Pear rootstock self-rooting rapid propagation method

Abstract

The invention belongs to the technical field of plant propagation, and particularly relates to a pear rootstock self-rooting rapid propagation method. The method comprises the following steps that firstly, 7-10 days before pear sprouting, base cryptobuds on a female parent tree are stimulated to germinate, and branches in a juvenile phase state are obtained after the cryptobuds germinate; secondly, sterilizing the branches in the juvenile stage, cutting the branches into branch sections, and inserting the branch sections into an MS culture medium for primary culture to obtain primarily cultured branch sections; then inserting the branch segments subjected to primary culture into a proliferation culture medium for proliferation culture to obtain adventitious shoots; then, the adventitious shoots are subjected to activation culture, and activated adventitious shoots are obtained; and finally, dipping the activated adventitious tips in a cutting rooting agent, and inserting the dipped adventitious tips into a cutting medium for cutting culture to obtain the self-rooted propagation seedlings of the pear rootstocks. The propagation method is suitable for pear rootstocks, can solve the problem of industrial application of pear rootstock varieties, and promotes modernization improvement of the pear industry.

Description

Technical Field

[0001] The invention belongs to the technical field of plant propagation, and in particular relates to a method for rapid self-root propagation of a pear rootstock. Background Art

[0002] Rootstocks are of great significance to fruit tree production. They can improve the resistance of fruit trees, promote early fruiting and high yield, improve fruit quality, and adjust tree structure. The key to the industrial application of rootstocks is to obtain a large number of high-quality self-rooted seedlings. At present, some fruit trees quickly obtain self-rooted rootstock seedlings mainly through propagation methods such as cuttings, layering and tissue culture. However, in Pyrus plants, there are problems such as difficulty in rooting cuttings, layering and tissue culture, and low or extremely low propagation efficiency, which affects the industrial application of excellent rootstocks. Therefore, it is particularly important to provide a method for self-rooted propagation of pear rootstocks. Summary of the invention

[0003] The purpose of the invention is to provide a method for rapid self-root propagation of pear rootstocks, which can improve the rooting rate of cuttings, increase the survival rate of transplanting, and solve the problem of industrial application of pear rootstock varieties.

[0004] The invention provides a method for rapid self-root propagation of pear rootstock, comprising the following steps:

[0005] 7-10 days before the pear buds sprout, stimulate the buds at the base of the mother tree to germinate and obtain branches in the juvenile state;

[0006] Sterilizing the juvenile branches and cutting them into branch segments, inserting the branch segments into MS culture medium for primary culture, and obtaining primary cultured branch segments;

[0007] inserting the primary cultured branch segments into a proliferation culture medium for proliferation culture to obtain adventitious shoots;

[0008] activating and culturing the adventitious shoots to obtain activated adventitious shoots;

[0009] The activated adventitious shoots are dipped in a cutting rooting agent, and after dipping, the adventitious shoots are inserted into a cutting medium for cutting culture to obtain pear rootstock self-root propagation seedlings.

[0010] As a preferred embodiment, the mother tree includes a perennial pear rootstock plant or a perennial grafted pear tree.

[0011] As a preferred solution, the method for stimulating the germination of latent buds at the base of the mother tree is: germination of the mother tree is accelerated by ring cutting or ring stripping to make the latent buds germinate;

[0012] The position of the ring cutting or ring stripping is 5 to 12 cm above the root neck of the perennial pear rootstock or 5 to 12 cm above the root neck of the rootstock below the grafting point of the perennial grafted pear tree.

[0013] As a preferred embodiment, the length of the branches in the juvenile state is 20 to 30 cm; the length of the branch segments is 1.3 to 1.7 cm; and the branch segments are inserted into the MS culture medium with the buds facing upward.

[0014] As a preferred embodiment, the temperature of the primary culture is 23-27°C, the illumination duration of the primary culture is 16h / d, the illumination intensity of the primary culture is 2000-2500lx, and the time of the primary culture is 20-30d.

[0015] As a preferred embodiment, the proliferation medium uses MS medium as the basic medium and further includes the following components in concentrations: 0.2 mg / L IBA, 1.0 mg / L 6-BA, 30 g / L sucrose and 7.5 g / L agar;

[0016] The temperature of the proliferation culture is 23-27° C., the illumination duration of the proliferation culture is 16 h / d, the illumination intensity of the proliferation culture is 2000-2500 lx, and the time of the proliferation culture is 20-30 days.

[0017] As a preferred embodiment, the activation culture medium is a proliferation medium;

[0018] The temperature of the activation culture is 23-27°C, the illumination time of the activation culture is 16h / d, the illumination intensity of the activation culture is 2000-2500lx, and the activation culture time is 15d;

[0019] The activation culture was performed three times, and the height of the adventitious shoots was adjusted to 1.4-1.6 cm after three activation cultures.

[0020] As a preferred embodiment, the cutting rooting agent includes 2000 mg / L IBA, 2000 mg / L NAA or 2000 mg / L IAA;

[0021] The dipping time is 50 to 70 seconds;

[0022] Before the dipping, the method further includes cutting the base of the adventitious tip into an incision with an angle of 45°.

[0023] As a preferred embodiment, the cutting matrix includes a combined matrix of vermiculite and perlite; the volume ratio of the vermiculite to the perlite is 1:1.

[0024] As a preferred solution, the cutting depth is 1 / 3 of the adventitious shoot;

[0025] The temperature of the cutting culture is 25-27° C., the relative humidity of the cutting culture is ≥90%, the illumination duration of the cutting culture is 16 h / d, and the illumination intensity of the cutting culture is 2000-2500 lx.

[0026] As a preferred embodiment, the cuttings include tissue culture bottle cuttings, plug tray cuttings or seedbed cuttings.

[0027] Beneficial effect: The present invention provides a method for rapid self-root propagation of pear rootstock, comprising the following steps: first, 7 to 10 days before pear budding, stimulating the germination of hidden buds at the base of the mother tree, and obtaining branches in the juvenile state after the hidden buds germinate; secondly, sterilizing the branches in the juvenile state and cutting them into branch segments, inserting the branch segments into MS culture medium for primary culture, and obtaining primary cultured branch segments; then inserting the primary cultured branch segments into proliferation culture medium for proliferation culture to obtain adventitious shoots; then activating the adventitious shoots to obtain activated adventitious shoots; finally, dipping the activated adventitious shoots in a cutting rooting agent, inserting the adventitious shoots into a cutting medium for cutting culture after dipping, and obtaining pear rootstock self-root propagation seedlings. The method of the present invention is suitable for pear rootstock, can significantly improve the cutting rooting rate of pear rootstock, and has a high survival rate of transplantation, thereby solving the problem of industrial application of pear rootstock varieties and promoting the modernization and upgrading of the pear industry.

[0028] The examples show that the branches obtained by using the base hidden buds in the present invention are in the juvenile state, with low dedifferentiation degree, strong root regeneration ability, and high transplant survival rate. According to statistics, the cutting rooting rate of the branches of the present invention is relatively high, reaching 81.67% to 91.26%, which is significantly better than the cutting rooting rate of adult branches. And according to the statistics of the proliferation coefficient, it is found that under the optimal proliferation culture medium conditions, the proliferation coefficient of Pyrus betulaeformis 'Qingzhen V101' germplasm is 4.47. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0030] Figure 1 The rootstock germplasm in Examples 2 to 3 is self-rooted propagation, wherein A to D are self-rooted propagation in tissue culture bottles; E to F are self-rooted propagation in plug trays; and G is self-rooted propagation in seedbeds;

[0031] Figure 2 Schematic diagram of a self-root propagation method suitable for pear rootstock. DETAILED DESCRIPTION

[0032] The invention provides a method for rapid self-root propagation of pear rootstock, comprising the following steps:

[0033] 7-10 days before the pear buds sprout, stimulate the buds at the base of the mother tree to germinate and obtain branches in the juvenile state;

[0034] Sterilizing the juvenile branches and cutting them into branch segments, inserting the branch segments into MS culture medium for primary culture, and obtaining primary cultured branch segments;

[0035] inserting the primary cultured branch segments into a proliferation culture medium for proliferation culture to obtain adventitious shoots;

[0036] activating and culturing the adventitious shoots to obtain activated adventitious shoots;

[0037] The activated adventitious shoots are dipped in a cutting rooting agent, and after dipping, the adventitious shoots are inserted into a cutting medium for cutting culture to obtain pear rootstock self-root propagation seedlings.

[0038] The present invention stimulates the germination of hidden buds at the base of the mother tree 7 to 10 days before the pears sprout, and obtains branches in the juvenile state. As a specific embodiment, the method for stimulating the germination of hidden buds at the base of the mother tree is: germination is accelerated by ring cutting or ring stripping, and the selected position is 5 to 12 cm above the root neck of the perennial plant of the rootstock or 5 to 12 cm above the root neck of the rootstock under the grafting point of the perennial grafted pear tree, and the ring cutting requires more than 2 to 3 circles, and the ring cutting width is 0.8 to 1.2 cm. After ring cutting or ring stripping treatment, the germination of hidden buds in the juvenile state of the rootstock can be promoted; after the hidden buds germinate, support rods are used to support them to prevent the hidden bud branches from breaking; the branches obtained by the germination of hidden buds are branches in the juvenile state.

[0039] As a specific embodiment, the number of days before the pear sprouts can be 7d, 8d, 9d or 10d; as a specific embodiment, the female parent tree includes a perennial pear rootstock plant or a perennial grafted pear tree; as a specific embodiment, the distance above the root neck (below the grafting point) can be 5.0cm, 5.1cm, 5.2cm, 5.3cm, 5.4cm, 5.5cm, 5.6cm, 5.7cm, 5.8cm, 5.9cm, 6.0cm, 6.1cm, 6.2cm, 6.3cm, 6.4cm, 6.5cm, 6.6cm, 6.7cm, 6.8cm, 6.9cm, 7.0cm, 7.1cm, 7.2cm, 7.3cm, 7.4cm, 7.5cm, 7.6cm, 7.7cm, 7.8cm, 7.9cm, 8.0cm, 8.1cm, 8.2cm, 8.3cm, 8.4cm, 8. 5cm, 8.6cm, 8.7cm, 8.8cm, 8.9cm, 9.0cm, 9.1cm, 9.2cm, 9.3cm, 9.4cm, 9.5cm, 9.6cm, 9.7cm, 9. 8cm, 9.9cm, 10.0cm, 10.1cm, 10.2cm, 10.3cm, 10.4cm, 10.5cm, 10.6cm, 10.7cm, 10.8cm, 10.9c m, 11.0cm, 11.1cm, 11.2cm, 11.3cm, 11.4cm, 11.5cm, 11.6cm, 11.7cm, 11.8cm, 11.9cm or 12.0cm; as a specific implementation, the number of circles of the ring cutting can be 2 circles or 3 circles; as a specific implementation, the width of the ring cutting can be 0.8cm, 0.9cm, 1.0cm, 1.1cm or 1.2cm.

[0040] When the branches in the juvenile state grow to 20 to 30 cm, the present invention sterilizes the branches in the juvenile state and cuts them into branch segments, inserts the branch segments into MS culture medium for primary culture, and obtains primary cultured branch segments. As a specific implementation method, the present invention collects branches in the juvenile state of the rootstock hidden buds (referred to as explants), rinses them with running water for 2 hours, and then treats them with 70% alcohol for 30 seconds; after treatment, the explants are cut into 4.8-5.2 cm with surgical scissors, and then sterilized with 0.1% HgCl2 or 2% NaClO in a clean bench for 5 minutes to 10 minutes; after sterilization, the explants are cut into 1.3-1.7 cm branch segments in a sterile culture dish with sterile scissors and tweezers in a clean bench, and it is necessary to ensure that there are buds in the branch segments and both ends are new incisions or cuts, and then rinsed with sterile distilled water for 3 times to remove the damage of the sterilizer; finally, the branch segments are inserted into MS culture medium with the buds facing upwards after absorbing the water with sterile absorbent paper, and primary culture is carried out in a culture room to obtain primary cultured branch segments. As a preferred embodiment, the temperature of the primary culture in the present invention is 23-27°C, the illumination duration of the primary culture is 16h / d, the illumination intensity of the primary culture is 2000-2500lx, and the time of the primary culture is 20-30d.

[0041] As a specific embodiment, the length of the juvenile state branches can be 20cm, 21cm, 22cm, 23cm, 24cm, 25cm, 26cm, 27cm, 28cm, 29cm or 30cm; as a specific embodiment, the length of the explant cut can be 4.8cm, 4.9cm, 5.0cm, 5.1cm or 5.2cm; as a specific embodiment, the sterilization time can be 5min, 6min, 7min, 8min, 9min or 1 0min; as a specific embodiment, the length of the branch segment can be 1.3cm, 1.4cm, 1.5cm, 1.6cm or 1.7cm; as a specific embodiment, the temperature of the primary culture can be 23℃, 24℃, 25℃, 26℃ or 27℃; as a specific embodiment, the light intensity of the primary culture can be 2000lx, 2010lx, 2020lx, 2030lx, 2040lx, 2050lx, 2060lx, 2070lx, 20 80lx, 2090lx, 2100lx, 2110lx, 2120lx, 2130lx, 2140lx, 2150lx, 2160lx, 2170lx, 2180lx, 2190lx, 2200lx, 22 10lx, 2220lx, 2230lx, 2240lx, 2250lx, 2260lx, 2270lx, 2280lx, 2290lx, 2300lx, 2310lx, 2320lx, 2330lx, 23 As a specific embodiment, the time of the primary culture is 20d, 21d, 22d, 23d, 24d, 25d, 26d, 27d, 28d, 29d or 30d.

[0042] The present invention inserts the primary cultured branch segments into a proliferation culture medium for proliferation culture to obtain adventitious shoots. As a preferred embodiment, the proliferation culture medium uses MS culture medium as a basic culture medium and also includes the following components at the following concentrations: 0.2 mg / L IBA, 1.0 mg / L 6-BA, 30 g / L sucrose and 7.5 g / L agar; the temperature of the proliferation culture is 23-27°C, the illumination time of the proliferation culture is 16 h / d, the illumination intensity of the proliferation culture is 2000-2500 lx, and the time of the proliferation culture is 20-30 days.

[0043] As a specific embodiment, the temperature of the proliferation culture can be 23°C, 24°C, 25°C, 26°C or 27°C; as a specific embodiment, the light intensity of the proliferation culture can be 2000lx, 2010lx, 2020lx, 2030lx, 2040lx, 2050lx, 2060lx, 2070lx, 2080lx, 2090lx, 2100lx, 2110lx, 2120lx, 2130lx, 2140lx, 2150lx, 2160lx, 2170lx, 2180lx, 2190lx, 2200lx, 2210lx, 2220lx, 2230lx, 2240lx, 2250lx lx, 2400lx, 2410lx, 2420lx, 2430lx, 2440lx, 2450lx, 2460lx, 2470lx, 2480lx, 2490lx or 2500lx; as a specific embodiment, the time of the proliferation culture is 20d, 21d, 22d, 23d, 24d, 25d, 26d, 27d, 28d, 29d or 30d.

[0044] As a specific embodiment, the optimal proliferation medium for 'Qingzhen V101' germplasm is: MS medium as the basic medium, and also includes the following concentrations of components: 0.2mg / L IBA, 1.0mg / L 6-BA, 30g / L sucrose and 7.5g / L agar, and the proliferation coefficient is 4.47.

[0045] After obtaining the adventitious shoots, the present invention dips the adventitious shoots in a cutting rooting agent, and after dipping, the adventitious shoots are inserted into a cutting medium for cutting culture to obtain pear rootstock self-root propagation seedlings. The present invention preferably cuts the base of the adventitious shoot into an incision with an angle of 45° and then dips it. As a specific embodiment, the present invention dips the adventitious shoots in a cutting rooting agent for 50 to 70 seconds; as a specific embodiment, the dipping time can be 50 seconds, 51 seconds, 52 seconds, 53 seconds, 54 seconds, 55 seconds, 56 seconds, 57 seconds, 58 seconds, 59 seconds, 60 seconds, 61 seconds, 62 seconds, 63 seconds, 64 seconds, 65 seconds, 66 seconds, 67 seconds, 68 seconds, 69 seconds or 70 seconds. The cutting rooting agent includes 2000 mg / L of IBA, 2000 mg / L of NAA or 2000 mg / L of IAA. As a specific implementation manner, before dipping, the adventitious shoots obtained by the proliferation culture are activated and cultured on a proliferation culture medium, and the culture period is shortened to 15 days. The height of the subcultured adventitious shoots is adjusted to 1.0 cm to 1.5 cm through the proliferation culture; then, after three consecutive activation cultures, the state of the rootstock germplasm explants is adjusted; after the third culture of the rootstock germplasm, the height of the adventitious shoots is adjusted to 1.5 cm; the temperature, illumination time and illumination intensity of the activation culture are the same as those of the proliferation culture and will not be repeated here; as a specific implementation manner, the height of the adventitious shoots can be increased to 1.0 cm, 1.1 cm, 1.2 cm, 1.3 cm, 1.4 cm or 1.5 cm.

[0046] As a specific implementation, the cutting matrix includes a matrix of vermiculite and perlite in a volume ratio of 1:1. As a specific implementation, before using the matrix, the matrix needs to be sterilized in a high-temperature and high-pressure sterilizer, and the sterilized matrix is ​​mixed with sterile water to achieve a ball that can be held in the hand without dripping and falls apart when lightly touched, which is about 30% of the maximum water holding capacity of the matrix.

[0047] The cutting depth is 1 / 3 of the adventitious shoot, and a pear rootstock self-root propagation seedling is obtained after cutting culture. The cutting culture temperature is 25-27°C, the relative humidity of the cutting culture is ≥90%, the lighting duration of the cutting culture is 16h / d, and the lighting intensity of the cutting culture is 2000-2500lx; the cutting includes tissue culture bottle cutting, plug tray cutting or seedling bed cutting.

[0048] As a specific embodiment, the temperature of the cutting culture can be 25°C, 26°C or 27°C; as a specific embodiment, the relative humidity of the cutting culture can be 90%, 91%, 92%, 93%, 94% or 95%; as a specific embodiment, the light intensity of the cutting culture can be 2000lx, 2010lx, 2020lx, 2030lx, 2040lx, 2050lx, 2060lx, 2070lx, 2080lx, 2090lx, 2100lx, 2110lx, 2120lx, 2130lx, 2140lx, 2150lx, 2160lx, 2170lx x, 2180lx, 2190lx, 2200lx, 2210lx, 2220lx, 2230lx, 2240lx, 2250lx, 2260lx, 2270lx, 2280lx, 2290lx, 2300lx, 2310lx, 2320lx, 2330lx, 23 40lx, 2350lx, 2360lx, 2370lx, 2380lx, 2390lx, 2400lx, 2410lx, 2420 lx, 2430lx, 2440lx, 2450lx, 2460lx, 2470lx, 2480lx, 2490lx or 2500lx.

[0049] As a specific implementation method, the method of tissue culture bottle cutting can be: ① put the matrix into the tissue culture bottle, and compact it to make the thickness of the matrix 1.5 cm; ② take out the adventitious shoots obtained by activation culture from the tissue culture bottle, clean the culture medium at the base with distilled water, and then dry the surface moisture with sterile filter paper or absorbent paper; ③ use a scalpel to cut off the blunt end of the base of the adventitious shoot to produce a new wound, the incision angle is 45°, and then dip it in the cutting rooting agent for 1 minute; ④ immediately after the dipping is completed, insert the adventitious shoot into the matrix of the tissue culture bottle with surgical forceps, the depth is 1 / 3 of the adventitious shoot; ⑤ cover the lid of the tissue culture bottle and place it in the culture room for culture.

[0050] As a specific implementation method, the method of plug tray cuttings can be: ① fill the plug tray with the matrix and compact it by hand to a thickness of ≥3cm; ② take out the adventitious shoots obtained by activation culture from the tissue culture bottle, clean the culture medium at the base with distilled water, and then dry the surface moisture with sterile filter paper or absorbent paper; ③ use a scalpel to cut off the blunt end of the base of the adventitious shoot to produce a new wound, with an incision angle of 45°, and then dip it in the cutting rooting agent for 1 minute; ④ immediately after the dipping, insert the adventitious shoot into the matrix of the plug tray with surgical forceps, with a depth of 1 / 3 of the adventitious shoot; ⑤ after the cutting is completed, culture it in an artificially controlled environment, and the moisture content of the matrix is ​​supplemented by mist spraying; as a specific implementation method, the thickness can be 3cm, 4cm, 5cm or 6cm.

[0051] As a specific implementation method, the method of seedbed cuttings can be: ① evenly spread the matrix on the seedbed and compact it by hand, with a thickness of ≥3cm; ② take out the adventitious shoots obtained by activation culture from the tissue culture bottle, clean the culture medium at the base with distilled water, and then dry the surface moisture with sterile filter paper or absorbent paper; ③ use a scalpel to cut off the blunt end of the base of the adventitious shoot to produce a new wound, with an incision angle of 45°, and then dip it in the cutting rooting agent for 1 minute; ④ immediately after the dipping, insert the adventitious shoot into the matrix of the seedbed with surgical forceps, with a depth of 1 / 3 of the adventitious shoot; ⑤ after the cutting is completed, culture it in an artificially controlled environment, and the moisture content of the matrix is ​​supplemented by mist spraying; as a specific implementation method, the thickness can be 3cm, 4cm, 5cm or 6cm.

[0052] As a specific implementation, taking the Pyrus betula 'Qingzhen V101' germplasm as an example, when the cutting rooting agent is 2000 mg / L IBA, the cutting rooting rate is 84.33%; when the cutting rooting agent is 2000 mg / L NAA, the cutting rooting rate is 81.67%; when the cutting rooting agent is 2000 mg / L IAA, the cutting rooting rate is 84.33%.

[0053] As a specific implementation, taking Pyrus betulata 'Qingzhen V101' germplasm as an example, when the matrix is ​​a combination matrix of vermiculite and perlite in a volume ratio of 1:1, the rooting rate of cuttings is 89.67%.

[0054] The adventitious shoot cuttings are 20d to 30d long, and can be transplanted after the cuttings take root. The transplanting can be directly transplanted into containers such as non-woven bags, or directly transplanted into the field. When the seedlings reach a thickness of 0.8cm and a height of 100cm, variety grafting can be performed. As a specific embodiment, the number of days for the adventitious shoot cuttings can be 20d, 21d, 22d, 23d, 24d, 25d, 26d, 27d, 28d, 29d or 30d.

[0055] In order to further illustrate the present invention, a method for rapid self-root propagation of a pear rootstock provided by the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0056] Unless otherwise specified, the present invention has no special requirements for the preparation raw materials, and commercially available products known to those skilled in the art can be used.

[0057] IBA used in the embodiments of the present invention was purchased from Sangon Biotech (Shanghai) Co., Ltd. with the item number A600725; 6-BA was purchased from Sangon Biotech (Shanghai) Co., Ltd. with the item number A600743; NAA was purchased from Sangon Biotech (Shanghai) Co., Ltd. with the item number A600729; IAA was purchased from Sangon Biotech (Shanghai) Co., Ltd. with the item number A600723; HgCl2 was purchased from Sinopharm Chemical Reagent Co., Ltd. with the item number 10013616CK; NaClO was purchased from Sangon Biotech (Shanghai) Co., Ltd. with the item number A501944.

[0058] Example 1 Selection of propagation material

[0059] Selection of rootstock: Choose 'Qingzhen V101' (https: / / zzzyk.qau.edu.cn / content / Rootstock / 6528bc7d3c37438d9c0c5c5ff2266e56), a perennial grafted tree.

[0060] Hidden bud germination of rootstock: 10 days before the budding of pear, the budding method is ring cutting. The selected location is 10 cm above the root neck of the rootstock under the grafting point, and the ring cutting is 3 circles with a width of 1 cm. After treatment, the budding of hidden buds in the juvenile state of the rootstock can be promoted. After the hidden buds germinate, support rods are used to support them to prevent the hidden bud branches from breaking. The branches obtained from the budding of hidden buds are branches in the juvenile state, which will be used for subsequent research.

[0061] When the branches in the juvenile state grow to 20 cm, collect the branches from the hidden buds (branches in the juvenile state) and the upper one-year-old branches (branches in the adult state), dip them in 2000 mg / L IBA (indolebutyric acid) for 1 minute, insert them into the vermiculite matrix after dipping, and count the rooting rate after 30 days. The results are shown in Table 1. The study found that the cutting rooting rate of branches in the juvenile state was relatively high, at 63.33%, which was significantly better than the cutting rooting rate of 2.00% of adult branches. However, the cutting rooting rate of 63.33% is still difficult to meet the needs of the industry, and the cutting rooting rate is unstable and the standard error is high. Therefore, in order to better serve the industry, subsequent technical research was carried out.

[0062] Table 1 Effects of branches in different states on rooting of cuttings

[0063] Cutting rooting rate (%) Childhood state branches 63.33±8.82A Adult branches 2.00±1.53B

[0064] Note: The least significant difference (LSD) method was used to analyze the differences. Letters in the same column indicate significant differences between treatment combinations at 0.05.

[0065] Example 2 Selection of proliferation culture medium

[0066] In order to improve the industrial application efficiency of high-quality rootstocks, tissue culture technology was optimized and research on rootstock self-rooting breeding technology was carried out.

[0067] Tissue culture is divided into two processes: primary culture and proliferation culture.

[0068] Primary culture: According to the method of Example 1, the salt-alkali tolerant rootstock 'Qingzhen V101' was germinated to promote the germination of the base hidden buds, and the branches in the juvenile state were obtained. The branches in the juvenile state (explants) with the hidden buds of the rootstock were collected, rinsed with running water for 2 hours, and then treated with 70% alcohol for 30 seconds. After treatment, the explants were cut into 5cm with surgical scissors, and then sterilized with 2% NaClO by volume for 8 minutes in the clean bench. After sterilization, sterile scissors and tweezers were used in the clean bench to cut the explants into 1.5cm branches in a sterile culture dish. It is necessary to ensure that there are buds in the branches, and both ends are new incisions or cuts, and then rinsed with sterile distilled water 3 times to remove the damage of the sterilizer. Finally, after absorbing the water with sterile absorbent paper, the branches were inserted into MS culture medium with the buds facing up and cultured in the culture room (temperature 25°C, light duration 16h / d, light intensity 2000lx).

[0069] Table 2 Screening of germplasm bud proliferation medium

[0070]

[0071] Note: The least significant difference (LSD) method was used to analyze the differences. Letters in the same column indicate significant differences between treatment combinations at 0.05.

[0072] Proliferation culture: Proliferation culture of different rootstock germplasms has different requirements for culture medium components, and it is necessary to screen the culture medium components to improve the proliferation efficiency. After 25 days of primary culture, the 'Qingzhen V101' that has undergone primary culture was used with MS medium as the basic culture medium to evaluate the effects of IBA (indolebutyric acid) and 6-BA (6-benzylaminopurine) on its bud proliferation culture, and screened the suitable type of bud proliferation culture medium. The setting of the culture medium is shown in Table 2. The culture conditions are conventional tissue culture conditions, temperature 25°C, light duration 16h / d, light intensity 2000lx, and culture time 30d. The adventitious shoots obtained by proliferation culture are as follows Figure 1 As shown in A.

[0073] The results in Table 2 show that the proliferation coefficients of adventitious buds induced by 'Qingzhen V101' on different culture media are different (adventitious bud proliferation coefficient = number of proliferation buds / number of inoculated buds). The best proliferation medium for 'Qingzhen V101' is: MS + IBA 0.2mg / L + 6-BA 1.0mg / L + 30g / L sucrose + 7.5g / L agar, with a proliferation coefficient of 4.47.

[0074] Example 3 Tissue culture adventitious shoot cutting technology

[0075] According to the method of Example 2, the optimal proliferation medium of 'Qingzhen V101' was used for culturing, and adventitious shoots after proliferation and culture of Pyrus betulaeformis 'Qingzhen V101' were obtained.

[0076] Activation of rootstock germplasm explants: Before tissue culture adventitious shoot cuttings, the adventitious shoots obtained by proliferation culture are activated on the 'Qingzhen V101' optimal proliferation medium. The culture cycle is shortened to 15 days, and the adventitious shoots of successive proliferation culture are used to increase their height.

[0077] Then, after three consecutive activation cultures (according to the activation method of stock germplasm explants), the state of the stock germplasm explants was adjusted. After the third activation culture of the stock germplasm, the height of the adventitious shoot was adjusted to about 1.5 cm. The culture conditions were conventional tissue culture conditions, temperature 25°C, light duration 16h / d, and light intensity 2000lx.

[0078] Preparation of cutting rooting agent: The cutting rooting agent was set with the hormones and concentrations in Table 3. The adventitious shoots of 'Qingzhen V101' were used as cuttings and vermiculite was used as the matrix. The cutting rooting effect of the rooting agent was evaluated in a tissue culture bottle. The results showed that when the concentration of NAA, IBA or IAA was 2000 mg / L, the cutting rooting rate was the highest, reaching 81.67.00%, 84.33% and 84.33% respectively (the results are shown in Table 3).

[0079] Table 3 Effects of different hormones on rooting of cuttings of pear rootstock germplasm 'Qingzhen V101'

[0080] deal with Hormone concentration and type Cutting rooting rate (%) T1 0 4.67±0.88dd T2 NAA1000mg / L 52.67±4.06c T3 NAA2000mg / L 81.67±2.73ab T4 NAA3000mg / L 64.67±2.73c T5 IBA1000mg / L 65.00±3.51c T6 IBA2000mg / L 84.33±2.40a T7 IBA3000mg / L 60.67±2.73c T8 IAA1000mg / L 63.67±1.76c T9 IAA2000mg / L 84.33±1.76a T10 IAA3000mg / L 53.67±4.06c T11 IBA1000mg / L+NAA100mg / L 68.00±4.36bc T12 IBA100mg / L+NAA1000mg / L 66.33±4.37bc

[0081] Note: The least significant difference (LSD) method was used to analyze the differences. Letters in the same column indicate significant differences between treatment combinations at 0.05.

[0082] Preparation of cutting medium: river sand, vermiculite, perlite, vermiculite + perlite (volume ratio = 1:1) are selected as the medium, and then sterilized in a high-temperature and high-pressure sterilizer for use. Before using the medium, mix the medium with sterile water to achieve a ball that can be held in the hand without dripping and dispersed by light touch, which is about 30% of the maximum water holding capacity of the medium. The adventitious shoots of 'Qingzhen V101' were used as cuttings, and 2000 mg / L IBA was used as a cutting rooting agent. The effect of the medium on cutting rooting was evaluated in a tissue culture bottle. The results showed that the rooting effect of 'Qingzhen V101' was the best when it was cut in the combined medium of vermiculite + perlite, and the cutting rooting rate was 89.67% (see Table 4 for the results).

[0083] Table 4 Effects of different substrates on rooting of cuttings of pear rootstock germplasm 'Qingzhen V101'

[0084] deal with Cutting medium type Cutting rooting rate (%) T1 River sand 74.33±1.86c T2 Vermiculite 83.67±1.45b T3 Perlite 73.33±2.03c T4 Vermiculite+Perlite 89.67±1.45a T5 peat soil 77.00±2.52c

[0085] Note: The least significant difference (LSD) method was used to analyze the differences. Letters in the same column indicate significant differences between treatment combinations at 0.05.

[0086] Cuttings in tissue culture bottles: ① Place the matrix (a mixed matrix of vermiculite and perlite in a volume ratio of 1:1) into the tissue culture bottle and compact it to make the thickness of the matrix 1.5 cm. ② Take out the adventitious shoots obtained by activated culture from the tissue culture bottle, clean the culture medium at the base with distilled water, and then dry the surface moisture with sterile filter paper or absorbent paper. ③ Use a scalpel to cut off the blunt end of the base of the adventitious shoot to create a new wound with an incision angle of 45°, and then dip it in the rooting agent (2000 mg / L NAA) for 1 minute. ④ After the dipping is completed, immediately insert the adventitious shoot into the matrix of the tissue culture bottle with surgical forceps, with a depth of 1 / 3 of the adventitious shoot. ⑤ Cover the lid of the tissue culture bottle and place it in the culture room for culture (see Figure 1 (middle B~D).

[0087] Plug tray cuttings: ① Fill the plug tray with the matrix (peat soil) and compact it by hand to a thickness of 4 cm. ② Take out the adventitious shoots obtained from the pre-culture from the tissue culture bottle, clean the culture medium at the base with distilled water, and then dry the surface moisture with sterile filter paper or absorbent paper. ③ Use a scalpel to cut off the blunt end of the base of the adventitious shoot to create a new wound with an incision angle of 45°, and then dip it in the rooting agent (2000 mg / L NAA) for 1 minute. ④ After the dipping is completed, immediately insert the adventitious shoot into the matrix of the plug tray with surgical tweezers, with a depth of 1 / 3 of the adventitious shoot. ⑤ After the cuttings are completed, culture them in an artificially controlled environment. The moisture content of the matrix is ​​supplemented by mist spraying. The culture temperature is 25°C, the relative humidity is 90%, and the light intensity is 2000lx (see Figure 1 Middle E~F) ， The rooting rate of plug-tray cuttings was 82.32%.

[0088] Seedbed cuttings: ① Spread the matrix (a mixed matrix of vermiculite and perlite in a volume ratio of 1:1) evenly on the seedbed and compact it by hand to a thickness of 5cm. ② Take out the adventitious shoots obtained from pre-culture from the tissue culture bottle, clean the culture medium at the base with distilled water, and then dry the surface moisture with sterile filter paper or absorbent paper. ③ Use a scalpel to cut off the blunt end of the base of the adventitious shoot to create a new wound, with an incision angle of 45°, and then dip it in the rooting agent (2000mg / L NAA) for 1min. ④ After the dipping is completed, immediately insert the adventitious shoot into the matrix of the seedbed with surgical forceps, with a depth of 1 / 3 of the adventitious shoot. ⑤ After the cuttings are completed, culture them in an artificially controlled environment. The moisture content of the matrix is ​​supplemented by mist spraying. The culture temperature is 27°C, the relative humidity is 90%, and the light intensity is 2000lx (see Figure 1Middle G), the rooting rate of cuttings in the seedbed was 91.26%.

[0089] (4) Cutting transplanting: After 30 days of cuttings, the adventitious shoots can be transplanted after the cuttings have taken root. They can be transplanted directly into containers such as non-woven bags, or directly into the field. When the seedlings reach a diameter of 0.8 cm and a height of 100 cm, they can be grafted.

[0090] It can be seen that the method of the present invention can significantly improve the rooting rate of cuttings of pear rootstocks, and the survival rate of transplanting is high. The breeding method of the present invention is suitable for pear rootstocks, can solve the problem of industrial application of pear rootstock varieties, and promote the modernization and improvement of the pear industry.

[0091] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for rapid self-root propagation of pear rootstock, characterized in that: The following steps are involved: 7-10 days before the pear buds sprout, stimulate the buds at the base of the mother tree to germinate and obtain branches in the juvenile state; Sterilizing the juvenile branches and cutting them into branch segments, inserting the branch segments into MS culture medium for primary culture, and obtaining primary cultured branch segments; inserting the primary cultured branch segments into a proliferation culture medium for proliferation culture to obtain adventitious shoots; activating and culturing the adventitious shoots to obtain activated adventitious shoots; The activated adventitious shoots are dipped in a cutting rooting agent, and after dipping, the adventitious shoots are inserted into a cutting medium for cutting culture to obtain pear rootstock self-root propagation seedlings.

2. The method according to claim 1, characterized in that The mother tree comprises a perennial pear rootstock plant or a perennial grafted pear tree; The method for stimulating the germination of latent buds at the base of the mother tree is: germination of the mother tree is accelerated by ring cutting or ring stripping to make the latent buds germinate; The position of the ring cutting or ring stripping is 5 to 12 cm above the root neck of the perennial pear rootstock or 5 to 12 cm above the root neck of the rootstock below the grafting point of the perennial grafted pear tree.

3. The method according to claim 1 or 2, characterized in that: The length of the branches in the juvenile state is 20 to 30 cm; the length of the branch segments is 1.3 to 1.7 cm; and the branch segments are inserted into the MS culture medium with the buds facing upward.

4. The method according to claim 1, characterized in that: The temperature of the primary culture is 23-27° C., the illumination duration of the primary culture is 16 h / d, the illumination intensity of the primary culture is 2000-2500 lx, and the time of the primary culture is 20-30 d.

5. The method according to claim 1 or 4, characterized in that: The proliferation medium uses MS medium as the basic medium and also includes the following components in concentrations: 0.2 mg / L IBA, 1.0 mg / L 6-BA, 30 g / L sucrose and 7.5 g / L agar; The temperature of the proliferation culture is 23-27° C., the illumination duration of the proliferation culture is 16 h / d, the illumination intensity of the proliferation culture is 2000-2500 lx, and the time of the proliferation culture is 20-30 days.

6. The method according to claim 1, characterized in that The culture medium for activation culture is a proliferation culture medium; The temperature of the activation culture is 23-27°C, the illumination time of the activation culture is 16h / d, the illumination intensity of the activation culture is 2000-2500lx, and the activation culture time is 15d; The activation culture was performed three times, and the height of the adventitious shoots was adjusted to 1.4-1.6 cm after three activation cultures.

7. The method according to claim 1 or 6, characterized in that: The cutting rooting agent includes 2000 mg / L IBA, 2000 mg / L NAA or 2000 mg / L IAA; The dipping time is 50 to 70 seconds; Before the dipping, the method further includes cutting the base of the adventitious tip into an incision with an angle of 45°.

8. The method according to claim 1, characterized in that The cutting matrix comprises a combined matrix of vermiculite and perlite; the volume ratio of the vermiculite to the perlite is 1:

1.

9. The method according to claim 1 or 8, characterized in that: The depth of the cutting is 1 / 3 of the adventitious shoot; The temperature of the cutting culture is 25-27° C., the relative humidity of the cutting culture is ≥90%, the illumination duration of the cutting culture is 16 h / d, and the illumination intensity of the cutting culture is 2000-2500 lx.

10. The method according to claim 1, characterized in that The cuttings include tissue culture bottle cuttings, plug tray cuttings or seedbed cuttings.

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