CN114731797A - Method for cultivating tree-shaped Chinese rose by using seedling stock - Google Patents

Abstract

A method for cultivating tree-shaped Chinese rose by using seedling stock. The method comprises the steps of selecting 5 pairs of SSR primers, accurately measuring the affinity of the scion and the rootstock by adopting an SSR molecular marker technology, and then selecting the scion with strong affinity to finish the cultivation of the dendriform China rose. The method also utilizes native seedlings of the rosa chuanxiong hort rosa multiflora as the rootstocks, so that the damage to wild rosa chuanxiong hort can be effectively reduced, the grafting survival rate of the rootstocks and the scions can be greatly improved, and the ornamental life of the dendriform rosa chinensis can be scientifically prolonged.

Description

Method for cultivating tree-shaped Chinese rose by using seedling stock

Technical Field

The invention belongs to the technical field of woody flower landscaping application, and particularly relates to a method for seedling stock breeding, grafting affinity determination and grafting and modeling.

Background

China rose (Rosa hybrida) is a plant of the Rosa genus (Rosa) of the Rosaceae family (Rosaceae). A dendriform Chinese rose, also called a Chinese rose tree, a tree Chinese rose, a lollipop Chinese rose and the like, which is a novel Chinese rose type cultivated by a rose stock with a vertical main branch through gardening means (measures such as cuttage or sowing, root cultivation, stem cultivation, grafting, pruning, molding and the like). Compared with common flowers, the crown of the dendriform Chinese rose is higher off the ground, has better ventilation property and is not easy to be infected by plant diseases and insect pests. The appearance is unique, noble and elegant, and well-arranged, and is novel in visual effect. At present, with the continuous upgrading of aesthetic requirements of consumers, the dendriform China rose becomes the leading role of courtyards and potted flowers at home and abroad.

At present, three types of domestic tree-shaped Chinese roses, namely Japanese Rosa multiflora (Rosa multiflora), aucklandia japonica (Rosa banksiae) and Rosa odorata (Rosa odorata), are mainly used as rootstocks of the tree-shaped Chinese roses. The Japanese rosa multiflora is mainly used for cultivating rootstocks through cutting propagation, so that the cultivated rootstocks are weak in later thickening capacity, and the height of main branches is not enough, so that the sizes of the flower crowns of the tree-shaped rosa chinensis cultivated by the rosa multiflora are limited. The application of the woody fragrant flowers and the fragrant Chinese roses as the rootstocks is mainly to cultivate tree-shaped Chinese roses by directly transplanting the woody fragrant flowers and the fragrant Chinese roses to cities from the field. The thickness and height of the main trunk of the woody fragrant flower serving as the stock are superior, but the uprightness of the woody fragrant flower is poor, and the outer skins of the main branches of the woody fragrant flower are seriously cracked after being transplanted for two years, so that the subsequent ornamental property and the service life of the woody fragrant flower are influenced. The aromatic Chinese rose is used as a stock, although the thickness, the height and the uprightness of the main trunk of the aromatic Chinese rose have advantages, the aromatic Chinese rose is only suitable for Yunnan climate because of belonging to the special species of Yunnan, the false survival phenomenon appears after the aromatic Chinese rose is transplanted outside provinces and grafted, the death rate of the scions reaches more than 90 percent after 1 to 2 years, and the whole tree-shaped Chinese rose is dead. The application of the woody fragrant flowers and the fragrant Chinese roses as the rootstocks not only has unsatisfactory subsequent ornamental value and survival rate, but also has destructive effect on the field environment and ecology of people. In conclusion, in the current market, the three kinds of tree-shaped Chinese rose stocks have the defects of weak sustainability and the like. Therefore, the development and utilization of the rose seedling with the existing stress resistance and strong adaptability in China as the rootstock of the dendriform rose are imperative.

For this reason, from 2008, we developed intensive and detailed research work on the distribution and characteristics of wild rose germplasm resources in China every year, and the research work is continued to the present. Aiming at the work of domestic operability of tree-shaped Chinese rose rootstocks, related experimental works such as wild resource collection, protection and preservation, selfing and fructification, sowing and breeding, design management, grafting experiments, later-stage management and horticultural trait observation are regularly developed. The comprehensive analysis is carried out according to various aspects of the market on the rootstock, such as strong adaptability, good stress resistance, long ornamental life and the like. The results show that the Rosa chuanxiong hort rose (Rosa soulieana) selected by the invention has the advantages of good stress resistance, developed root system, fast growth, strong vitality and the like, can be used as a good stock for production and popularization of the tree-shaped Rosa chinensis in future, and can gradually replace the stock types with weak sustainability in the existing market.

Disclosure of Invention

In order to solve the technical problems that the existing arborescent China rose cultivation adaptability is narrow, the judgment of the affinity of the stock and the scion depends on experience, the operability is weak, the grafting survival rate is low, and the ornamental life is short, the invention provides the method for cultivating arborescent China rose by utilizing the seedling stock with wide adaptability, and the method has the characteristics of strong operability, high survival rate, long ornamental life and the like.

The technical scheme of the invention is as follows:

a method for cultivating tree-shaped Chinese roses by utilizing seedling stocks is characterized by comprising the following steps: setting the rosa plant seedling stock, selecting a Chinese rose scion with strong affinity with the rosa plant seedling stock, and grafting the selected Chinese rose scion with the set seedling stock.

Further, preferably, the setting of the rosa plant seedling rootstock is that only the lateral buds at the top of the main branch and the main branch of the rosa plant seedling rootstock are reserved when the rosa plant seedling rootstock grows to the height of 50cm-150cm, and the lateral buds and the rest lateral buds of the rosa plant seedling rootstock are cut off completely by using a sterilized cutter.

Further, preferably, when the diameter of the main branch of the set rosa plant seedling stock grows to be more than 1.5cm, the selected rosa chinensis scion is grafted with the rosa plant seedling stock.

Further, preferably, in the grafting, a large flower type Chinese rose scion is grafted to a rosa plant seedling rootstock with a main branch diameter of more than or equal to 3cm, a middle flower type Chinese rose scion is grafted to a rosa plant seedling rootstock with a main branch diameter of more than or equal to 2cm, and a small flower type Chinese rose scion is grafted to a rosa plant seedling rootstock with a main branch diameter of more than or equal to 1.5 cm; the large-flower type Chinese rose scions refer to Chinese rose scions with the flower diameter larger than 9cm, the medium-flower type Chinese rose scions refer to Chinese rose scions with the flower diameter of 4-9 cm, and the small-flower type Chinese rose scions refer to Chinese rose scions with the flower diameter of less than 4 cm.

Further, preferably, the new lateral branches and lateral buds growing from the set rosa plant seedling rootstocks are cut off by a sterilized cutter 10-14 days before grafting.

Further, preferably, the step of selecting the rose scions with strong affinity with the rosa plant rootstocks is to perform accurate determination on the rose scions and the rosa plant rootstocks by using an SSR molecular marker technology, the accurate determination is to perform PCR on DNAs extracted from leaf samples of the rosa plant rootstocks and the rose scions by using 5 pairs of SSR primers respectively, calculate Nei's similarity coefficients between the samples by using NTSYS-PC 2.0 analysis software, and select the rose scions with Nei's similarity coefficient being equal to or greater than 0.4 between every two of the rosa scions and the rosa plant rootstocks as the rose scions with strong affinity with the rosa plant rootstocks;

the 5 pairs of SSR primers are SSR01 primers, SSR02 primers, SSR03 primers, SSR04 primers and SSR05 primers;

the SSR01 primer consists of an SSR01 upstream primer and an SSR01 downstream primer, and the nucleotide sequence of the SSR01 upstream primer is shown as SEQ ID NO: 1, the nucleotide sequence of the SSR01 downstream primer is shown as SEQ ID NO: 2 is shown in the specification;

the SSR02 primer consists of an SSR02 upstream primer and an SSR02 downstream primer, and the nucleotide sequence of the SSR02 upstream primer is shown as SEQ ID NO: 3, the nucleotide sequence of the SSR02 downstream primer is shown as SEQ ID NO: 4 is shown in the specification;

the SSR03 primer consists of an SSR03 upstream primer and an SSR03 downstream primer, and the nucleotide sequence of the SSR03 upstream primer is shown as SEQ ID NO: 5, the nucleotide sequence of the SSR03 downstream primer is shown as SEQ ID NO: 6 is shown in the specification;

the SSR04 primer consists of an SSR04 upstream primer and an SSR04 downstream primer, and the nucleotide sequence of the SSR04 upstream primer is shown as SEQ ID NO: 7, the nucleotide sequence of the SSR04 downstream primer is shown as SEQ ID NO: 8 is shown in the specification;

the SSR05 primer consists of an SSR05 upstream primer and an SSR05 downstream primer, and the nucleotide sequence of the SSR05 upstream primer is shown as SEQ ID NO: 9, the nucleotide sequence of the SSR05 downstream primer is shown as SEQ ID NO: shown at 10.

Further, preferably, the breeding process of the rosa plant seedling rootstock is as follows: carrying out cold Tibetan processing, sterilization processing, water absorption processing, germination accelerating processing, sowing and seedling planting on rosa plant seeds in sequence, and specifically comprising the following steps:

1) refrigerator processing: selecting selfing seeds of the rose plants in the current year, and refrigerating the selfing seeds for 45 to 60 days at constant temperature of 4 ℃ and relative air humidity of 70 to 90 percent in dark light in autumn or winter;

2) and (3) sterilizing the seeds: soaking the mixture for 15 to-25 min by using a potassium permanganate solution with the concentration of 3 to 5 thousandth w/w at the beginning of 2 months in the next year, and washing the mixture for 2 to 3 times by using sterile water;

3) carrying out water absorption treatment on seeds: soaking the sterilized seeds in sterile water at the temperature of 95-100 ℃ for 4-5 min, then putting the seeds in sterile water at room temperature, changing the water every night, and soaking for 3-5 days;

4) and (3) carrying out germination accelerating treatment on the seeds: putting the seeds subjected to water absorption treatment on a germination accelerating substrate, covering 2-3 cm of sterilized wet sand on the seeds, wherein the germination accelerating environment is that the relative air humidity is 80-95%, and the temperature of the germination accelerating environment is 18-23 ℃; the germination accelerating matrix is formed by mixing sterilized perlite, humus soil and peat according to the volume ratio of 1:1: 1.

5) Sowing seeds: when the seeds subjected to the pregermination treatment begin to sprout, putting the seeds into a hole tray filled with a sowing substrate, and sowing the seeds in a mode of one hole for one seed; the beginning of germination accelerating seeds is that the seed shells crack or the germination is seen; the seeding matrix is formed by mixing sterilized perlite and humus according to the volume ratio of 1: 1;

6) planting seedling: and after sowing, transplanting seedlings with developed root systems into flowerpots or root control containers with the calibers of 30-50 cm for planting, wherein soil in the flowerpots or the root control containers is formed by mixing garden soil and humus soil according to the volume ratio of 1: 1.

Further, preferably, after the interface between the rosa plant seedling rootstock and the rosa chinensis scion is healed, the tree-shaped rosa chinensis is modeled, wherein the modeling is as follows: 1) every month, removing the sprouts and branches of the non-scions on the seedling stocks; 2) carrying out small pruning once each at 5 months and 9 months every year, wherein the small pruning is to prune 10-20 cm of withered flowering branches of the Chinese rose scions; 3) carrying out major pruning once every 12 months, wherein the major pruning for the large flower type Chinese rose scions is as follows: pruning the flowering branches of the large-flower type Chinese rose scions to the positions with the diameters of 8-10 mm; the major pruning for the medium-flower Chinese rose scions is as follows: pruning the flowering branches of the medium-flower type Chinese rose scions to positions with the diameter of 5-7 mm; the major pruning for the small flower type Chinese rose scions is as follows: pruning the flowering branches of the small-flower type Chinese rose scions to the positions with the diameters of 3-4 mm.

Further, preferably, the grafting is bud grafting, and when the bud grafting is carried out, the axillary bud of the Chinese rose scion is selected to grow to be full and not to germinate.

Further, preferably, the rosa plant seedling stock is a rosa yunnanensis seedling stock.

The invention also provides a group of SSR primers for screening the Chinese rose scions with strong affinity with the rosa plant parenchyma rootstocks, wherein the SSR primers comprise an SSR01 primer, an SSR02 primer, an SSR03 primer, an SSR04 primer and an SSR05 primer;

the SSR01 primer consists of an SSR01 upstream primer and an SSR01 downstream primer, and the nucleotide sequence of the SSR01 upstream primer is shown as SEQ ID NO: 1, the nucleotide sequence of the SSR01 downstream primer is shown as SEQ ID NO: 2 is shown in the specification;

the SSR02 primer consists of an SSR02 upstream primer and an SSR02 downstream primer, and the nucleotide sequence of the SSR02 upstream primer is shown as SEQ ID NO: 3, the nucleotide sequence of the SSR02 downstream primer is shown as SEQ ID NO: 4 is shown in the specification;

the SSR03 primer consists of an SSR03 upstream primer and an SSR03 downstream primer, and the nucleotide sequence of the SSR03 upstream primer is shown as SEQ ID NO: 5, the nucleotide sequence of the SSR03 downstream primer is shown in SEQ ID NO: 6 is shown in the specification;

the SSR04 primer consists of an SSR04 upstream primer and an SSR04 downstream primer, and the nucleotide sequence of the SSR04 upstream primer is shown as SEQ ID NO: 7, the nucleotide sequence of the SSR04 downstream primer is shown as SEQ ID NO: 8 is shown in the specification;

the SSR05 primer consists of an SSR05 upstream primer and an SSR05 downstream primer, and the nucleotide sequence of the SSR05 upstream primer is shown as SEQ ID NO: 9, the nucleotide sequence of the SSR05 downstream primer is shown as SEQ ID NO: shown at 10.

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

1. the invention firstly proposes that 5 pairs of SSR primers are used, molecular marker technology is combined with analysis software to carry out accurate determination on the affinity between the seedling rootstock and the Chinese rose scion, NTSYS-PC 2.0 analysis software is used for calculating and selecting the Chinese rose scion material with Nei's similarity coefficient being larger than or equal to 0.4 between the Chinese rose scion and the seedling rootstock of the rosa genus plant as the Chinese rose scion with strong affinity with the seedling rootstock of the rosa genus plant, and the Chinese rose scion material is grafted with the seedling rootstock of the rosa genus plant, so that the grafting survival rate of the rootstock and the scion can be greatly improved (the survival rate reaches 89-100 percent), and the ornamental life of tree-shaped Chinese rose can be scientifically prolonged.

2. The method selects the rosa chuanxiong maxim seedling as the tree-shaped rosa chinensis stock, has strong suitability, the grafted tree-shaped rosa chinensis is suitable for being planted in the whole China, and has the advantages of high selfing and setting rate in a wild environment, large yield, contribution to breeding of the stock, no damage to the field environment and ecology, good stress resistance, developed root system, fast growth (2-3 years of main branch and trunk molding), long ornamental life after grafting (still keeping good ornamental character after 10 years of grafting) and the like, and effectively overcomes the defects of low seed yield, poor stress resistance, underdeveloped root system, slow growth (3-5 years of main branch and trunk molding), short ornamental life after grafting (losing ornamental character after 5 years of grafting) and the like of the tree-shaped rosa chinensis stock variety in the current market.

3. The rosa yunnanensis selected by the invention is a domestic and native wild rose germplasm resource as a seedling stock, has strong adaptability and breeding capability, can continuously provide stock seedlings for domestic and foreign markets, and does not have the risk of foreign neck clamping in the aspects of provenance and technology in large-scale production and sales markets.

4. The invention carries out standardized, scientific and detailed summarization on the later-stage plastic model of the grafted dendriform China rose: the method is characterized in that the method is to prune slightly in spring and autumn and prune greatly in winter, so that the completeness and the fullness of the crown width of the flower can be scientifically and efficiently modeled, a better ornamental effect can be achieved in the full-bloom period, and the operability is strong.

SEQ ID NO: shown in FIG. 1 is the nucleotide sequence of the primer upstream of SSR 01.

SEQ ID NO: shown in 2 is the nucleotide sequence of the primer downstream of SSR 01.

SEQ ID NO: 3 shows the nucleotide sequence of the primer upstream of SSR 02.

SEQ ID NO: 4 shows the nucleotide sequence of the primer downstream of SSR 02.

SEQ ID NO: 5 shows the nucleotide sequence of the primer upstream of SSR 03.

SEQ ID NO: shown in FIG. 6 is the nucleotide sequence of the primer downstream of SSR 03.

SEQ ID NO: shown in 7 is the nucleotide sequence of the primer upstream of SSR 04.

SEQ ID NO: shown in FIG. 8 is the nucleotide sequence of the downstream primer of SSR 04.

SEQ ID NO: shown in FIG. 9 is the nucleotide sequence of the primer upstream of SSR 05.

SEQ ID NO: 10 shows the nucleotide sequence of the primer downstream of SSR 05.

Detailed Description

The following examples are given without specific indication of conventional methods. Reagents, kits, all biological materials of the Sichuan Yunnan rose and Chinese rose scion varieties and the like related in the embodiments can be purchased from commercial sources.

The terms herein:

seedling rootstock: the rootstock cultivated by the seeding method is called seedling rootstock.

NTSYS-PC 2.0 analytical software: the molecular bioinformatics analysis software is mainly used for carrying out species bioinformatics correlation analysis on molecular markers such as SSR (simple sequence repeat) and ISSR (inter simple sequence repeat).

Nei's similarity coefficient: is a distance coefficient or similarity coefficient used to describe the difference or degree of similarity between two binary variables. The Nei's similarity coefficient is automatically generated after the data is input by the software analyzed by NTSYS-PC 2.0.

And (3) PCR: polymerase Chain Reaction (PCR), which is called PCR for short, is a molecular biology technique.

Large-flower Chinese rose scions, namely large-flower scion varieties: refers to the Chinese rose scions with the diameter of the flower being more than 9 cm.

Middle-flowered Chinese rose scions, namely, middle-flowered scions varieties: is a Chinese rose scion with the diameter of 4-9 cm.

Small flower type Chinese rose scions, namely small flower type scion varieties: is a Chinese rose scion with the diameter of the flower less than 4 cm.

Dark light: without light irradiation.

Seedlings with developed root system: has a main root, and at least 20 lateral roots around the main root.

Example 1 cultivation of a Tree-like China rose of Large, Medium and Small flower types Using Rosa Citrinii

(1) Breeding the rosa seedling stock: selecting selfing seeds of the Yunnan rose in the current year, and carrying out the following operations on the seeds:

1) and (3) performing cold storage treatment on the seeds: and (4) refrigerating in dark for 60 days at constant temperature of 4 ℃ and humidity of 80% in 11 days 12 and 2008.

2) And (3) sterilizing the seeds: soaking in potassium permanganate solution with concentration of 4% w/w (mass percent) for 20min, and washing with sterile water for 3 times in the next year, 2 months and 9 days.

3) Carrying out water absorption treatment on seeds: soaking the sterilized seeds in sterile water with a high temperature of 97 ℃ for 5min, putting the seeds in sterile water with room temperature, changing water every night, and soaking for 5 days.

4) Carrying out germination accelerating treatment on seeds: putting the seeds subjected to water absorption treatment on a germination accelerating substrate, covering 2-3 cm of sterilized wet sand on the seeds, wherein the water content of the wet sand is 10-15%, the germination accelerating environment is air relative humidity 85%, the temperature of the germination accelerating environment at night is 18 ℃, and the temperature of the germination accelerating environment at day is 22 ℃. The germination accelerating matrix is formed by mixing sterilized perlite, humus soil and peat according to the volume ratio of 1:1:1, and the sterilization mode is 121.3 ℃ under the condition of high pressure of 0.25MPa and 20 min.

5) Sowing seeds: when the germination accelerating seeds begin to sprout, putting the seeds into a hole tray filled with a sowing substrate, and sowing the seeds in a hole-by-seed mode; the germination accelerating seeds begin to germinate by cracking of seed shells or germination of seeds, the seeding matrix is formed by mixing sterilized perlite and humus according to the volume ratio of 1:1, and the sterilization mode is 121.3 ℃ under the condition of high pressure of 0.25MPa and 20 min.

6) Planting seedling: after sowing, transplanting seedlings with developed root systems into root control containers (the root control containers are commercial products) with the caliber of 40cm or flowerpots with the caliber of 40cm, wherein soil in the root control containers or the flowerpots is garden soil and humus soil which are mixed according to the volume ratio of 1: 1. The cultivated Yunnan rose seedling is the Yunnan rose seedling stock.

(2) Shaping of seedling rootstocks

When the grown Sichuan Yunnan rose seedling grows to 50-150 cm, only the main branch and the lateral buds on the top of the main branch are kept, and the lateral buds and the rest lateral buds are cut off by a sterilized cutter (the sterilization mode of the cutter is 121.3 ℃ under the condition of high pressure of 0.25MPa, the temperature is 20min, and the sterilization mode of the cutter is the same as the mode), and the daily fertilization and watering management culture are continuously carried out.

(3) Determination of affinity of seedling stock and scion

SSR molecular assay and data analysis: the invention designs 5 pairs of SSR primers shown in the table 1, and commissions Shanghai Czeri Biotechnology Limited to synthesize the 5 pairs of SSR marker primers provided by the invention for later use. The test operation is carried out according to the instruction of a plant DNA extraction kit produced by Beijing TransGen Biotech company, after leaf sample DNAs of the rosa chuanxiong hort seed rootstocks and 60 parts of all the rosa chinensis scions (shown in Table 5) are extracted one by one, the PCR amplification test is respectively carried out on the leaf sample DNAs of the rosa chuanxiong hort seed rootstocks and the leaf sample DNAs of each rosa chinensis scion in Table 5 by using each pair of SSR primers in Table 1 according to the steps of the instruction of the PCR kit produced by Beijing TransGen Biotech company. PCR amplification reaction of 10. mu.l of each material, in which ddH₂O5.84. mu.l, 10 XBuffer (containing Mg)²⁺) 1. mu.l of 2mM dNTP, 0.5. mu.l of 0.25nM forward primer, 0.5. mu.l of 0.25nM downstream primer, 0.16. mu.l of Taq enzyme (2.5U/. mu.l), 1. mu.l of Chinese rose material DNA (20 ng); the rose material is a rosa chuanxiong maxim seedling stock leaf sample or a rosa chinensis scion leaf sample; reaction procedure: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at annealing temperature for 30s, extension at 72 ℃ for 90s for 40 cycles, final extension at 72 ℃ for 10min, and cooling at 4 ℃. The steps of preparing the gel, loading the sample, performing electrophoresis and dyeing are carried out one by one according to the conventional operation steps.

TABLE 1 primer information for SSR molecular test for PCR

And (3) repeatedly amplifying each DNA material for 3 times by each pair of SSR primers, quantifying a stably-appearing band (no matter the intensity) in an electrophoresis result to be 1 and quantifying no band to be 0 according to the migration distance and the size of each amplified fragment, and counting to form a 0, 1 matrix. Examples are: taking SSR01 primers as an example, PCR amplification, electrophoresis and staining are respectively carried out on the China rose material No. 1 and No. 2 in the Table 2, and the counting is carried out by quantifying the stably appeared bands (no matter the intensity) in the electrophoresis result to be 1 and the quantification without bands to be 0 according to the migration distance and the size of each amplified fragment. Combining the primer information of the molecular test of each pair of SSR primers in the table 1, wherein 1 material is amplified to form 1 strip at the positions of 101 bp, 108bp and 124bp respectively, and the 1 strip is marked as 1, 1 and 1; material No. 2 amplified 1 band at 101 and 124bp, respectively, and no band at 108, and was recorded as 1, 0, 1 (since no band was amplified at 108bp, it was recorded as 0).

Nei's similarity coefficient between samples was calculated using the NTSYS-PC 2.0 analysis software (software generated automatically) and the results are shown in Table 3. Selecting a rose scion material with a Nei's similarity coefficient between the rose scion and the seedling rootstock being not less than 0.4, namely the rose scion with strong affinity with the seedling rootstock of the rosa genus plant (see table 3), wherein 5 varieties in total of 2 (water lily, flos chrysanthemi indici), 7 (attritor of the peaches), 8 (pearl oyster shell), 9 (meadowrue roses) and 10 (star-language star application) in table 3 are the rose scions with strong affinity with the seedling rootstock of the rosa chuanxiong, so as to prepare subsequent operation.

In the design of SSR primers, 50 pairs of SSR primers are designed in total, and SSR primer amplification band polymorphism screening is carried out on 60 parts of Rosa wild species and cultivated variety materials in the SSR primer pair 5 one by one, and the result shows that: some primers in 50 pairs of SSR primers can not amplify bands in some Rosa materials, the number and the positions of bands amplified by some primers in all tested materials are the same, no polymorphism exists, 5 pairs of primers designed by us are finally screened (see table 1), each pair of the 5 pairs of SSR primers can amplify at least one band in 60 Rosa wild species and cultivated variety materials in table 5, and the number and the positions of the bands have obvious diversity (see tables 3 and 4), so that Nei's similarity coefficients among the materials can be calculated by software, and the 5 pairs of SSR primers provided by the invention have obvious specificity and polymorphism.

A large number of experiments are also carried out in the earlier stage aiming at the Nei's similarity coefficient among materials. After the calculated Chinese rose scion variety with Nei's similarity coefficient smaller than 0.4 with the Chinese rose seedling stock is grafted with the Chinese rose seedling stock, the survival rate is smaller than 80%; after the Chinese rose scion variety with the Nei's similarity coefficient being greater than or equal to 0.4 is grafted with the rosa chuanxiong hort seedling stock, the survival rate reaches 89-100%. The survival rates of the 5 screened Chinese rose scions with strong affinity with the Chinese rose seedling stocks (Nei's similarity coefficient ≧ 0.4) after being grafted with the Chinese rose seedling stocks are respectively as follows: 89% of No. 2 (Zingiberaceae), 98% of No. 7 (the day of the peach), 92% of No. 8 (first-grade Zhuyi), 91% of No. 9 (meadowrue) and 100% of No. 10 (star-and-star-wish). The ornamental life is prolonged by more than 3 years on average.

(4) Preparation of scions and rootstocks

And selecting the scion variety with the Nei's similarity coefficient not less than 0.4 in 5 months, and performing subsequent grafting and other operations when the axillary buds of the scion grow to be full and not germinate. And (3) cutting off newly grown lateral branches and lateral buds of the shaped rosa yunnanensis seedling stock by using a sterilized cutter 12 days before grafting.

(5) Grafting of dendriform China rose

In 2011 in 12 months (in winter or early spring), the seedling stock and the scion are grafted by adopting a bud grafting mode: 1) cutting a shield-shaped cut of 2-3 cm from top to bottom along a 40-degree oblique angle at the position 3cm below the top of the main branch of the seedling stock by using a sterilized cutter, and reserving an opening below the cut; 2) respectively taking down healthy disease-free and full ungerminated axillary buds of different Chinese rose scion varieties by the same method; 3) the fresh-keeping film is quickly embedded into the seedling stock interface, is tightly attached, is fastened and wound by a ring from top to bottom by using a fresh-keeping film, and ensures that bud points are exposed in the air; 4) and (3) keeping the relative humidity of air to be more than 70%, and removing the preservative film as soon as possible after the budding wound is healed. According to the method, 2 axillary buds are grafted at the position which is 3cm downwards around the top of the main branch of the seedling stock. Wherein, the large flower type scion variety is grafted to the seedling stock with the diameter of the main branch of 3cm or more, and the middle flower type scion variety is grafted to the seedling stock with the diameter of the main branch of 2cm or more. The small flower type scion variety is grafted to a seedling stock with the diameter of a main branch of 1.5 cm.

(6) Modeling of tree-shaped Chinese rose

After the joints of the seedling stocks and the Chinese rose scions are healed and grow normally, management and cultivation are carried out according to daily management. The tree-shaped Chinese rose is shaped as follows: 1) every month, removing the sprouts and branches of the non-scions on the seedling stocks, and if the grafted axillary buds are dead, performing supplementary grafting according to the steps; 2) carrying out small pruning once each at 5 months and 9 months every year, wherein the small pruning is to remove 10-20 cm of withered flowering branches of the Chinese rose scions; 3) a major cut is made 12 months per year, which is: and (3) trimming for a large flower type scion variety: pruning the flowering branches of the large-flower type Chinese rose scions to the positions with the diameters of 8-10 mm; and (3) pruning aiming at the medium-pattern scion variety: pruning the flowering branches of the medium-flower type Chinese rose scions to positions with the diameter of 5-7 mm; and (3) trimming for a small flower type scion variety: pruning the flowering branches of the small-flower type Chinese rose scions to the positions with the diameters of 3-4 mm.

By using the tree-shaped Chinese rose shaping mode, the four-season flowering quantity and the disease and pest resistance of the tree-shaped Chinese rose are obviously improved. Compared with the same dendriform China rose without the dendriform China rose shaping mode (the types and the ages of the stocks are the same, the varieties of the scions are the same, and the grafting time is consistent), the former increases the annual flowering number by 40-65%, reduces the pesticide application frequency by 2-3 times, and reduces the dosage by 40-50%.

TABLE 210 parts of China rose scion material

Numbering	Name of breed	Type (B)	Numbering	Name of breed	Type (B)
						1	Yun Mei (Chinese rose)	Middle flower type	6	Soft fragrant red	Middle flower type
2	All-grass of Jasminum Armandii	Big flower type	7	The day of the peach	Middle flower type
						3	Spring Sichun tea	Middle flower type	8	One-product Zhu clothing	Middle flower type
4	Yu Ling Long	Small flower type	9	Root of Whiteflower Pond	Big flower type
						5	Peony rose	Big flower type	10	Star language wish	Small flower type

Note: in the table 2, 10 parts of materials are all Chinese varieties, which cover large, medium and small flower types, and have more outstanding ornamental value.

TABLE 310 Nei's similarity coefficient between the rose scion material and the rootstock

Note: 11 in table 3 represents the Yunnan rose, which is the stock; 1-10 represent different Chinese rose scions, and the variety names of the Chinese rose scions correspond to the numbers in the table 2 one by one; the number of Nei's similarity coefficient between the Chinese rose scion and the rootstock is not less than 0.4 is highlighted in a manner of being marked and thickened.

TABLE 4 amplification of 60 Rosa material in Table 5 with 5 pairs of primers of the invention

Primer and method for producing the same	Allelic factor	Number of polymorphic sites	Polymorphic site Rate
				SSR01	12	11	91.7％
SSR02	8	8	88.9％
				SSR03	12	11	91.7％
SSR04	16	15	93.8％
				SSR05	12	11	91.7％

TABLE 560 parts of Rosa plant Material tested

Serial number	Name of Material	Serial number	Name of Material
				1	Soft fragrant red R.hybrida 'Ruanxianghong'	31	Star wish R.hybrida 'Xingyuxingan'
2	Azalea ' Yingrihehua ' of Ziziphora arvensis '	32	Four season flour R.hybrid 'Four seasides Powder'
				3	Si Chun R.hybrida 'Sichun'	33	Treasure R.hybrida 'Treasure'
4	Chinese blue lotus scholar R.hybrida 'Qinglianxueshi'	34	Allatin R.hybrida 'Aladdin'
				5	All-grass of Red chocolate R.hybrida 'Yipinzhuyi'	35	Love of R.hybrida 'Four Love'
6	Yulinglong R.hybrid 'Yuulinglong'	36	Peng R.hybrida 'Pengpeng'
				7	Peony rose R.hybrida 'Mudanyueji'	37	All-grass of Ardisia crenata R.hybrida 'Alfred'
8	Moon Red R. chinensis ' Slater's climmson China '	38	Apricot flower Village R.hybrida 'Apricot Village'
				9	Radix et rhizoma Smilacis Glabrae R.hybrida 'Jinfenlian'	39	Arwaki R.hybrid 'Youth'
10	Lunar meal R.chinensis 'Pallida'	40	Four season Rose R.hybrid 'Four seasides Rose'
				11	R.praelucens of Dahurian Rose fruit	41	Blue object language R.hybrida 'Lansewuyu'
12	Flos aucklandiae flower R.banksiae	42	Autumn carmine R.hybrida 'Autumn Rouge'
				13	Damascona r	43	Yuncao R.hybrida 'Yuncaixia'
14	Fructus Rosae Laevigatae R	44	Shinetic wife R.hybrid ' The poet's wife '
				15	R. longicuspis of Rosa longifolia	45	Mickey R.hybrid 'Mickey'
16	Rosa microphylla R	46	Ligament R.hybrida 'Kizuna'
				17	R. pragmatica	47	Rubia cordifolia R.hybrid 'Princess Sissi'
18	Rose r	48	Comet R.hybrid 'Comet'
				19	R. omeiensis of rosa omeiensis	49	The peach is not the Japanese B.hybrida' Taozhiyaoyao’
20	Purple Chinese rose flower R	50	Cimba R.hybrida 'Simba'
				21	Roxburghi r	51	Caramel Antique R.hybrida 'Caramel Antique'
22	China rose flower perfume R	52	R.hybrida ' Huaye ' of Rosa multiflora '
				23	Seven sisters r	53	Dragon-Shao-Gem R.hybrida 'Longsharaoshi'
24	Rosa davurica R	54	Willow dancing autumn flower R.hybrida 'Liuwuquuhua'
				25	Multiflora of multiflora rose	55	Generous gardener. hybrida 'general garden'
26	Sky Terrace R.hybrida 'Saturn Terrace'	56	Kaili R.hybrida 'Carey'
				27	Yunnan red wine R.hybrida 'Dianhong'	57	Austin R.hybrid 'Austin'
28	' ink Red R.hybrid ' Crimsin Glory '	58	Spectrum R.hybrid 'Spectrum'
				29	Vegetarian season R.hybrid 'Suji'	59	Mi phenanthrene R.hybrida 'MiFi'
30	Cloud rose R.hybrid 'Yunmei'	60	Sun flower balcony R.hybrida 'Sunflower balcony'

The 5 tree-shaped Chinese rose finished products grafted by the method are transplanted to the northwest Xinjiang Wulu wood city, the northern Changchun city, the southern Haikou city and the eastern Qingdao city in China, and the test planting experiment for 2 years is carried out, and the result shows that the survival rate is 100 percent, the viewing effect of the finished products is good, and the results show that: by adopting the method, the affinity between the rootstock and the scion is accurately measured, the operation is simple and convenient, and the survival rate is high. The seedling of the rosa chuanxiong hort has strong adaptability, is suitable for popularization in China east, west, south and north, and overcomes the defect of narrow adaptability range of the rosa chuanxiong hort rootstocks generally adopted in the prior art. Meanwhile, the wild condition selfing setting rate of the rosa chuanxiong maxim is high, the yield is large, the wild condition selfing setting rate of the rosa chuanxiong maxim is favorable for breeding the seedling stock, the wild environment and the ecology are not damaged, and the wild condition selfing setting rate and the yield are high, the wild condition selfing setting rate and the wild condition selfing setting method have the advantages of good stress resistance, developed root systems, fast growth (2-3 years main branch and trunk molding), long ornamental life after grafting and the like, and effectively overcome the defects of poor stress resistance, insufficiently developed root systems, slow growth (3-5 years main branch and trunk molding), short ornamental life after grafting (losing ornamental characters after 5 years) and the like of tree-shaped rosa chuanxiong maxim stock varieties in the current market.

The distribution of wild resource of Rosa chinensis is centered on Yunnan and Sichuan. On the basis of surveying and comparing the adaptability and the selfing setting rate of 24 wild rose resources widely distributed in the southwest region and simultaneously testing the plant uprightness of the resources, the inventors find that the Sichuan Yunnan rose has strong adaptability, high selfing setting rate and good plant uprightness, integrates the three essential characteristics of a tree-shaped Chinese rose root stock, and is not the second choice of the tree-shaped Chinese rose root stock at present.

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Claims (10)

1. A method for cultivating tree-shaped Chinese roses by utilizing seedling stocks is characterized by comprising the following steps: setting the rosa plant seedling stock, selecting a Chinese rose scion with strong affinity with the rosa plant seedling stock, and grafting the selected Chinese rose scion with the set seedling stock.

2. The method for cultivating tree-shaped China rose by using a seedling rootstock according to claim 1, wherein: the setting of the rosa plant seedling stock is that only the main branch and the lateral buds at the top of the main branch of the rosa plant seedling stock are reserved when the rosa plant seedling stock grows to the height of 50-150 cm, and the lateral branches and the rest lateral buds of the rosa plant seedling stock are cut off by a sterilized cutter.

3. The method for cultivating tree-shaped Chinese roses by using seedling stocks according to claim 1, wherein the method comprises the following steps: and grafting the selected Chinese rose scion and the rosa plant seedling stock when the diameter of the main branch and the trunk of the shaped rosa plant seedling stock grows to be more than 1.5 cm.

4. The method for cultivating tree-shaped China rose by using seedling rootstocks according to claim 3, wherein: in the grafting, a large-flower Chinese rose scion is grafted to a rosa plant seedling stock with a main branch diameter of more than or equal to 3cm, a middle-flower Chinese rose scion is grafted to a rosa plant seedling stock with a main branch diameter of more than or equal to 2cm, and a small-flower Chinese rose scion is grafted to a rosa plant seedling stock with a main branch diameter of more than or equal to 1.5 cm; the large-flower type Chinese rose scions refer to Chinese rose scions with the flower diameter larger than 9cm, the medium-flower type Chinese rose scions refer to Chinese rose scions with the flower diameter of 4-9 cm, and the small-flower type Chinese rose scions refer to Chinese rose scions with the flower diameter of less than 4 cm.

5. The method for cultivating tree-shaped China rose by using a seedling rootstock according to claim 1, wherein: and (3) cutting off new lateral branches and lateral buds of the set rosa plant seedling rootstocks by using a sterilized cutter 10-14 days before grafting.

6. The method for cultivating tree-shaped China rose by using a seedling rootstock according to claim 1, wherein: the selection of the Chinese rose scions with strong affinity with the rosa plant parenchyma rootstocks is to perform accurate determination on the Chinese rose scions and the rosa plant parenchyma rootstocks by using an SSR molecular marker technology, the accurate determination is to perform PCR on DNA (polymerase chain reaction) extracted from leaf samples of the rosa plant parenchyma rootstocks and the Chinese rose scions by using 5 pairs of SSR primers respectively, calculate Nei's similarity coefficients between the samples by adopting NTSYS-PC 2.0 analysis software, and select Chinese rose scion materials with Nei's similarity coefficients being not less than 0.4 between every two of the Chinese rose scions and the rosa plant parenchyma rootstocks, namely the Chinese rose scions with strong affinity with the rosa plant parenchyma rootstocks;

the 5 pairs of SSR primers are SSR01 primers, SSR02 primers, SSR03 primers, SSR04 primers and SSR05 primers;

the SSR01 primer consists of an SSR01 upstream primer and an SSR01 downstream primer, and the nucleotide sequence of the SSR01 upstream primer is shown as SEQ ID NO: 1, the nucleotide sequence of the SSR01 downstream primer is shown as SEQ ID NO: 2 is shown in the specification;

the SSR02 primer consists of an SSR02 upstream primer and an SSR02 downstream primer, and the nucleotide sequence of the SSR02 upstream primer is shown as SEQ ID NO: 3, the nucleotide sequence of the SSR02 downstream primer is shown as SEQ ID NO: 4 is shown in the specification;

the SSR03 primer consists of an SSR03 upstream primer and an SSR03 downstream primer, and the nucleotide sequence of the SSR03 upstream primer is shown as SEQ ID NO: 5, the nucleotide sequence of the SSR03 downstream primer is shown in SEQ ID NO: 6 is shown in the specification;

the SSR04 primer consists of an SSR04 upstream primer and an SSR04 downstream primer, and the nucleotide sequence of the SSR04 upstream primer is shown as SEQ ID NO: 7, the nucleotide sequence of the SSR04 downstream primer is shown as SEQ ID NO: 8 is shown in the specification;

the SSR05 primer consists of an SSR05 upstream primer and an SSR05 downstream primer, and the nucleotide sequence of the SSR05 upstream primer is shown as SEQ ID NO: 9, the nucleotide sequence of the SSR05 downstream primer is shown as SEQ ID NO: shown at 10.

7. The method for cultivating tree-shaped Chinese roses by using seedling stocks according to claim 1, wherein the method comprises the following steps: the breeding process of the rosa plant seedling rootstock comprises the following steps: carrying out cold Tibetan processing, sterilization processing, water absorption processing, germination accelerating processing, sowing and seedling planting on rosa plant seeds in sequence, and specifically comprising the following steps:

1) refrigerator treatment: selecting selfing seeds of the rose plants in the current year, and refrigerating the selfing seeds for 45 to 60 days at constant temperature of 4 ℃ and relative air humidity of 70 to 90 percent in dark light in autumn or winter;

2) and (3) sterilizing the seeds: soaking the mixture for 15 to-25 min by using a potassium permanganate solution with the concentration of 3 to 5 thousandth w/w at the beginning of 2 months in the next year, and washing the mixture for 2 to 3 times by using sterile water;

3) carrying out water absorption treatment on seeds: soaking the sterilized seeds in sterile water at the temperature of 95-100 ℃ for 4-5 min, then putting the seeds in sterile water at room temperature, changing the water every night, and soaking for 3-5 days;

4) and (3) carrying out germination accelerating treatment on the seeds: putting the seeds subjected to water absorption treatment on a germination accelerating substrate, covering 2-3 cm of sterilized wet sand on the seeds, wherein the germination accelerating environment is that the relative air humidity is 80-95%, and the temperature of the germination accelerating environment is 18-23 ℃; the germination accelerating matrix is formed by mixing sterilized perlite, humus soil and peat according to the volume ratio of 1:1: 1.

5) Sowing seeds: when the seeds subjected to the pregermination start to sprout, putting the seeds into a hole tray provided with a sowing substrate, and sowing the seeds in a hole-by-seed mode; the beginning of germination accelerating seeds is that the seed shells crack or the germination is seen; the seeding matrix is formed by mixing sterilized perlite and humus according to the volume ratio of 1: 1;

6) planting seedling: and after sowing, transplanting seedlings with developed root systems into flowerpots or root control containers with the calibers of 30-50 cm for planting, wherein soil in the flowerpots or the root control containers is formed by mixing garden soil and humus soil according to the volume ratio of 1: 1.

8. The method for cultivating tree-shaped China rose by using a seedling rootstock according to claim 1 or 3, wherein the method comprises the following steps: after the interface between the rosa plant seedling stock and the rosa chinensis scion is healed, performing modeling on the dendriform rosa chinensis, wherein the modeling comprises the following steps: 1) every month, removing the sprouts and branches of the non-scions on the seedling stocks; 2) carrying out small pruning once each at 5 months and 9 months every year, wherein the small pruning is to prune 10-20 cm of withered flowering branches of the Chinese rose scions; 3) carrying out major pruning once every 12 months, wherein the major pruning for the large flower type Chinese rose scions is as follows: pruning the flowering branches of the large-flower type Chinese rose scions to the positions with the diameters of 8-10 mm; the major pruning for the medium-flower Chinese rose scions is as follows: pruning the flowering branches of the medium-flower type Chinese rose scions to positions with the diameter of 5-7 mm; the major pruning for the small flower type Chinese rose scions is as follows: pruning the flowering branches of the small-flower type Chinese rose scions to the positions with the diameters of 3-4 mm.

9. The method for cultivating tree-shaped Chinese rose using seedling rootstock according to any one of claims 1 to 9, wherein: the rosa genus plant seedling stock is a rosa chuanxiong maxim seedling stock.

10. A group of SSR primers for screening Chinese rose scions with strong affinity with rosa plant seedling stocks is characterized in that: the SSR primers are an SSR01 primer, an SSR02 primer, an SSR03 primer, an SSR04 primer and an SSR05 primer;

the SSR01 primer consists of an SSR01 upstream primer and an SSR01 downstream primer, and the nucleotide sequence of the SSR01 upstream primer is shown as SEQ ID NO: 1, the nucleotide sequence of the SSR01 downstream primer is shown in SEQ ID NO: 2 is shown in the specification;

the SSR02 primer consists of an SSR02 upstream primer and an SSR02 downstream primer, and the nucleotide sequence of the SSR02 upstream primer is shown as SEQ ID NO: 3, the nucleotide sequence of the SSR02 downstream primer is shown as SEQ ID NO: 4 is shown in the specification;

the SSR03 primer consists of an SSR03 upstream primer and an SSR03 downstream primer, and the nucleotide sequence of the SSR03 upstream primer is shown as SEQ ID NO: 5, the nucleotide sequence of the SSR03 downstream primer is shown as SEQ ID NO: 6 is shown in the specification;

the SSR04 primer consists of an SSR04 upstream primer and an SSR04 downstream primer, and the nucleotide sequence of the SSR04 upstream primer is shown as SEQ ID NO: 7, the nucleotide sequence of the SSR04 downstream primer is shown as SEQ ID NO: 8 is shown in the specification;

the SSR05 primer consists of an SSR05 upstream primer and an SSR05 downstream primer, and the nucleotide sequence of the SSR05 upstream primer is shown as SEQ ID NO: 9, the nucleotide sequence of the SSR05 downstream primer is shown as SEQ ID NO: shown at 10.