dormancy period of the Chinese toon tree, including: When pruning during the growing season, use methods such as heading back, shortening, and letting go. When pruning during the dormant period, use thinning methods; When managing fertilizer and water for parent trees, apply compound fertilizer to the parent trees in April each year. The mass ratio of nitrogen, phosphorus and potassium in the compound fertilizer is 15:15:15, and the amount of fertilizer applied to each parent tree is 2 kg.

7. The method according to claim 1, characterized in that, Step (4) includes the following steps: (4-1) Select parent trees as male and female parents according to the breeding objectives. During the full bloom period of the male parent, collect mature pollen and screen, dry and store the collected pollen in sequence. (4-2) Before the anthers of the female parent split open, the female parent is emasculated, and the pollen collected in step (4-1) is used to perform artificial pollination on the female parent; after artificial pollination, the female parent is bagged.

8. The method according to claim 7, characterized in that, In step (4-1), the pollen collection method is as follows: cut off the inflorescence, with a 3-5cm one-year-old branch at the base of the cut inflorescence; use a paintbrush or brush to brush the pollen from the anthers; The pollen screening method is as follows: use a sieve with an aperture of 0.25 mm or less to screen the pollen; the material that passes through the sieve is the pollen. Use a desiccator to dry the selected pollen; when storing the pollen, place it in a desiccator and store it at 4°C.

9. The method according to claim 7, characterized in that, In step (4-2), during artificial pollination, a pollination solution is used for liquid spray pollination. The pollination solution contains: 2-5 g / L of pollen, 5 wt%-10 wt% of sucrose, 0.005%-0.02% of Tween 20 by volume, 50-100 mg / L of boric acid, 300-500 mg / L of calcium chloride, and 1-5 mg / L of water extract of Toona sinensis leaves; the pH of the pollination solution is 5.5-6.

5.

10. The method according to claim 9, characterized in that, The pollination solution contains: 3 g / L pollen, 7 wt% sucrose, 0.015% Tween 20 (volume fraction), 80 mg / L boric acid, 400 mg / L calcium chloride, and 3 mg / L Chinese toon leaf water extract; the pH of the pollination solution is 6.

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