CN114303939B - Eucalyptus artificial crossbreeding method based on female parent dwarfing - Google Patents

Abstract

The invention discloses a female parent dwarf-based eucalyptus artificial crossbreeding method, which is characterized by comprising the following steps of: (1) screening excellent single plants; (2) stock cultivation; (3) grafting; (4) cultivating grafted plants; (5) pollen collection; (6) pollen storage; (7) artificial control pollination; (8) post-pollination management; (9) fruit harvesting; (10) fruit treatment. The dwarf plants are grafted by the method, flowering is carried out in the second year after grafting, the dwarf plants can be used as female parents for artificial control pollination on the ground, a great amount of manpower and material resources are saved, a great number of hybrid seeds are harvested, and the dwarf plants can also be used as male parents for collecting pollen; the dwarf breeding garden is constructed, so that the force of hybridization breeding can be increased, and the orthogonal, backcross and test cross can be carried out simultaneously, thereby obtaining a hybridization offspring population with wider genetic basis, and having milestone significance for accelerating the eucalyptus breeding process.

Description

Eucalyptus artificial crossbreeding method based on female parent dwarfing

Technical Field

The invention relates to the technical field of tree breeding and eucalyptus cultivation, in particular to a female parent dwarf-based eucalyptus artificial hybridization breeding method.

Background

Eucalyptus is native to australia and belongs to the general names of Myrtaceae (Myrtaceae), cupboards (Angophora cav.), parasol (Corymbia K.D. Hill & L.A.S. Johnson) and Eucalyptus (Eucalyptus L' Herit) tree species. Eucalyptus is abundant in resources, and there are 808 species and 137 subspecies or varieties, counting 945 taxonomic groups. The growth is fast, the application is wide, and the tree is one of three widely accepted artificial forest tree species in the world. The method starts to lead in China sporadically from 1890, and is implemented in Dongmen forest farm in 1982 'the Australian technology cooperation Dongmen eucalyptus demonstration forest project', and a large number of lead experiments are systematically carried out, so that six suitable tree species of Eucalyptus robusta, eucalyptus grandis, eucalyptus barks, eucalyptus robusta and Eucalyptus robusta are selected. After 1986, extensive artificial crossbreeding was performed between nine parent tree species of Eucalyptus urophylla, eucalyptus grandis, eucalyptus rimonia, eucalyptus cyrtophylla, and part of the hybrid, and 1200 hybrid combinations were developed, of which 905 combinations obtained seeds. A group of excellent F1 generation single plants which grow rapidly, have large genetic gain and strong stress resistance are bred from the filial generation, the clone is established by asexual propagation methods such as cuttage, tissue culture and the like, and the excellent clone is screened out by clone comparison measurement and regional test, so that the method is popularized and afforested in a large scale in the south China. The popularization of the excellent filial generation clone has profound influence on the development of forestry in China.

Artificial control of pollination is one of the important methods if eucalyptus growth and quality are to be continuously increased. It can be determined according to subjective intention of human body-breeding goal, and can obtain excellent hybridization combination which does not exist in nature so as to produce hybridization filial generation with greater genetic gain. Along with the development of a eucalyptus asexual seedling raising technology, the development of asexual forestry by adopting a mode of sexual breeding and asexual utilization becomes reality, and the importance of artificial hybridization breeding is more prominent.

To obtain good progeny hybrids, it is first necessary to select the good parent. The parental selection requires that the following conditions be met: the hybrid parent has affinity; the parent has desirable superior traits; the parent is a selected excellent single plant; the advantages and disadvantages of the parent and the mother are complementary. The problem is that the majority of individuals with superior parental performance are large trees that are nearly mature, which necessitates manual control pollination at crowns of more than ten meters high, typically by building scaffolding or using overhead vehicles, as shown in fig. 1. The artificial control pollination is a complex and fine work, and has long time consumption and low workload, so that the difficulty of artificial cross breeding of eucalyptus is high, the cost is high, and the quantity of obtained hybrid seeds is limited. In order to solve the problems, the main method is to dwarf the parents so as to achieve the aim of pollination on the ground. The dwarfing technique includes grafting, high-altitude layering and chemical agent such as paclobutrazol. The high-altitude layering is carried out on the tree crown, so that the difficulty is high. Whereas paclobutrazol dwarfing method can be used for young trees, is not suitable for passing through the preferred big tree. Grafting dwarfing is relatively easy to implement, but the technical requirement is quite high, whether the grafting can survive or not, and whether the grafting can bloom in time after survival is a constraint factor. Therefore, the scion selection, grafting method and grafted plant cultivation become key technical links

In the prior art, the research on eucalyptus hybridization breeding is relatively more, and the exploration on the artificial control pollination of the female parent dwarfed eucalyptus is relatively less. Xue Peng et al, research on artificial hybridization breeding technology of eucalyptus, show that dwarfing hybrid gardens are built after grafting, 4-6 plants of each dwarfing material are helpful for collecting pollen of male parent, and parent combination is selected to increase diversity of filial generation; the main purpose is genetic diversity research, in the experimental exploration phase, and the paclobutrazol is used for controlling high growth. Paclobutrazol is a plant growth retardant, and has obvious application effect in herbaceous plants by interfering physiological activities in the plant body to control the growth and development of the plant. In woody plants such as eucalyptus, the concentration of paclobutrazol is difficult to grasp, the effect of controlling high growth is not obvious when the concentration is low, and the plant is malformed or even dies when the concentration is Gao Zao. More importantly, the plant dwarfing agent is used for dwarfing plants targeted for breeding, can intuitively show female parent character changes such as plant dwarfing, fruiting increase and the like, but does not have adverse effects on the generation and even have mutation, and is not clear. Zeng Ji et al, in the initial report of the research on the dwarf artificial hybridization technology of eucalyptus, show that the growth of the lateral branches is increased by cutting off the top at the height of 1 m of the stock, removing the top advantages, fixing the lateral branches on a manually arranged steel wire rope, pressing the height of the branches to achieve the aim of dwarf artificial hybridization breeding, grafting, and carrying out artificial control pollination after the scion survives. The problem is that the diameter is larger when the height of the stock reaches 1 meter, and the lateral branches of the stock consume water and nutrients, so that the grafting survival rate is low. In addition, because eucalyptus is a fast-growing tree species, when the stock is left to be 1 meter high, nutrients of lateral branches are obtained, and the extension is fast. However, the top dominance removal of the side branches is insufficient to control plant height growth, so that the dwarfing effect of eucalyptus is limited.

Disclosure of Invention

Aiming at the technical problems, the invention provides a female parent dwarfing eucalyptus artificial hybridization breeding method, which grafts the fruiting female branches of the crowns of the fine individual trees of the fine families such as the tail eucalyptus, the grandis eucalyptus, the round-angle eucalyptus and the rough-skin eucalyptus onto the stock of a seedling cup, and transplants the stock of the seedling cup into a dwarfing breeding garden for cultivation after survival. When the female parent reaches the flowering phase, the pollinating is manually controlled on the ground by using the excellent single plant pollen of excellent families such as eucalyptus urophylla, eucalyptus grandis, eucalyptus angustifolia, eucalyptus robusta and the like, so that the corresponding hybrid seeds are obtained.

In order to achieve the above purpose, the technical scheme provided by the invention is as follows:

a female parent dwarfing-based eucalyptus artificial hybridization breeding method, which comprises the following operation steps:

(1) Screening of good individuals: calculating family accumulation and annual average growth amount by a statistical method through breast diameter and tree height which are continuously measured for many years, analyzing and comparing to select excellent families, selecting 1-2 excellent single plants with optimal phenotypic characters from the excellent families with mature ages, numbering, and cultivating the excellent single plants as scions; the method for determining the excellent family is to perform growth quantity (chest diameter,High tree height, accumulation amount), dry shape, resistance (wind resistance, disease resistance and insect resistance) and other characters, constructing an excellent family and single plant selection index equation, comprehensively scoring 20% before ranking to be used as a selected family, calculating genetic gains of the selected family and single plant, and screening excellent single plant; specifically, the average accumulation rank is first examined (annual average accumulation of 30-45 m) ³ ·hm ^-2 ) Then, the chest diameter, the tree height and other ranges of the excellent single plant are changed along with the growth age, the annual average chest diameter range of the 6-year-old excellent single plant is 2.5-3.5 cm, and the annual average tree height range is 3.5-4.5 m;

(2) Stock cultivation: sowing and cultivating seedlings of the same tree species as the single plant with excellent scion, selecting good-growing seedlings after germination, transplanting the seedlings into seedling cups with the diameter of 20cm, and culturing for later use;

(3) Grafting: in spring or autumn, climbing a ladder or directly on the ground by using a special high-altitude sampler, collecting the fruiting parent branch of the excellent single plant of the scion in the step (1) as the scion, soaking the branch cut part with water for carrying back, selecting the well-cultivated stock in the step (2) in a nursery, cutting the stock until the diameter of the stock is more than or equal to 0.35cm, cutting the stock to 10-15 cm, trimming and grafting the fruiting parent branch onto the stock, and adopting a split grafting method, a butt grafting method and a cutting grafting method according to the size of the stock (when the scion is as large as the stock, the butt grafting method and the split grafting method are adopted, and when the scion is small and the stock is large, the cutting grafting method is adopted, so that the cambium alignment of the stock and the scion is ensured, and after the alignment, the grafting is completed by tightly binding the interface by using a sealing film; wherein, the scion can leave leaves by a split grafting method and a butt grafting method; the scion is a segment with a section, and leaves are not left; grafting with fruiting parent branches, enabling the grafted eucalyptus tree to bloom in the next year, and trimming the top of the trunk of the dwarf eucalyptus tree in the later period to control the length of the trunk;

(4) Cultivating grafted plants;

(5) Collecting pollen;

(6) Pollen storage;

(7) Manual control pollinating: when the situation that the grafted plants transplanted to the dwarf breeding garden in the step (4) grow up starts to bloom is observed, artificial control pollination is started;

(8) Management after pollination: reducing human and animal interference after pollination, performing windproof and rainproof management on plants convenient for centralized management, preventing and controlling falling or mildew, removing isolation bags 7-14 days after the last pollination, frequently observing the growth of branches and the development condition of fruits in four to six months until the fruits are ripe, and when competing branches affecting the growth of pollination branches are found, timely shearing off, ensuring the development of the fruits, simultaneously performing the work of pest control and the like;

(9) Harvesting fruits: timely harvesting when the fruit is observed to expand and crack in mature seasons (generally 3-4 months of the second year of hybridization), counting fruit setting rate, and filling in artificial hybridization breeding files including parent condition, pollen collection and emasculation, pollination time, fruit setting rate, seed number and the like;

(10) Fruit treatment: after picking fruits, airing the fruits on bamboo basket (kraft paper), after the fruits are cracked, collecting seeds separated from the fruits, and obtaining seeds of hybrid first generation (F1), and storing the seeds for later use after strict marking.

Preferably, the eucalyptus fine single plant selection standard in the step (1) is divided into two stages; first stage selection criteria: (a) no pest characteristics: no obvious insect pest characteristics; (B) dry straightening: the dry index was scored into 5 grades: bending for 1 minute; in the average straightness range, a plurality of trunks are arranged at 2 minutes; within the average straightness range, a single trunk is 3 minutes; slightly curved, single trunk, 4 minutes; the shape of the trunk is straight, the trunk is single, and the trunk is 5 minutes; (C) little branching: the branch indexes are scored according to 3 grades according to the branch size; big branch 1, middle branch 2 and small branch 3; (D) cumulative growth index: average annual accumulation of 30-45 m ³ ·hm ^-2 The method comprises the steps of carrying out a first treatment on the surface of the Second stage selection criteria: (E) bark and wood properties: mainly considers the very important wood basic density of the pulp wood tree species, and requires the wood basic density index in the rotation period (6 years) to be 500-600 kg/m ³ The method comprises the steps of carrying out a first treatment on the surface of the (F) germination capacity and cutting viability: as a asexual propagation material, the selected wood must have strong germination capacity, and the survival rate of the rod insertion must not be too low, at least more than 70%.

Preferably, the fruiting parent branch in the step (3) is a branch which grows out in the year and becomes fruiting branch, flowers and fruits in the 2 nd year.

Preferably, the grafted plants in the step (4) are cultivated by placing grafted seedlings into a semi-automatic temperature and humidity control spray irrigation system for management and protection, controlling the humidity to be more than 70%, preventing and controlling plant diseases and insect pests, after 7-14 days, starting sprouting and growing gradually, timely removing the sprouting on the stock to prevent the stock from competing for nutrients and moisture, after the grafting opening is completely healed, applying fertilizer appropriately to promote growth, planting the grafted seedlings into a dwarfing breeding garden after the new buds grow to 10-15 cm, and timely developing tending management such as weeding and fertilization after planting, wherein the plant row spacing is 5.0×5.0m, and flowering is started after one year.

Preferably, the pollen in the step (5) is collected as the pollen is collected by a special high-altitude sampler when the flower bud capsule cap of the selected excellent male parent is whitened and falls off, the branches are picked up by the special high-altitude sampler, the leaves are removed, the branch incision is quickly inserted into water for culture, the branches are brought back to a laboratory, the flower buds which are already opened are sheared off, the branches are transferred to new water for culture, the pollen is removed when the plastic cap falls off completely, the flower silk is exposed, the flower anther starts to release pollen, the flower anther is placed in a glass dryer, the glass dryer is dried for 24-48 hours, and the sieved pollen is filled into a small bottle after sterilization and drying treatment.

Preferably, the pollen in step (6) is stored as: in order to solve the problem of the shortage of parent-parent flowering period in artificial hybridization breeding among different tree species and varieties, the pollen obtained in the step (5) is stored in a special refrigerator or a cold storage, and the pollen is easy to lose activity at normal temperature, so that the requirement on the storage temperature is strict, and the temperature is maintained at-4 ℃.

Preferably, the artificial pollination in step (7) comprises the following items (a) (b) (c);

(a) Pollen germination test: in order to perform artificial control pollination by utilizing the pollen stored in the step (6), pollen activity needs to be detected, and a pollen germination test is carried out, the specific method is that a small amount of pollen is extracted at 26 ℃, and the pollen is cultured on a culture medium of 1.5 percent agar and 35 percent sucrose and 100 to 250ppm boric acid for 24 hours, when the pollen germination rate is less than or equal to 30 percent, the pollen activity is low or the pollen activity is lost, and the pollen is not used for pollination any more; the germination rate of pollen is 30-60%, the pollen can be used for pollination, and the pollination amount of a single stigma is increased to achieve the pollination effect; the germination rate of pollen is more than or equal to 60 percent, the pollen can be used for pollination, and the pollination amount of a single stigma can be less;

(b) Emasculation and bagging isolation: when female parent flower buds are light yellow and calyx rings are cracked, the flower with the opened nearby branches and the cyan flower buds are cut off in the optimal period, then a surgical scissors or a blade is used for cutting off flower bud capsule covers to be removed, the flower bud capsules can also be directly taken off by hands, stamens are removed by using forceps or a blade, pollen at the column heads and the flower cup bases is brushed by using a fine brush, the column heads are washed by distilled water or sterile water so as to prevent pollen pollution, after the emasculation is finished, a sulfuric acid paper bag or a cotton cloth bag is sleeved for isolation, the cloth bag is supported by a wire spiral frame, the two ends of the cloth bag are tied on the branches, cotton is padded at the contact position of the wire and the branches so as to avoid scratching the branches, and the sulfuric acid paper bag is bound by cotton threads, so that the use is convenient but the ventilation property is poor;

(c) Control pollination: and (3) 5-6 days after emasculation, when the stigma is observed to be expanded and white mucus exists, opening one end of the bagging, dipping the pollen detected in the step (6) by using a medical cotton swab, lightly smearing the pollen on the stigma, wherein each cotton swab is only used for one hybridization combination, preventing pollen pollution, ensuring that the pollen is uniformly smeared on the stigma, and in order to improve the pollination success rate, pollinating once again 1-2 days after the first pollination, cutting off one third of the upper end of the stigma, smearing the pollen at the incision at one time, then bagging and isolating, and marking the pollination completion including pollination date, pollen information, pollination bud number and the like.

Compared with the prior art, the invention has the following beneficial effects:

(1) The invention uses the branches of the excellent single plants in the excellent family of eucalyptus to graft, belongs to asexual propagation, flowers and fruits after the branches sprout, can completely maintain the hereditary character of the excellent tree, and has more reliable hereditary character of the female parent and no risk of variation compared with the method for treating young plants by using chemical agent paclobutrazol;

(2) The invention adopts the grafting of the crowns of the excellent single plants in the excellent family of eucalyptus to grafting the fruit tree branches, the physiological age of the scions is in a mature state, after the grafting survival, the sprouting strips grow and develop, and can bloom and fruit in time, and the biological characteristics of the fruit shape and the like are consistent with those of the excellent single plants of eucalyptus;

(3) After dwarfing female parent, artificial control pollination is carried out on dwarfed eucalyptus, emasculation, pollination, bagging isolation and seed collection can be carried out on the ground, and the artificial hybridization breeding method based on dwarfed eucalyptus greatly saves manpower and material resources, does not need a lifter or a scaffold, greatly improves the pollination amount of one person in one day, simultaneously avoids the risk of high-altitude operation, and has the advantages of more harvested seeds and low breeding cost;

(4) The dominant effect of the eucalyptus hybrid progeny is that the excellent hybrid progeny generated by utilizing dominant gene recombination is selected; according to the eucalyptus artificial hybridization breeding method based on the dwarf female parent, because the quantity of the dwarf female parent harvested hybrid seeds is increased, the quantity of individuals with high genetic gain in the hybrid progeny is increased, a larger population is provided for screening the hybrid progeny, the probability of selecting excellent single plants is greatly increased, and the advantage of hybridization breeding can be better reflected;

(5) The whole set of female parent dwarf hybridization breeding technology is obtained, 36 excellent hybrid first-generation (F1) clone is cultivated by the method, 12 excellent varieties are selected, 1 of the excellent varieties is determined to be fine varieties by the Guangxi Zhuang autonomous region forest fine variety committee, so that the method has good application value;

(6) The dwarf plants grafted by the method can be used as female parent for pollination and can also be used as male parent for collecting pollen; the dwarf breeding garden is constructed, so that the force of hybridization breeding can be increased, and the orthogonal, backcross and test cross can be carried out simultaneously, thereby obtaining a hybridization offspring population with wider genetic basis, and having milestone significance for accelerating the eucalyptus breeding process.

Drawings

Figure 1 is a prior art high altitude pollination.

FIG. 2 is a view of the dwarfed eucalyptus pollination site obtained in accordance with the present invention.

FIG. 3 shows a grafted seedling according to the method of the present invention.

FIG. 4 shows the flowering phase of the dwarf female parent of Eucalyptus rimonia according to the present invention.

FIG. 5 is a pollen collection method of the present invention.

FIG. 6 shows the inventive method of flower and branch bagging isolation.

FIG. 7 is a schematic illustration of hybrid seed treatment and sun-drying according to the method of the present invention.

Detailed Description

The following detailed description, in conjunction with the accompanying drawings, describes in detail, but it is to be understood that the scope of the invention is not limited to the specific embodiments. The raw materials and reagents used in the examples were commercially available unless otherwise specified.

The eucalyptus fine single plant selection standard in the step (1) of each embodiment is divided into two stages; first stage selection criteria: (a) no pest characteristics: no obvious insect pest characteristics; (B) dry straightening: the dry index was scored into 5 grades: bending for 1 minute; in the average straightness range, a plurality of trunks are arranged at 2 minutes; within the average straightness range, a single trunk is 3 minutes; slightly curved, single trunk, 4 minutes; the shape of the trunk is straight, the trunk is single, and the trunk is 5 minutes; (C) little branching: the branch indexes are scored according to 3 grades according to the branch size; big branch 1, middle branch 2 and small branch 3; (D) cumulative growth index: average annual accumulation of 30-45 m ³ ·hm ^～2 The method comprises the steps of carrying out a first treatment on the surface of the Second stage selection criteria: (E) bark and wood properties: the important wood basic density of the pulp wood tree species is mainly considered, and the wood basic density index in the rotation period (6 years) is required to be 500-600 kg/m1; (F) germination capacity and cutting viability: as a asexual propagation material, the selected wood must have strong germination capacity, and the survival rate of the rod insertion must not be too low, at least more than 70%.

Example 1

A female parent dwarfing-based eucalyptus artificial hybridization breeding method comprises the following specific operation steps:

(1) Screening of good individuals: in the eucalyptus urophylla family test forest, according to the eucalyptus excellent single plant optimization standard, the family accumulation and the annual average growth are calculated through a statistical method by using the chest diameter and the tree height which are measured for a plurality of years continuously, and an excellent family is selected after analysis and comparison,then selecting 1-2 excellent single plants with optimal phenotype characters from mature excellent families, numbering, and cultivating the excellent single plants as scions; the excellent single plant with the best phenotypic character is an individual with high tree height, maximum breast diameter and small trunk straight and sharp angle; the specific mode of determining the excellent families is to perform genetic variation analysis of growth (breast diameter, tree height, accumulation), dry shape, resistance (wind resistance, disease resistance and insect resistance) material property and other characters among families, construct an excellent family and single plant selection index equation, comprehensively score 20% of the top ranking as an optional family (namely the top 20 of the top ranking 100), calculate genetic gains of the optional family and single plants, screen the excellent single plants, and look at the average accumulation ranking (30-45 m of annual average accumulation ³ ·hm ^-2 ) Then, the chest diameter, the tree height and other ranges of the excellent single plant are changed along with the growth age, the annual average chest diameter range of the 6-year-old excellent single plant is 2.5-3.5 cm, and the annual average tree height range is 3.5-4.5 m;

(2) Stock cultivation: sowing eucalyptus urophylla seeds, cultivating seedlings, selecting good-growing seedlings after germination, transplanting the seedlings into seedling cups with the diameter of 20cm, and culturing for later use;

(3) Grafting: in spring, climbing a ladder or directly on the ground by using a special high-altitude sampler for seed collection, collecting the fruiting parent branch of the excellent single plant of the scion in the step (1) as the scion (the fruiting parent branch is the fruiting branch which grows out in the year and flowers and fruits in the 2 nd year), soaking the notch of the branch with water to bring back, selecting the well-cultivated stock in the step (2) by using a nursery, cutting the stock to 10 cm with the ground diameter more than or equal to 0.35cm, trimming and grafting the fruiting parent branch onto the stock by using a split grafting method, ensuring the alignment of cambium of the stock and the scion, and binding the interface by using a sealing film after alignment to finish grafting; grafting with fruiting parent branches, enabling the grafted eucalyptus tree to bloom in the next year, and trimming the top of the trunk of the dwarf eucalyptus tree in the later period to control the length of the trunk;

(4) Cultivating grafted plants: placing grafted seedlings into a semi-automatic temperature-control and humidity-control spray irrigation system for management and protection, controlling the humidity to be more than 70%, performing pest control, starting sprouting and growing up gradually after 7 days, timely removing the sprouting on the stock to prevent the stock from competing for nutrients and moisture, after the grafting opening is completely healed, applying a proper amount of additional fertilizer to promote growth, when the new bud grows to 15 cm and the growth is stable, planting the grafted seedlings into a dwarfing breeding garden, performing tending management such as weeding and fertilization in time after planting, and starting flowering after one year;

(5) Pollen collection: taking eucalyptus grandis as a hybrid male parent, taking off flowers and branches by a special high-altitude sampler when the flower and bud capsule covers of the single plant of the good male parent are selected to be whitened and fall off according to the flowering period of eucalyptus grandis, taking off leaves, quickly inserting branch cuts into water for culture, taking back to a laboratory, shearing off the opened flowers and then transferring the flowers and branches to new water for culture, taking off the anther when the plastic cover falls off completely and the anther is exposed, placing the anther in a glass dryer, drying for 24 hours, sieving the anther by a 100-mesh sample sieve, and putting the sieved pollen into a sterilized and dried vial;

(6) Pollen storage: storing the pollen obtained in the step (5) in a special refrigerator, wherein the pollen is easy to lose activity at normal temperature, so that the pollen has strict requirement on storage temperature, and the temperature is maintained at-4 ℃;

(7) Manual control pollinating: when the situation that the grafted plants transplanted to the dwarf breeding garden for growing starts to bloom is observed, artificial control pollination is carried out; comprising the following (a) (b) (c);

(a) Pollen germination test: in order to perform artificial control pollination by utilizing the pollen stored in the step (6), pollen activity needs to be detected, and pollen germination test is carried out, the specific method is that a small amount of pollen is extracted at 26 ℃, and the pollen is cultured on a culture medium of 1.5% agar, 35% sucrose and 200ppm boric acid for 24 hours, when the pollen germination rate is less than or equal to 30%, the pollen activity is low or the pollen activity is lost, and the pollen is not used for pollination; the germination rate of pollen is 30-60%, the pollen can be used for pollination, and the pollination amount of a single stigma is increased to achieve the pollination effect; the germination rate of pollen is more than or equal to 60 percent, the pollen can be used for pollination, and the pollination amount of a single stigma can be less;

(b) Emasculation and bagging isolation: when female parent flower buds are light yellow and calyx rings are cracked, flowers with opened nearby branches and cyan flower buds are cut off, then a surgical scissors or a blade is used for cutting off flower bud capsule covers to be separated off, stamens are removed by forceps, pollen at the column heads and the flower cup base parts are brushed off by a fine brush, the column heads are washed by sterile water to prevent pollen pollution, white cotton bags are sleeved for isolation after emasculation is finished, iron wire spiral frames are used for supporting, two ends of each iron wire are tied on the branches, and cotton is padded at the contact positions of the iron wires and the branches so as to avoid scratching the branches;

(c) Control pollination: after emasculation, 5 days later, when the stigma is observed to be expanded and white mucus exists, one end of the bagging is opened, the pollen detected in the step (6) is dipped by a medical cotton swab and lightly smeared on the stigma, each cotton swab is only used for one hybridization combination, so that pollen pollution is prevented, the pollen is ensured to be uniformly smeared on the stigma, each cotton swab is only used once, pollination is carried out once again 2 days later after the first pollination, then bagging is carried out for isolation, and marks including pollination date, pollen information, pollination bud number and the like are made after the pollination is completed;

(8) Management after pollination: reducing human and animal interference after pollination, facilitating centralized management of plants, carrying out windproof and rainproof management, preventing and controlling falling or mildew, removing isolation bags 7 days after the last pollination, frequently observing the growth of branches and the development condition of fruits in four to six months until the fruits are ripe, and when competing branches affecting the growth of pollination branches are found, timely shearing off, ensuring the development of the fruits, simultaneously carrying out the work of pest control and the like;

(9) Harvesting fruits: timely harvesting when the fruit is observed to expand and crack in the mature season (3-4 months of the second year of hybridization), counting the fruit setting rate, and filling in an artificial hybridization breeding file including parent condition, pollen collection and emasculation, pollination time, fruit setting rate, seed number and the like;

(10) Fruit treatment: after picking fruits, placing the fruits on bamboo basket (kraft paper), airing, after the fruits are cracked, collecting seeds separated from the fruits, and obtaining seeds of the first filial generation (F1) of eucalyptus urophylla, and storing for later use after strict marking.

Example 2

A female parent dwarfing-based eucalyptus artificial hybridization breeding method comprises the following specific operation steps:

(1) Screening of good individuals: in a eucalyptus urophylla family test forest, calculating family accumulation and annual average growth through a statistical method according to the chest diameter and tree height measured for many years continuously according to the eucalyptus urophylla excellent single plant selection standard, analyzing and comparing to select excellent families, selecting excellent single plants with optimal phenotype characters from the excellent families of mature ages, numbering, and cultivating the excellent single plants as scions; the excellent single plant with the best phenotypic character is an individual with high tree height, maximum breast diameter and small trunk straight and sharp angle; the specific mode of determining the excellent families is to perform genetic variation analysis of growth (breast diameter, tree height, accumulation), dry shape, resistance (wind resistance, disease resistance and insect resistance) material property and other characters among families, construct an excellent family and single plant selection index equation, comprehensively score 20% of the top ranking as an optional family (namely the top 20 of the top ranking 100), calculate genetic gains of the optional family and single plants, screen the excellent single plants, and look at the average accumulation ranking (30-45 m of annual average accumulation ³ ·hm ^-2 ) Then, the chest diameter, the tree height and other ranges of the excellent single plant are changed along with the growth age, the annual average chest diameter range of the 6-year-old excellent single plant is 2.5-3.5 cm, and the annual average tree height range is 3.5-4.5 m;

(2) Stock cultivation: sowing eucalyptus urophylla seeds, cultivating seedlings, selecting good-growing seedlings after germination, transplanting the seedlings into seedling cups with the diameter of 20cm, and culturing for later use;

(3) Grafting: in autumn, climbing a ladder or directly putting the tree on the ground by using a special high-altitude sampler for seed collection, collecting the fruiting parent branch of the excellent single plant of the scion in the step (1) as the scion (the fruiting parent branch is the fruiting branch which grows out in the year and is flowering and fruiting branch in the 2 nd year), soaking the branch cut part with water for carrying back, picking up the well cultivated stock in the step (2) by using a nursery, cutting the stock until the ground diameter is more than or equal to 0.35cm, cutting the stock to 10 cm, trimming and grafting the fruiting parent branch onto the stock by using a cutting grafting method, ensuring that the cambium of the stock and the scion is aligned, binding the interface by using a sealing film, taking the branch section without leaves as the scion, sealing the upper end after grafting by using the sealing film, wrapping the scion by using a layer of agricultural film, preventing water loss, and finishing grafting; grafting with fruiting parent branches, enabling the grafted eucalyptus tree to bloom in the next year, and trimming the top of the trunk of the dwarf eucalyptus tree in the later period to control the length of the trunk;

(4) Cultivating grafted plants: placing grafted seedlings into a semi-automatic temperature-control and humidity-control spray irrigation system for management and protection, controlling the humidity to be more than 70%, performing pest control, starting sprouting and growing up gradually after 14 days, timely removing the sprouting on the stock to prevent the stock from competing for nutrients and moisture, after the grafting opening is completely healed, applying a proper amount of additional fertilizer to promote growth, when the new bud grows to 10 cm and the growth is stable, planting the grafted seedlings into a dwarfing breeding garden, performing tending management such as weeding and fertilization in time after planting, and starting flowering after one year;

(5) Pollen collection: taking eucalyptus urophylla as a hybrid male parent, according to the flowering stage of eucalyptus urophylla, when the flower bud capsule of a single plant of the selected excellent male parent is about to fall off when the flower bud capsule of the single plant is whitened, taking off the flower branches by a special high-altitude sampler, removing leaves, quickly inserting branch cuts into water for culture, taking the cut branches back to a laboratory, shearing off the opened flower buds, transferring the cut branches to new water for culture, taking off the flower anther when the flower anther begins to release pollen after the plastic cover falls off completely, placing the flower anther in a glass dryer, drying for 48 hours, sieving the flower buds by a 200-mesh sample sieve, and putting the sieved pollen into a small bottle after sterilization and drying treatment;

(6) Pollen storage: the pollen obtained in the step (5) is stored in a special refrigeration house, and the pollen is easy to lose activity at normal temperature, so that the pollen has strict requirements on the storage temperature, and the temperature is maintained at-4 ℃;

(7) Manual control pollinating: when the situation that the grafted plants transplanted to the dwarf breeding garden for growing starts to bloom is observed, artificial control pollination is carried out; comprising the following (a) (b) (c);

(a) Pollen germination test: in order to perform artificial control pollination by utilizing the pollen stored in the step (6), pollen activity needs to be detected, and a pollen germination test is carried out, the specific method is that a small amount of pollen is extracted at 26 ℃, and is cultured on a culture medium of 1.5% agar, 35% sucrose and 250ppm boric acid for 24 hours, when the pollen germination rate is less than or equal to 30%, the pollen activity is low or the pollen activity is lost, and the pollen is not used for pollination; the germination rate of pollen is 30-60%, the pollen can be used for pollination, and the pollination amount of a single stigma is increased to achieve the pollination effect; the germination rate of pollen is more than or equal to 60 percent, the pollen can be used for pollination, and the pollination amount of a single stigma can be less;

(b) Emasculation and bagging isolation: when female parent flower buds are light yellow and calyx rings are cracked, flowers with opened nearby branches and cyan flower buds are cut off, then a surgical scissors or a blade is used for cutting off flower bud capsule covers to be separated off, stamens are removed by the blade, pollen at the column heads and the flower cup bases is brushed off by a fine brush, the column heads are washed by distilled water to prevent pollen pollution, white cotton cloth bags are sleeved for isolation after emasculation is finished, iron wire spiral frames are used for supporting, two ends of each iron wire are tied on the branches, and cotton is padded at the contact positions of the iron wires and the branches so as to prevent the branches from being scratched;

(c) Control pollination: 6 days after emasculation, when the stigma is observed to be expanded and white mucus exists, opening one end of the bagging, dipping the pollen detected in the step (6) with a medical cotton swab, lightly smearing the pollen on the stigma, wherein each cotton swab is only used for one hybridization combination to prevent pollen pollution, ensuring that the pollen is uniformly smeared on the stigma, each cotton swab is only used once, pollinating once again after 1 day after the first pollination, then bagging and isolating, and marking the pollen after the pollination is completed, including pollination date, pollen information, pollination bud number and the like;

(8) Management after pollination: reducing human and animal interference after pollination, facilitating centralized management of plants, carrying out windproof and rainproof management, preventing and controlling falling or mildew, removing isolation bags 14 days after the last pollination, frequently observing the growth of branches and the development condition of fruits in four to six months until the fruits are ripe, and when competing branches affecting the growth of pollination branches are found, timely shearing off, ensuring the development of the fruits, simultaneously carrying out the tasks of disease and pest control and the like;

(9) Harvesting fruits: timely harvesting when the fruit is observed to expand and crack in the mature season (3-4 months of the second year of hybridization), counting the fruit setting rate, and filling in an artificial hybridization breeding file including parent condition, pollen collection and emasculation, pollination time, fruit setting rate, seed number and the like;

(10) Fruit treatment: after picking fruits, placing the fruits on bamboo basket (kraft paper), airing, after the fruits are cracked, collecting seeds separated from the fruits, and obtaining seeds of first generation (F1) of Eucalyptus urophylla hybrid, and storing for later use after strict marking.

Stability of detection after hybridization:

sowing and cultivating the obtained F1 generation seeds into eucalyptus seedlings, and establishing F1 hybrid offspring to measure forests (the planting density is 83 plants/mu), wherein the plant row spacing is 2m multiplied by 4m. The growth amount measurement of breast diameter, tree height, single plant volume and the like is carried out continuously for 6 years, and the genetic variation analysis is carried out by combining the properties of dry shape, resistance (wind resistance, disease resistance, insect resistance), wood resistance and the like to verify whether the F1 filial generation is stable.

The following table shows the results of artificial control pollination of the female parent of the dwarf eucalyptus urophylla and the male parent of the eucalyptus grandis (excellent single plant pollen) obtained by the method of the invention:

the female parent dwarfing eucalyptus artificial hybridization breeding method has a relatively high fruit setting rate and a relatively high number of seeds per fruit, and the female parent dwarfing eucalyptus artificial hybridization breeding method has good expression results in eucalyptus urophylla, eucalyptus urophylla and eucalyptus urophylla.

In the technical link of pollination, the fruit setting rate of one-time pollination after cutting off one third of the stigma is more than the number of seeds per fruit, and is superior to the scheme of directly pollination 2 times on the stigma. However, from the perspective of biological reproduction evolution, stigma has a screening effect on pollen. The dominant pollen tube can pass through and enter the ovary preferentially after germination. After the column cap is cut off, the column cap is not good or bad, and all the column cap enters the ovary. Although the number of seeds produced is large, the seed ratio of the genetic gene may be decreased.

The grafting method in the step (3) is characterized in that a crack is split in the middle of the cross section of the stock, the scion is cut into a wedge shape and then inserted into the crack of the cross section of the stock, the cambium is aligned, and then the cambium is bound and fixed by a sealing film. The butt grafting method is to cut the cross sections of the scion and the stock into a horse ear shape, then cling to each other, ensure that the cambium is bound and fixed by a sealing film after being aligned. The cutting method is to cut a slit on the outer side of the cross section of the stock to form a longitudinal section, cut a part of the scion, form a longitudinal section in the same way, and then tightly attach the two longitudinal sections together to ensure that the cambium is bound and fixed by a sealing film after being aligned; the method is suitable for the situation that the stock is large and the scion is small. The three methods are used comprehensively, so that the types of fruiting parent branches with different diameters can be fully utilized, and the success rate of grafting of good single plants of the female parent is improved.

The foregoing descriptions of specific exemplary embodiments of the present invention are presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application to thereby enable one skilled in the art to make and utilize the invention in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.

Claims (5)

1. A female parent dwarfing-based eucalyptus artificial hybridization breeding method, which is characterized by comprising the following operation steps:

(1) Screening of good individuals: calculating family accumulation and annual average growth amount by a statistical method through breast diameter and tree height which are continuously measured for many years, analyzing and comparing to select excellent families, selecting 1-2 excellent single plants with optimal phenotypic characters from the excellent families with mature ages, and cultivating the excellent single plants as scions; the determination mode of the excellent family is to carry out genetic variation analysis on chest diameter, tree height, accumulation, dry shape, wind resistance, disease resistance and insect resistance characteristics among families, construct an excellent family and single plant selection index equation and comprehensively evaluateThe top 20% of the ranking is used as a selected family, the genetic gain of the selected family and the single plants is calculated, and excellent single plants are screened; specifically, firstly, ranking average accumulation amount, then, breast diameter and tree height, wherein the annual average breast diameter range of a 6-year-old excellent single plant is 2.5-3.5 cm, and the annual average tree height range is 3.5-4.5 m; wherein, the eucalyptus excellent single plant selection standard comprises two stages; first stage selection criteria: (a) no pest characteristics: no obvious insect pest characteristics; (B) dry straightening: the dry index was scored into 5 grades: bending for 1 minute; in the average straightness range, a plurality of trunks are arranged at 2 minutes; within the average straightness range, a single trunk is 3 minutes; slightly curved, single trunk, 4 minutes; the shape of the trunk is straight, the trunk is single, and the trunk is 5 minutes; (C) little branching: the branch indexes are scored according to 3 grades according to the branch size; big branch 1, middle branch 2 and small branch 3; (D) cumulative growth index: average annual accumulation of 30-45 m ³ ·hm ^-2 The method comprises the steps of carrying out a first treatment on the surface of the Second stage selection criteria: (E) bark and wood properties: the important wood basic density of the pulp wood tree species is mainly considered, and the wood basic density index in 6 years of the round-robin period is required to be 500-600 kg/m1; (F) germination capacity and cutting viability: as asexual propagation material, the selected wood must have strong germination capacity, and the cutting survival rate must not be too low, at least more than 70%;

(2) Stock cultivation: sowing and cultivating seedlings of the same tree species as the single plant with excellent scion, selecting seedlings with good growth vigor after germination, transplanting and cultivating for later use;

(3) Grafting: in spring or autumn, collecting the fruiting parent branch of the excellent single plant of the scion in the step (1) as the scion, soaking the branch cut with water for carrying back, selecting the stock cultivated in the step (2) in a nursery, cutting the stock to 10-15 cm in diameter, trimming and grafting the fruiting parent branch onto the stock, and selecting different methods according to the stock size, a split grafting method, a butt grafting method and a cutting grafting method and according to the scion size and the relative size of the stock: when the scions are as large as the stocks, a butt grafting method and a split grafting method are used; when the scion is small and the stock is large, a cutting grafting method is used to ensure that cambium alignment of the stock and the scion is carried out, and the grafting is completed by binding the interface after alignment; grafting with fruiting parent branches, enabling the grafted eucalyptus tree to bloom in the next year, and trimming the top of the trunk of the dwarf eucalyptus tree in the later period to control the length of the trunk;

(4) Cultivating grafted plants;

(5) Pollen collection, namely collecting flowers and branches by a high-altitude sampler when the flower bud capsule covers of the selected single plant of the excellent male parent turn white and fall off, removing leaves, inserting branch cuts into water for culture, taking back, cutting off the opened flowers and then transferring the flowers and buds to new water for culture, and taking out the flowers and the flowers out when the flowers and the flowers begin to release pollen, drying for 24-48 hours, and sieving with a 100-200-mesh sample separating sieve;

(6) Pollen storage;

(7) Manual control pollinating: when the plants in the step (4) are observed to start flowering, artificial control pollination is started;

(8) Management after pollination: reducing human and animal interference after pollination, performing windproof and rainproof management on plants convenient for centralized management, preventing falling or mildew, removing isolation bags 7-14 days after the last pollination, observing the growth of branches and the development condition of fruits in four to six months until the fruits are ripe, and timely cutting out competitive branches affecting the growth of pollination branches when the competitive branches are found;

(9) Harvesting fruits: harvesting in time when the fruit is observed to expand and is about to crack in the mature season of the fruit, which is generally 3-4 months of the second year of hybridization;

(10) Fruit treatment: and after picking fruits, airing the fruits, and after cracking the fruits, collecting seeds separated from the fruits to obtain the seeds of the first filial generation (F1).

2. The method for female-dwarf-based eucalyptus artificial hybridization breeding according to claim 1, wherein: the fruiting parent branch in the step (3) is a branch which grows out in the year and becomes a fruiting branch, flowers and fruits in the 2 nd year.

3. The method for female-dwarf-based eucalyptus artificial hybridization breeding according to claim 1, wherein: and (4) cultivating the grafted plants, namely placing the grafted seedlings into a semi-automatic temperature and humidity control spray irrigation system for management, controlling the humidity to be more than 70%, preventing and controlling plant diseases and insect pests, enabling scions to start to sprout and grow gradually after 7-14 days, timely removing sprouts on the stock to prevent the scions from competing for nutrients and moisture, applying a proper amount of fertilizer after the grafting openings heal completely, promoting growth, planting the grafted seedlings into a dwarfing breeding garden after new buds grow to 10-15 cm, enabling plant row spacing to be 5.0x5.0m, timely developing tending management such as weeding and fertilization after planting, and beginning flowering after one year.

4. The method for female-dwarf-based eucalyptus artificial hybridization breeding according to claim 1, wherein: the pollen in step (6) is stored as: the temperature was maintained at-4 ℃.

5. The method of female stunted eucalyptus artificial cross-breeding according to claim 1, wherein the artificial pollination in step (7) comprises the following items (a) (b) (c);

(a) Pollen germination test: in order to perform artificial control pollination by using the pollen stored in the step (6), pollen activity needs to be detected, and pollen germination test is carried out, the specific method is that a small amount of pollen is extracted at 26 ℃, and the pollen is cultured on a culture medium of 1.5% agar and 35% sucrose and 100-250 ppm boric acid for 24 hours, when the pollen germination rate is less than or equal to 30%, the pollen activity is low or the pollen activity is lost, and the pollen is not used for pollination; the germination rate of pollen is 30% -60%, the pollen can be used for pollination, and in order to achieve the pollination effect, the pollination amount of a single stigma is increased; the germination rate of pollen is more than or equal to 60 percent, the pollen can be used for pollination, and the pollination amount of a single stigma can be less;

(b) Emasculation and bagging isolation: when the flower bud of the female parent is light yellow and the calyx ring is cracked, cutting off flowers with opened nearby branches and cyan flower buds, cutting off flower bud capsules to be dropped off, removing stamens, brushing pollen on the head of the flower and the base of the flower cup, flushing the head of the flower, and bagging and isolating after emasculation is finished;

(c) Control pollination: and (3) 5-6 days after emasculation, when the stigma is observed to expand and white mucus exists, opening one end of the bagging, dipping the pollen detected in the step (6), smearing the pollen on the stigma, and in order to improve the pollination success rate, pollinating once again 1-2 days after the first pollination, wherein the other pollination mode is to cut off one third of the upper end of the stigma, smear the pollen at the cut at one time, then isolate the bagging, and marking the pollination date, pollen information, the number of pollinated buds and the like after the pollination is finished.

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