Tissue culture method of pear rootstock

Abstract

The invention belongs to the technical field of plant biology, and particularly relates to a tissue culture method of pear rootstock. According to the invention, stage-specific dynamic light quality regulation and control and a combined strategy based on novel plant growth regulating substances are integrated. The method achieves significant breakthrough in key indexes such as proliferation efficiency, rooting quality, tissue culture seedling robustness and transplanting survival rate.

Description

Technical Field

[0001] The present invention belongs to the field of plant biotechnology, and particularly relates to a tissue culture method for pear rootstocks. Background Art

[0002] Pears (Pyrus spp.) are important economic fruit trees in China, and their cultivated varieties generally rely on grafting onto rootstocks to improve stress resistance and adaptability. Pyrus betulifolia and Pyrus calleryana, as the mainstream semi-dwarfing rootstocks for pears in China, have advantages such as cold resistance, salt-alkali tolerance, and strong grafting affinity. Traditional rootstock propagation relies on seed sowing, which has defects such as unstable genetic traits, long propagation cycles (3 - 4 years to become seedlings), and high risks of pest and disease transmission.

[0003] Tissue culture technology can achieve virus-free, industrialized, and rapid propagation of rootstocks, which is crucial for promoting the standardized development of the pear industry. However, long-term tissue culture of pear rootstocks has faced technical bottlenecks such as low proliferation efficiency, difficult rooting, and unstable transplanting survival rates, severely restricting industrial application. Summary of the Invention

[0004] To solve the above problems, the present invention provides a tissue culture method for pear rootstocks, comprising the following steps:

[0005] (1) Explant selection and disinfection: Select the current-year semi-lignified branches on a healthy mother plant of the pear rootstock and disinfect them.

[0006] (2) Primary culture: Cut the disinfected stem segments into small segments with 1 - 2 axillary buds and inoculate them onto the initiation medium; culture under the condition of alternating red light and far-red light, with a photoperiod of 12 - 14 h / d and a temperature of 23 / 18 °C for 4 weeks.

[0007] (3) Proliferation culture: Cut and transfer the aseptic buds induced in the primary culture to the proliferation medium; culture under the condition of a high proportion of blue light and red light combination, with a photoperiod of 14 - 16 h / d and a temperature of 23 / 18 °C, and subculture every 4 - 5 weeks to obtain cluster buds.

[0008] [[ID=2D]](4) Rooting culture: Select healthy aseptic seedlings, immerse the base in a solution containing IBA / NAA + SNP for 30 - 60 minutes; take them out and inoculate them onto the rooting medium; culture under the condition of mainly red light, with a photoperiod of 10 - 12 h / d, a temperature of 23 / 18 °C, and a relative humidity of 60 - 70% for 3 - 5 weeks.

[0009] (5) Acclimatization and transplantation: After the roots are well-developed, acclimatize the seedlings in vitro for 3 - 5 days; the acclimatization process is to carefully take out the tissue culture seedlings, wash the root medium, and transplant them into the sterilized substrate; maintain high humidity and weak light at the initial stage, gradually reduce the humidity and increase the light intensity, and transition to normal greenhouse management.

[0010] Furthermore, it also includes strong seedling culture, and the steps are as follows:

[0011] Cut the longer single bud in the proliferated cluster buds and transfer it to the strong seedling culture medium. The photoperiod is 12 h / d, and culture for 1 - 2 generations to make the seedlings strong.

[0012] Furthermore, the components of the strong seedling culture medium include QL basal medium and 6 - BA.

[0013] Furthermore, the components of the initiation medium in step (2) include basic QL medium, 6 - BA, and antioxidant.

[0014] Furthermore, the antioxidant in step (2) is citric acid.

[0015] Furthermore, the components of the proliferation medium in step (3) include QL medium, 6 - BA, GR24, and spermidine.

[0016] Furthermore, the components of the rooting medium in step (4) include 1 / 2 QL medium, melatonin, and AC.

[0017] The present invention has the following beneficial effects:

[0018] The present invention uses a low R:FR ratio to simulate a shaded environment, promotes the elongation of explants (shoot tips or stem segments with axillary buds), effectively reduces the browning rate in the initiation stage, and preliminarily breaks the axillary bud dormancy. GR24 can strongly promote the occurrence of lateral branches (buds), and in synergistic action with 6 - BA, greatly increases the number of effective proliferated buds and reduces malformed buds. Detailed Embodiments

[0019] Now, various exemplary embodiments of the present invention will be described in detail. In the examples, the methods are all conventional methods unless otherwise specified, and the reagents are all conventional commercially available reagents or reagents prepared by conventional methods unless otherwise specified. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0020] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0021] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention has been described only in terms of preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with those documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0022] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the description of the present invention, which will be apparent to those skilled in the art. Other embodiments obtained from the description of the present invention will be apparent to those skilled in the art. The description and examples of the present invention are merely exemplary.

[0023] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0024] Example 1: Tissue culture method using Pyrus betulifolia as rootstock

[0025] 1. Explant disinfection and primary culture

[0026] Material preparation: Select healthy, pest- and disease-free current-year semi-lignified branches from the Pyrus betulifolia mother orchard, and cut stem segments with axillary buds (about 1.5 - 2 cm in length).

[0027] Disinfection steps: Rinse with running water for 30 min; soak in 75% ethanol for 30 s; disinfect with 0.1% HgCl2 (containing 1 drop of Tween-20) by shaking for 8 min; rinse 5 times with sterile water, 2 min each time.

[0028] Medium formula (primary initiation):

[0029] Basic medium: QL (Quoirin & Lepoivre)

[0030] Additive: 6-BA 0.3 mg / L

[0031] Anti-browning agent: 0.1 g / L activated carbon (AC) + 100 mg / L citric acid

[0032] Sucrose 30 g / L

[0033] Agar 6.5 g / L

[0034] pH 5.8.

[0035] Culture conditions:

[0036] Light: Red light (630 nm): far-red light (730 nm) = 4:5 (photosynthetic photon flux density is 40 μmol·m -2 ·s -1 ), photoperiod 12 h / d;

[0037] Temperature: 23 ± 1 °C during the day / 18 ± 1 °C at night;

[0038] Humidity: 70–80%;

[0039] Result: After 4 weeks of culture, the axillary bud germination rate reached 85%, and the browning rate was < 10%.

[0040] 2. Proliferation culture

[0041] Medium formula (proliferation):

[0042] Basic medium: QL

[0043] Additives: 6-BA 0.8 mg / L; GR24 (strigolactone analogue) 0.3 μM; spermidine 10 mg / L; sucrose 30 g / L; agar 6.5 g / L; pH 5.8.

[0044] Culture conditions:

[0045] Light: Blue light (450 nm): red light (660 nm) = 2.5:1 (total light intensity 60 μmol·m -2 ·s -1 ), photoperiod 16 h / d;

[0046] Temperature: 23 ± 1 °C during the day / 18 ± 1 °C at night;

[0047] Humidity: 75%.

[0048] Operation: Cut the primary germinated buds into single-bud stem segments (with 1–2 leaf primordia), transfer them to the proliferation medium, and inoculate 4–5 in each bottle.

[0049] Result: Subculture once every 4 weeks, the proliferation coefficient reached 6.8 ± 0.3 (the traditional method is 3.5–4.2), and the bud seedlings were robust with short internodes.

[0050] 3. Seedling strengthening culture (key pretreatment)

[0051] Medium formula (seedling strengthening):

[0052] Basic medium: QL

[0053] Additives: 6-BA 0.2 mg / L; sucrose 20 g / L; agar 6.5 g / L;

[0054] pH 5.8

[0055] Cultivation conditions:

[0056] Light: white light (400–700 nm) + 15% UV-B (310 nm, dose 0.1 W / m 2 ), photoperiod 12 h / d;

[0057] Temperature: the same as the proliferation stage;

[0058] Humidity: 70%;

[0059] Operation: Transfer single seedlings with a height > 2 cm in the proliferated clustered buds and culture for 2 weeks.

[0060] Result: The stem thickness of the seedlings increased by 25%, the chlorophyll content increased by 18%, and lignin accumulation was significant.

[0061] 4. Root induction

[0062] Pre-rooting treatment:

[0063] Take aseptic seedlings (3–4 cm in height) after selecting strong seedlings, immerse the base in a solution containing the following components for 45 min:

[0064] IBA 1.0 mg / L + SNP (sodium nitroprusside, NO donor) 10 μM

[0065] Solvent: sterile deionized water

[0066] Rooting medium formula:

[0067] Basic medium: 1 / 2 MS (halved salts)

[0068] Additives:

[0069] Melatonin 1.0 μM

[0070] Activated carbon (AC) 0.8 g / L (adsorb phenolic substances)

[0071] Sucrose 20 g / L

[0072] Agar 6.0 g / L

[0073] pH 5.8

[0074] Cultivation conditions:

[0075] Light: red light (660 nm) accounts for 80% (light intensity 50 μmol·m -2 ·s -1 ), photoperiod 10 h / d

[0076] Temperature: 23 ± 1 °C during the day / 18 ± 1 °C at night

[0077] Humidity: 65%

[0078] Result: After 4 weeks of culture, the rooting rate was 96.2%, the average number of roots was 4.5 ± 0.6, the root length was > 3 cm, and the root system was thick and without vitrification.

[0079] 5. Acclimatization and transplantation

[0080] Acclimatization:

[0081] Uncap the rooted seedlings and acclimatize them under natural indoor light for 4 days (humidity gradually decreases from 90% to 70%).

[0082] Transplantation substrate:

[0083] Sterilized peat soil: vermiculite: perlite = 1:1:1 (volume ratio), pH 6.0–6.5.

[0084] Transplantation management:

[0085] Wash the agar from the roots and implant them into a plug tray (50 holes);

[0086] Cover with a transparent film to keep moisture (humidity > 85%), temperature 22–25°C;

[0087] Gradually uncover the film for ventilation after 7 days, and apply 1 / 4 MS nutrient solution after 14 days.

[0088] Result: The transplantation survival rate was 92.5% (the control traditional method was 75–80%).

[0089] Example 2: Verification with Pyrus calleryana as the rootstock (adjustment of key parameters)

[0090] Note: For different rootstock varieties, fine-tune the hormone concentration and photoperiod to reflect the universality of the scheme.

[0091] Proliferation stage:

[0092] The concentration of GR24 was reduced to 0.2 μM (Pyrus calleryana is more sensitive to strigolactone), and 6-BA was maintained at 0.8 mg / L.

[0093] Rooting stage:

[0094] The pre-soaking solution was changed to 0.8 mg / L NAA + 15 μM SNP (Pyrus calleryana responds better to NAA).

[0095] Result:

[0096] The proliferation coefficient was 6.2, the rooting rate was 94.7%, and the transplantation survival rate was 90.3%.

[0097] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A tissue culture method for pear rootstocks, characterized in that, It includes the following steps: (1) Explant selection and disinfection: Select the current-year semi-lignified branches on the robust mother plants of pear rootstocks and disinfect them; (2) Primary culture: Cut the disinfected stem segments into small segments with 1-2 axillary buds and inoculate them onto the initiation medium; Cultivate for 4 weeks under the condition of alternating red light and far-red light, with a photoperiod of 12-14 h / d and a temperature of 23 / 18 °C; (3) Proliferation culture: Cut and transfer the sterile buds induced in the primary stage to the proliferation medium; Cultivate under the condition of a combination of high-proportion blue light and red light, with a photoperiod of 14-16 h / d and a temperature of 23 / 18 °C, and subculture every 4-5 weeks to obtain cluster buds; (4) Rooting culture: Select robust sterile seedlings, immerse the bases in a solution containing IBA / NAA + SNP for 30-60 minutes; After taking them out, inoculate them onto the rooting medium; Cultivate for 3-5 weeks under the condition of mainly red light, with a photoperiod of 10-12 h / d, a temperature of 23 / 18 °C, and a relative humidity of 60-70%; (5) Acclimatization and transplantation: After the roots are well-developed, open the bottle for acclimatization for 3-5 days; The acclimatization process is to carefully take out the tissue culture seedlings, wash the root medium, and transplant them into the sterilized substrate; Keep high humidity and weak light at the initial stage, gradually reduce the humidity and increase the light intensity, and transition to normal greenhouse management.

2. The method according to claim 1, wherein It also includes strong seedling culture, and the steps are as follows: Cut the longer single buds from the proliferated cluster buds and transfer them to the strong seedling medium, with a photoperiod of 12 h / d, and culture for 1-2 generations to make the seedlings strong.

3. The method according to claim 2, wherein The components of the strong seedling medium include QL basal medium and 6-BA.

4. The method according to claim 1, characterized in that, The components of the initiation medium described in step (2) include basal QL medium, 6-BA, and antioxidant.

5. The method according to claim 4, wherein The antioxidant described in step (2) is citric acid.

6. The method according to claim 1, wherein The components of the proliferation medium described in step (3) include QL medium, 6-BA, GR24, and spermidine.

7. The method according to claim 1, wherein The components of the rooting medium described in step (4) include 1 / 2 QL medium, melatonin, and AC.