A method for selecting and breeding pear rootstock ND60 and asexual propagation method

Abstract

The present invention relates to a breeding method and asexual propagation method of a pear easy-to-root rootstock ND60. The present invention breeds an easy-to-root rootstock ND60 through a breeding process of browning index measurement, lenticel callus conversion rate determination, rooting induction rate and rooting number. Based on the above-mentioned easy-to-root rootstock D60, two asexual propagation methods of ND60, namely, cutting propagation and tissue culture propagation, have been developed. By applying the easy-to-root ND60 and the asexual propagation method provided by the present invention, the breeding cycle of pear rootstocks can be shortened from more than 20 years to 2-3 years, and the rooting coefficient of the cultivated pear easy-to-root rootstock ND60 can reach up to 2.98, and tissue culture and cutting propagation can achieve factory production.

Description

Technical Field

[0001] The invention relates to the field of plant biotechnology, in particular to a breeding method and an asexual propagation method for a pear easy-to-root rootstock ND60. Background Art

[0002] Pear is the third most popular fruit in my country. my country's pear production and planting area have consistently ranked first in the world. It is also a pillar industry that helps farmers increase their income and become rich. Pear production requires grafting scion varieties onto rootstocks to ensure the purity of the variety and resistance to adverse stress.

[0003] Existing rootstock breeding technology mostly relies on traditional hybridization, but pear is a perennial woody fruit tree with a long growth period and a breeding cycle of up to 20 to 30 years. The rootstock breeding cycle is even longer, and it requires many years of investigation and evaluation after grafting to breed. At present, almost all of my country's pear rootstocks are wild pear resources propagated by seeds, and the rootstock traits are severely separated, resulting in many problems such as incomplete garden establishment, unstable yield, and uneven varieties after grafting.

[0004] Therefore, it is urgent to develop rapid breeding technology for pear rootstocks, cultivate pear rootstock varieties that are easy to root and suitable for asexual factory propagation, and promote the modernization of the pear industry. Summary of the invention

[0005] In view of the defects in the existing technology of pear rootstock breeding, the purpose of the present invention is to provide a method for selecting and breeding pear easy-rooting rootstock ND60 and a method for asexual propagation. To achieve the above purpose, the technical solution adopted by the present invention is:

[0006] A method for breeding pear easy-rooting rootstock ND60, characterized by comprising the following steps:

[0007] Step 1, cutting off the semi-lignified branches of a pear tree in full flowering period after spring budding, immediately extracting the cross-cut stem segments at the base of the semi-lignified branches, extracting the cross-cut stem segments at the base of the semi-lignified branches again after 4 hours, and detecting the absorbance difference △X(850) of the cross-cut stem segments extracted twice at a spectral wavelength of 850nm 4h , screen for semi-lignified branches with browning index Y>0.80;

[0008] The browning index calculation model is shown as follows:

[0009] Y=1.334-2.557×△X(850) 4h ;

[0010] Step 2, culturing the semi-lignified branches newly grown from the natural or hybrid offspring group of mature plants to which the semi-lignified branches obtained in step 1 are derived after germination in the current season in the flowering period of large trees in the dark for 10 days, and selecting the semi-lignified branches with a conversion rate of lenticel dedifferentiation into callus tissue greater than 50%;

[0011] Step 3, take the mature plants to which the semi-lignified branches in step 2 belong, and the newly sprouted semi-lignified branches after germination in the current season, and after induction cultivation at room temperature, dark and 100% humidity for 45 days, select the semi-lignified branches with a rooting rate > 50% and a number of roots > 2, which is the ND60.

[0012] On the basis of the above scheme,

[0013] The varieties of the large pear trees in full bloom in step 1 include Pyrus betula, Pyrus yamanensis, Pyrus chinensis or Pyrus chuanxiensis.

[0014] On the basis of the above scheme,

[0015] The specific conditions for the dark-proof culture in step 2 are a temperature of 25° C. and a humidity of 100%.

[0016] On the basis of the above scheme,

[0017] The specific conditions for the induction cultivation described in step 3 are 100% humidity and 25-28°C temperature.

[0018] A method for asexual propagation of pear easy-rooting rootstock ND60, characterized in that it comprises the following steps:

[0019] Step 1, adding nano-triacontanol to a 2% NaClO aqueous solution and diluting the nano-triacontanol to a concentration of 1 g / L to obtain a nano-triacontanol dispersion;

[0020] Step 2, inserting the ND60 one-year-old branches into the ND60 cutting matrix, diluting the nano-triacontanol dispersion obtained in step 1 to 1 mg / L to obtain a nano-triacontanol dilution, and irrigating the ND60 one-year-old branches with the nano-triacontanol dilution to obtain an ND60 cutting clone propagation system;

[0021] On the basis of the above scheme,

[0022] The specific conditions of the irrigation conditions described in step 2 are: tidal irrigation twice a day, the irrigation height is flush with the upper surface of the substrate, the relative humidity of the environment is greater than 90%, and the temperature is 28°C-30°C.

[0023] A method for asexual propagation of pear easy-rooting rootstock ND60, characterized in that it comprises the following steps:

[0024] Step 1, adding nano triacontanol to a NaClO aqueous solution and diluting the solution to a nano triacontanol concentration of 1 g / L to obtain a nano triacontanol dispersion;

[0025] Step 2, adding the nanotriacontanol dispersion obtained in step 1 to the enrichment medium until the final concentration of the nanotriacontanol dispersion relative to the enrichment medium is 1 mg / L;

[0026] Step 3, preparing explants from ND60 one-year-old shoots, placing them in the propagation medium obtained in step 2 for tissue propagation, and culturing them under red and blue light for 14 / 10 hours to obtain the ND60 tissue culture asexual propagation system.

[0027] On the basis of the above scheme,

[0028] The specific conditions for the red-blue light culture for 14 / 10h in step 3 are: red-blue light ratio 1:1, light intensity 2000lx, and temperature 25±2°C.

[0029] The breeding method and asexual propagation method of the pear easy-to-root rootstock ND60 of the present invention have the following beneficial effects:

[0030] Compared with the pear rootstock breeding method with a cycle of more than 20 years, the present invention can shorten the pear rootstock breeding cycle to 2-3 years, and the ND60 rooting coefficient of the cultivated pear easy-to-root rootstock can reach up to 2.98, and tissue culture and cutting propagation can realize factory production. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The present invention has the following accompanying drawings:

[0032] Figure 1 It is a schematic diagram of the ND60 breeding process and application of the present invention;

[0033] Figure 2 This is a schematic diagram of browning index determination of the present invention;

[0034] Figure 3 This is a schematic diagram of the determination of lenticel callus conversion rate of the present invention;

[0035] Figure 4 This is the rooting situation of ND60 cutting propagation in the present invention;

[0036] Figure 5 This is the rooting situation of ND60 tissue culture propagation in the present invention. DETAILED DESCRIPTION

[0037] The present invention is further described in detail below in conjunction with the accompanying drawings.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0039] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0040] Unless otherwise specified, the following component concentrations are the final concentrations of the above reagents.

[0041] Example 1. Browning index of one-year-old branch cross section measured value Y>0.80, selection rate 20.64%

[0042] A total of 5039 natural populations and hybrid offspring populations of major pear rootstocks such as Pyrus betulaeformis, Pyrus yamanensis, Pyrus douxi and Pyrus chuanxiensis, which were more than 8 years old and in full bloom, were selected. One-year-old semi-lignified branches 45 days after spring budding were disinfected by soaking in 75% alcohol and 2% sodium hypochlorite, and then cut off at 10 cm below the morphological apical meristem of the branches. 10 μm thick stem segments at the base of the branches were cut crosswise 4 hours after cutting, fixed in a transmission spectrum detection dish, and the difference in absorbance between the spectral wavelength of 850 nm and 0 h was detected. 4h .

[0043] According to the absorbance difference of 10 μm thick stem segment at 850 nm at the base of the branch 4 hours after truncation, △X(850) 4h Based on Y = 1.334-2.557 × △X (850) 4h The browning index calculation model was used, and 1049 materials were screened out based on Y>0.80, with a selection rate of 20.64%.

[0044] Example 2. One-year-old branch lenticel callus conversion rate> 50%, selection rate 9.34%

[0045] The 1049 natural and hybrid offspring population materials of pear rootstocks obtained by screening in Example 1 were used as test materials, large trees in full flowering period were taken, 20 cm long annual branches containing semi-lignified stem segments with full buds were taken 45 days after budding, 10 stem segments were taken from each strain, the branch surface was wiped with 75% ethanol, rinsed with running water for 2 minutes and then wiped dry, and cultured for 10 days at a temperature of 25°C, a humidity of 100%, and a light-proof environment. The percentage of branches whose lenticels were dedifferentiated and converted into callus tissue was investigated to be >50%, and 98 candidate strains were screened, with a selection rate of 9.34%.

[0046] Example 3. One-year-old branch rooting rate>50%, root number>2, selection rate 1.02%

[0047] The 98 natural and hybrid offspring population materials of pear rootstocks obtained by screening in Example 2 were used as test materials. 45 days after the budding of the big tree in full flowering period, the newly grown semi-lignified green branches in the current growing season were collected at 10 cm to 30 cm from the lower end of the stem tip morphology, and cut into a stem segment of about 10 cm in length, containing 2 nodes, retaining 1 leaf at the top, and treated the lower end of the stem segment with IAA for 10 minutes, and induced under the conditions of 100% humidity and 25 to 28 degrees Celsius. After 45 days, the rooting rate>50% and the number of roots>2 were used as screening indicators, and 1 material was screened, named ND60, with a selection rate of 1.02%.

[0048] Example 4. Factory breeding method and application of ND60 clones

[0049] Take ND60 one-year-old branches at 5-10 nodes, 10 cm in length, with an oblique bottom and a flat top with 1-2 leaves left, filter and sterilize triacontanol with a suction filter tube, take 1 mL of 10% NaClO and add it to 4 mL of sterile water to dilute it to 2% NaClO, then take 1 mL of nanotriacontanol and add it to 4 mL of NaClO (2%) to dilute the nanotriacontanol to 1 g / L, and obtain a nanotriacontanol dispersion.

[0050] Cutting propagation: Take a 12cm long stem segment from the 5-10th node of the ND60 one-year-old branch morphologically downward, cut the base of the stem segment into a flat bevel and the top into a flat end, leaving a leaf curtain area of ​​3-4cm at the top of the stem segment 2 The leaves were immediately inserted into a culture matrix with a ratio of peat: vermiculite: sphagnum moss: pine needle soil of 4:1:1:1, the above nano-triacontanol dispersion was diluted with clean water to 1 mg / L, tidal irrigation was performed twice a day, the irrigation height was flush with the upper surface of the matrix, the relative humidity of the environment was >90%, the temperature was 28°C-30°C, and after 45 days of cultivation, the ND60 cutting asexual propagation system with a proliferation coefficient of 2.98 was obtained;

[0051] Tissue culture propagation: The above-mentioned nano-triacontanol dispersion was mixed into the propagation medium (formula: 1 / 2MS + 26g / L sucrose + 6.7g / L agar powder + 1mg / L 6-BA) to a final concentration of 1mg / L, cultured under red and blue light for 14 / 10h, red and blue light ratio of 1:1, light intensity of 2000lx, temperature of 25±2°C, and then cultivated for 45d to obtain the ND60 tissue culture asexual propagation system with a proliferation coefficient of 2.52.

[0052] The contents not described in detail in this specification belong to the prior art known to professional and technical personnel in this field.

Claims

1. A method for breeding pear easy-rooting rootstock ND60, characterized in that: The steps include: Step 1, cutting off the semi-lignified branches of a pear tree in full flowering period after spring budding, immediately extracting the cross-cut stem segments at the base of the semi-lignified branches, extracting the cross-cut stem segments at the base of the semi-lignified branches again after 4 hours, and detecting the absorbance difference △X(850) of the cross-cut stem segments extracted twice at a spectral wavelength of 850nm 4h , screen for semi-lignified branches with browning index Y>0.80; The browning index calculation model is shown as follows: Y=1.334-2.557×△X(850) 4h ; Step 2, culturing the semi-lignified branches newly grown from the flowering trees of the natural or hybrid offspring group of mature plants to which the semi-lignified branches obtained in step 1 sprout in the current season in the dark for 10 days, and selecting the semi-lignified branches with a conversion rate of lenticel dedifferentiation into callus tissue greater than 50% among the semi-lignified branches; Step 3, take the mature plants to which the semi-lignified branches in step 2 belong, and the newly sprouted semi-lignified branches after germination in the current season, and after induction cultivation at room temperature, dark and 100% humidity for 45 days, select the semi-lignified branches with a rooting rate >50% and a number of roots >2, which is the ND60.

2. The method for breeding a pear easy-to-root stock ND60 as claimed in claim 1, characterized in that: The varieties of the large pear trees in full bloom in step 1 include Pyrus betula, Pyrus yamanensis, Pyrus chinensis or Pyrus chuanxiensis.

3. The method for breeding a pear easy-to-root stock ND60 as claimed in claim 1, characterized in that: The specific conditions for the dark-proof culture in step 2 are a temperature of 25° C. and a humidity of 100%.

4. The method for breeding a pear easy-to-root stock ND60 as claimed in claim 1, characterized in that: The specific conditions for the induction cultivation described in step 3 are 100% humidity and 25-28°C temperature.

5. A method for asexual propagation of pear easy-rooting rootstock ND60, using the pear easy-rooting rootstock ND60 obtained by the breeding method of pear easy-rooting rootstock ND60 according to any one of claims 1 to 4, characterized in that: The steps include: Step 1, adding nano triacontanol to a NaClO aqueous solution and diluting the solution to a nano triacontanol concentration of 1 g / L to obtain a nano triacontanol dispersion; Step 2, inserting the ND60 one-year-old branches into the ND60 cutting matrix, diluting the nano-triacontanol dispersion obtained in step 1 to 1 mg / L, obtaining a nano-triacontanol dilution, and using the above-mentioned nano-triacontanol dilution to irrigate the above-mentioned ND60 one-year-old branches to obtain the ND60 cutting asexual propagation system.

6. The method according to claim 5, characterized in that: The specific conditions of the irrigation conditions described in step 2 are: tidal irrigation twice a day, the irrigation height is flush with the upper surface of the substrate, the relative humidity of the environment is greater than 90%, and the temperature is 28°C-30°C.

7. A method for asexual propagation of pear easy-rooting rootstock ND60, using the pear easy-rooting rootstock ND60 obtained by the breeding method of pear easy-rooting rootstock ND60 according to any one of claims 1 to 4, characterized in that: The steps include: Step 1, adding nano-triacontanol to a NaClO aqueous solution and diluting the nano-triacontanol to a concentration of 1 g / L to obtain a nano-triacontanol dispersion; Step 2, adding the nanotriacontanol dispersion obtained in step 1 to the enrichment medium until the final concentration of the nanotriacontanol dispersion relative to the enrichment medium is 1 mg / L; Step 3, preparing explants from ND60 one-year-old shoots, placing them in the propagation medium obtained in step 2 for tissue propagation, and culturing them under red and blue light for 14 / 10 h to obtain the ND60 tissue culture asexual propagation system.

8. The method according to claim 7, characterized in that: The specific conditions for the red-blue light illumination culture for 14 / 10 h in step 3 are: red-blue light ratio 1:1, light intensity 2000 lx, and temperature 25±2°C.

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