Method for inducing rooting of avocado cutting slips by using agrobacterium rhizogenes

Abstract

The invention relates to the field of asexual propagation of avocados, in particular to a method for inducing rooting of avocado cutting slips by utilizing agrobacterium rhizogenes. The method comprises the following steps: under open experimental conditions, taking current-year-old semi-lignified shoots of grafting plant scions of persea americana L. of 2-3 years old as experimental materials, taking high-temperature sterilized coco coir as a cutting medium, carrying out vacuum infiltration infection on the shoots in an agrobacterium rhizogenes K599 infection solution for 20 minutes, infecting wounds of seedlings, inducing rooting, and carrying out rooting. A system for inducing avocado cutting rooting by using agrobacterium rhizogenes is established. According to the method, the technical problem that the avocado cutting slips are difficult to root is solved, and the technical effect of rapidly breeding the avocado clonal rootstock seedlings is achieved. According to the method, clonal breeding, popularization and application of the excellent stock variety can be realized, a more convenient and efficient way is provided for breeding, popularization and application of the excellent persea americana stock variety, and the method has important significance for breeding, popularization and application of the excellent persea americana stock.

Description

Technical Field

[0001] The present invention relates to the field of avocado asexual reproduction, and particularly to a method for inducing root formation in avocado cuttings by using Agrobacterium rhizogenes. Background Art

[0002] Avocado (Persea americana L.) is an evergreen arbor fruit tree with high nutrition and high economic value. Also known as alligator pear or butter fruit, avocados are deeply loved by consumers due to their high health care value and good economic benefits. Currently, avocados are still being extensively planted, and a large number of avocado seedlings are needed. Avocados have high nutrient requirements and are vulnerable to root diseases such as root rot and brown root disease, so there is an urgent need for rootstocks with strong resistance and high nutrient absorption and utilization rates. However, the current avocado seedling cultivation method uses seedlings as rootstocks to graft clonal scions. Due to the immaturity of asexual reproduction techniques such as tissue culture and cutting, the clonalization of rootstocks cannot be achieved, which restricts the popularization and application of excellent rootstock varieties. The existing rooting powder-induced avocado cutting rooting technology has problems such as cumbersome operation, low rooting rate, poor root growth, and long rooting time. Although rooting powder combined with Agrobacterium rhizogenes has been used to induce root formation in the cuttings of existing woody plants, the method of using rooting powder to induce root formation in avocado cuttings has the following problems:

[0003] 1. Strict environmental requirements: The formation of adventitious roots is a direct determinant of the success of cutting, and the formation of adventitious roots is closely related to cutting substrates, exogenous hormones, and other environmental factors such as temperature and light. The existing cutting techniques mainly rely on hormones to promote root formation in cuttings and have strict requirements for the growth environment. 2. Long cycle, low rooting rate and cutting survival rate: Since avocados have relatively high contents of phenolic compounds and oxidase in their roots and are difficult-to-root tree species for cutting, the rooting cycle is long, increasing the risk of infection and necrosis, and the rooting rate is low and the root growth is poor.

[0004] Currently, there has been no report on the method of using Agrobacterium rhizogenes to induce root formation in avocado cuttings. Based on this, the present invention has preliminarily established a method for using Agrobacterium rhizogenes to induce root formation in avocado cuttings, which can achieve the clonal propagation, popularization and application of excellent rootstock varieties. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for using Agrobacterium rhizogenes to induce root formation in avocado cuttings to solve the problems existing in the above-mentioned prior art. In view of the bottlenecks and defects existing in the existing avocado cutting techniques, the present invention proposes a technical solution for using Agrobacterium rhizogenes to induce root formation in avocado cuttings, providing a more convenient and efficient way for the breeding, popularization and application of excellent avocado rootstocks.

[0006] To achieve the above purpose, the present invention provides the following solutions:

[0007] The present invention provides a method for inducing the rooting of avocado cuttings by using Agrobacterium rhizogenes, comprising the following steps:

[0008] Using the current-year semi-lignified new shoots of the scions of avocado grafted plants as cuttings, and subjecting the cuttings to vacuum infection with an Agrobacterium rhizogenes K599 infection solution to obtain treated cuttings; transferring the cuttings to coconut coir for cultivation;

[0009] The scions of the avocado grafted plants are scions of 2- to 3-year-old avocado grafted plants.

[0010] Preferably, the time of the vacuum infection is 15 min.

[0011] Preferably, the method for preparing the Agrobacterium rhizogenes K599 infection solution comprises the following steps:

[0012] Inoculating the activated Agrobacterium rhizogenes K599 into an LB liquid medium containing streptomycin for cultivation, followed by centrifugation and resuspension to obtain an Agrobacterium rhizogenes K599 bacterial solution;

[0013] Mixing the Agrobacterium rhizogenes K599 bacterial solution, 2-morpholinoethanesulfonic acid, and acetosyringone to obtain the Agrobacterium rhizogenes K599 infection solution.

[0014] Preferably, the final concentration of 2-morpholinoethanesulfonic acid in the Agrobacterium rhizogenes K599 infection solution is 10 mmol / L, and the final concentration of acetosyringone is 200 μmol / L.

[0015] Preferably, the final concentration of streptomycin in the LB liquid medium containing streptomycin is 50 mg / L.

[0016] The present invention provides the application of the above method in the asexual reproduction of avocados.

[0017] The present invention provides the application of the above method in shortening the rooting time of avocado cuttings.

[0018] The present invention provides the application of the above method in increasing the rooting rate of avocado cuttings.

[0019] The present invention provides the application of the above method in increasing the number of roots of avocado cuttings.

[0020] The present invention provides the application of the above method in increasing the root biomass of avocado cuttings.

[0021] The present invention discloses the following technical effects:

[0022] The present invention has improved two defects existing in the existing cutting technology of avocado (1. strict environmental requirements; 2. long cycle, low rooting rate and low cutting survival rate), and successfully realized a technical solution for inducing the rooting of avocado cuttings by Agrobacterium rhizogenes. Under open experimental conditions, the present invention uses the current-year semi-lignified new shoots of 2- to 3-year-old avocado grafted plants as experimental materials, and uses high-temperature sterilized coconut coir as the cutting substrate. After the new shoots are vacuum infiltrated in the Agrobacterium rhizogenes K599 infection solution for 20 minutes, the wounds of the infected seedlings are induced to root, and a system for inducing the rooting of avocado cuttings by Agrobacterium rhizogenes is established. The present invention solves the technical problem of difficult rooting of avocado cuttings and achieves the technical effect of quickly breeding avocado clonal rootstock seedlings. The present invention can realize the clonal breeding and popularization and application of excellent rootstock varieties, provides a more convenient and efficient way for the breeding, popularization and application of excellent avocado rootstock varieties, and has important significance for the breeding, popularization and application of excellent avocado rootstocks. The present invention mainly includes the following key points and technical effects:

[0023] (1) For different tree species, the present invention screened the Agrobacterium rhizogenes with the best rooting effect for avocado cuttings. Specifically, the current-year semi-lignified new shoots of 2- to 3-year-old avocado grafted plants were used as experimental materials. Comparing the rooting of cuttings induced by rooting powder, four Agrobacterium rhizogenes, namely MSU440, K599, Ar.Qual and C58C1, were used to infect the cuttings. The results showed that all four Agrobacterium rhizogenes shortened the rooting time of the cuttings, increased the rooting rate and root biomass of the cuttings, and finally screened out the Agrobacterium rhizogenes K599 with the best rooting effect for avocado cuttings; (2) The method provided by the present invention does not need to use rooting powder to soak, and only uses Agrobacterium rhizogenes K599 to infect the cuttings to achieve the rooting of avocado cuttings, and the rooting effect is equivalent to that of the combination of rooting powder and Agrobacterium rhizogenes; (3) The present invention screened the current-year lignified new shoots of different tree ages as cuttings, and found that the branches of 2- to 3-year-old avocado grafted plants were the best as cuttings; (4) Vacuum infiltration was adopted, which improved the infection rate of Agrobacterium rhizogenes, shortened the rooting time, increased the rooting rate and root growth; (5) The present invention changed to use high-temperature sterilized coconut coir as the cutting substrate, which is simpler, reduced the infection rate of cuttings and saved costs; (6) The present invention optimized the vacuum treatment time, and selected 20 minutes with the shortest average rooting time, the highest rooting rate, the most average number of roots and the highest root biomass to ensure the rooting of avocado. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0025] Figure 1 The specific process for inducing avocado cutting plants; among them, the parts circled in red are the parts that can be omitted;

[0026] Figure 2 The root systems of avocado cutting plants under different treatments; among them, A is the root system of the current-year semi-lignified new shoots of 2- to 3-year-old avocado grafted plants in Example 1 as avocado cutting plants; B is the root system of the current-year semi-lignified new shoots of 2- to 3-year-old avocado grafted plants in Example 2 as avocado cutting plants; C is the root system of the current-year semi-lignified new shoots of avocado grafted plants over 5 years old in Example 3 as avocado cutting plants. Detailed implementation manners

[0027] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be construed as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation manners of the present invention.

[0028] It should be understood that the terms used in the present invention are only for describing particular implementation manners and are not intended 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. Any 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 may be independently included or excluded from the range.

[0029] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0030] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the specification of the present invention, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0031] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0032] Example 1: Comparing the effects of different Agrobacterium rhizogenes on the rooting of avocado cuttings

[0033] The specific process of inducing avocado cuttings is as follows Figure 1 shown, and the specific steps are as follows:

[0034] (1) Preparation of avocado cuttings

[0035] Select the current-year semi-lignified new shoots of the scions of 2- to 3-year-old avocado grafted plants as cuttings, and use a sterile scalpel to cut off about 20 cm of new shoots, which are the avocado cuttings.

[0036] (2) Preparation of cutting substrate

[0037] Soak the coconut coir in water, place it in a fresh-keeping bag, and sterilize it with a high-temperature sterilizer, that is, sterilize it at 121°C for 15 min. Use the sterilized coconut coir as the cutting substrate, pack it into an 8 cm × 10 cm non-woven fabric bag, and place it in a seedling tray.

[0038] (3) Preparation of Agrobacterium rhizogenes infection solution

[0039] Activation of Agrobacterium rhizogenes: Use four strains, MSU440, K599, Ar.Qual, and C58C1. Streak the MSU440, K599, Ar.Qual, and C58C1 strains on an LB solid medium containing 50 mg / L Str (streptomycin), let the bacterial solution dry, seal the petri dish, and incubate it upside down in an incubator at 28°C for 48 h.

[0040] Preparation of Agrobacterium rhizogenes infection solution: Pick single colonies of the activated Agrobacterium rhizogenes MSU440, K599, Ar.Qual, and C58C1 strains and transfer them to a liquid LB medium (50 mg / L Str), and culture them at 28°C and 220 rmp for 48 h; centrifuge the cultured bacterial solution at 6000 rpm for 5 min to remove the supernatant, resuspend it with a sterile 10 mmol / L MgCl2 solution, and adjust the OD 600 value to 1.0; add 10 mmol / L 2-(N-morpholino)ethanesulfonic acid (MES) and 200 μmol / L acetosyringone (AS) to the bacterial solution, that is, the final concentration of 2-(N-morpholino)ethanesulfonic acid in the bacterial solution is 10 mmol / L, and the final concentration of acetosyringone is 200 μmol / L. Let it stand in the dark at room temperature for 3 h, which is the infection solution.

[0041] (4) Infection with Agrobacterium rhizogenes infection solution

[0042] The avocado scions were immersed in the infection solution of Agrobacterium rhizogenes, and then transferred to a vacuum pump for vacuuming for 20 min. At the same time, the avocado scions immersed in rooting powder (500 mg / kg ABT1, preparation method: first, 1 g of ABT1 was dissolved by stirring with 100 mL of 75% alcohol, and then diluted to 500 mg / kg with sterile water.) for 15 min were used as the control.

[0043] The treated cutting materials were inserted into the high-temperature sterilized coconut coir, sprayed with water several times a day and wrapped with plastic film to keep the substrate moist. After 2 days of dark treatment, they were transferred to light culture for 30 - 45 days to induce rooting and obtain cutting plants.

[0044] After 45 days of culture, the average rooting time, average number of roots, rooting rate, root biomass and cutting survival rate of different treatments were investigated. 80 cuttings were investigated for each treatment. The results are shown in Table 1, and the rooting situation is as shown in Figure 2 A in.

[0045] Table 1 Effects of different treatments on the root growth of avocado cutting plants

[0046]

[0047]

[0048] As can be seen from Table 1 and Figure 2 A in, the average rooting time of the treatment with Agrobacterium rhizogenes K599 was the shortest, followed by the treatments with MSU440, C58C1, Ar.Qual and rooting powder in turn; taking the rooting rate, average number of roots and root biomass as indicators, the treatment with Agrobacterium rhizogenes K599 was the best, followed by the treatments with MSU440, C58C1, Ar.Qual and rooting powder in turn.

[0049] Example 2 Comparison of the effects of single use of Agrobacterium rhizogenes and the combination of Agrobacterium rhizogenes and rooting powder on the rooting effect of avocado cuttings

[0050] (1) Preparation of avocado cuttings

[0051] The current-year semi-lignified new shoots of the scions of 2 - 3-year-old avocado grafted plants were selected as cuttings, and about 20 cm of new shoots were cut off with a sterile blade to obtain avocado cuttings.

[0052] (2) Preparation of cutting substrate and rooting solution

[0053] Preparation of cutting substrate: After soaking coconut coir with water, it was placed in a fresh-keeping bag and sterilized with a high-temperature sterilizer, that is, sterilized at 121 °C for 15 min. The sterilized coconut coir was used as the cutting substrate, filled into an 8 cm × 10 cm non-woven fabric bag and placed in a seedling tray.

[0054] Preparation of rooting solution: First, dissolve 1 g of ABT1 in 100 mL of 75% alcohol with stirring, and then add sterile water to dilute it to 500 mg / kg.

[0055] (3) Preparation of Agrobacterium rhizogenes infection solution

[0056] Activation of Agrobacterium rhizogenes: Select the K599 strain with the best effect on inducing rooting of avocado cuttings, streak it on a solid medium containing 50 mg / L Str (streptomycin), air-dry the bacterial solution, seal the petri dish, and incubate it upside down in a constant temperature incubator at 28 °C for 48 h.

[0057] Preparation of Agrobacterium rhizogenes infection solution: Pick single colonies of the activated Agrobacterium rhizogenes K599 strain and transfer them to a liquid LB medium (50 mg / L Str), and culture them at 28 °C and 220 rmp for 48 h; centrifuge the cultured bacterial solution at 6000 rpm for 5 min to remove the supernatant, resuspend it with a sterile 10 mmol / L MgCl2 solution, and adjust the OD 600 value to 1.0; add 10 mmol / L 2-(N-morpholino)ethanesulfonic acid (MES) and 200 μmol / L acetosyringone (AS) to the bacterial solution, and let it stand in the dark at room temperature for 3 h to obtain the infection solution.

[0058] (4) Infection with rooting solution and Agrobacterium rhizogenes infection solution

[0059] Treatment with rooting powder combined with Agrobacterium rhizogenes: Immerse the avocado scions in 500 mg / kg ABT1 for 15 min, then immerse the materials soaked with rooting powder in the infection solution of Agrobacterium rhizogenes K599 and transfer them to a vacuum pump for vacuuming for 20 min.

[0060] Treatment with single Agrobacterium rhizogenes: Immerse the avocado scions in the infection solution of Agrobacterium rhizogenes (the control group is immersed in 500 mg / kg ABT1), and then transfer them to a vacuum pump for vacuuming for 20 min.

[0061] Treatment with single rooting powder: Immerse the avocado scions in 500 mg / kg ABT1 for 15 min.

[0062] After that, insert the infected cutting materials into autoclaved coconut coir, spray water several times a day and wrap them with a plastic film to keep the substrate moist, culture them in the dark for 2 days and then transfer them to light for 30 - 45 days to induce rooting and obtain cutting plants.

[0063] After culturing for 45 d, investigate the average rooting time, average number of roots, rooting rate, root biomass and cutting survival rate of different treatments. Investigate 80 cuttings for each treatment. The results are shown in Table 2, and the rooting situation is as Figure 2 shown in B below.

[0064] Table 2 Effects of different treatments on root growth of avocado cutting plants

[0065]

[0066] As can be seen from B in Table 2 and Figure 2 the average rooting time is the shortest under the treatment of single Agrobacterium rhizogenes and the combination of rooting powder and Agrobacterium rhizogenes K599, followed by the treatment of single rooting powder; taking the rooting rate, average number of roots and root biomass as indicators, the treatment of single Agrobacterium rhizogenes and the combination of rooting powder and Agrobacterium rhizogenes K599 have the best effect, followed by the treatment of single rooting powder. At the same time, the results also show that the treatment of single Agrobacterium rhizogenes can achieve the same technical effect as the treatment of Agrobacterium rhizogenes combined with rooting powder. It can be seen that the method provided by the present invention does not need to soak with rooting powder, and only by infecting the cuttings with Agrobacterium rhizogenes can the rooting of avocado cuttings be realized, and the rooting effect is equivalent to that of the combination of rooting powder and Agrobacterium rhizogenes.

[0067] Example 3 Influence of avocado mother trees of different tree ages on the cutting effect of cuttings

[0068] Select the current-year semi-lignified new shoots of the scions of avocado grafted plants over 5 years old and conduct cuttings on them according to Example 1, and observe the survival rate of the current-year semi-lignified new shoots of the scions of avocado grafted plants over 5 years old. The results show that the scions of avocado grafted plants over 5 years old did not root, and the roots turned brown and died. Thus, the survival rate of the scions of avocado grafted plants over 5 years old is 0% ( Figure 2 C in

[0069] Example 4 Influence of different substrates on the cutting effect of cuttings

[0070] (1) Preparation of avocado cuttings

[0071] Select the current-year semi-lignified new shoots of the scions of 2-3-year-old avocado grafted plants as cuttings, and use a sterile scalpel to cut off about 20 cm of new shoots, which are the avocado cuttings.

[0072] (2) Preparation of cutting substrate

[0073] Soak the coconut coir with water, place it in a fresh-keeping bag and sterilize it with a high-temperature sterilizer, that is, sterilize it at 121 °C for 15 min. Use the sterilized coconut coir as the cutting substrate, pack it into an 8 cm × 10 cm non-woven bag, and place it in a seedling tray.

[0074] (3) Preparation of Agrobacterium rhizogenes infection solution

[0075] Activation of Agrobacterium rhizogenes: Four strains, namely MSU440, K599, Ar.Qual, and C58C1, were used. The MSU440, K599, Ar.Qual, and C58C1 strains were streaked on LB solid medium containing 50 mg / L Str (streptomycin), allowed to air dry the bacterial solution, sealed the petri dishes, and incubated upside down in a constant temperature incubator at 28 °C for 48 h.

[0076] Preparation of Agrobacterium rhizogenes infection solution: Single colonies of the activated Agrobacterium rhizogenes strains MSU440, K599, Ar.Qual, and C58C1 were picked and transferred to liquid LB medium (50 mg / L Str), and cultured at 28 °C and 220 rmp for 48 h; the cultured bacterial solution was centrifuged at 6000 rpm for 5 min to remove the supernatant, resuspended with sterile 10 mmol / L MgCl2 solution, and the OD 600 value was adjusted to 1.0; 10 mmol / L 2-(N-morpholino)ethanesulfonic acid (MES) and 200 μmol / L acetosyringone (AS) were added to the bacterial solution, and the mixture was allowed to stand in the dark at room temperature for 3 h to obtain the infection solution.

[0077] (4) Infection with Agrobacterium rhizogenes infection solution

[0078] The avocado scions were immersed in the Agrobacterium rhizogenes infection solution and then transferred to a vacuum pump for 20 min of vacuuming.

[0079] The treated cuttings were inserted into a sterilized substrate (coconut coir, vermiculite, peat soil, or sand), sprayed with water several times a day, and wrapped with a plastic film to keep the substrate moist. After 2 days of dark treatment, they were transferred to light culture for 30 - 45 days to induce root formation and obtain cuttings.

[0080] After 45 days of culture, the average rooting time, average number of roots, rooting rate, root biomass, and cutting survival rate of different treatments were investigated. 80 cuttings were investigated for each treatment, and the results are shown in Table 3.

[0081] Table 3 Effects of different treatments on the root growth of avocado cuttings

[0082] Treatment Average rooting time (d) Rooting rate (%) Average number of roots (pieces) Root biomass (g) Cutting survival rate (%) Coconut coir 15 95 15.9 2.34 93.75 Vermiculite 30 41.25 5.1 1.24 40 Peat soil 21 52.5 5.7 1.37 47.5 Sand 42 18.75 1.3 0.02 15

[0083] As can be seen from the data recorded in Table 3, the average rooting time was the shortest under coconut coir treatment, followed by peat soil, vermiculite, and sand; in terms of rooting rate, average number of roots, and root biomass as indicators, coconut coir had the best effect, followed by peat soil, vermiculite, and sand. Thus, it can be seen that the choice of substrate will affect the average rooting time, average number of roots, rooting rate, and root biomass of avocado, and coconut coir is the most suitable substrate for avocado.

[0084] Example 5 Effects of different infection times on the cutting effect of scions

[0085] (1) Preparation of avocado cuttings

[0086] Select the current-year semi-lignified new shoots of the scions of 2- to 3-year-old avocado grafted plants as cuttings. Use a sterile blade to cut off about 20 cm of new shoots, which are the avocado cuttings.

[0087] (2) Preparation of cutting substrate

[0088] Soak the coconut coir in water, place it in a fresh-keeping bag, and sterilize it with a high-temperature sterilizer, that is, sterilize it at 121 °C for 15 min. Use the sterilized coconut coir as the cutting substrate, pack it into an 8 cm × 10 cm non-woven fabric bag, and place it in a seedling tray.

[0089] (3) Preparation of Agrobacterium rhizogenes infection solution

[0090] Activation of Agrobacterium rhizogenes: Use four strains, MSU440, K599, Ar.Qual, and C58C1. Streak the MSU440, K599, Ar.Qual, and C58C1 strains on an LB solid medium containing 50 mg / L Str (streptomycin), let the bacterial liquid dry, seal the petri dish, and incubate it upside down in an incubator at 28 °C for 48 h.

[0091] Preparation of Agrobacterium rhizogenes infection solution: Pick single colonies of the activated Agrobacterium rhizogenes strains MSU440, K599, Ar.Qual, and C58C1 and transfer them to a liquid LB medium (50 mg / L Str), and culture them at 28 °C and 220 rmp for 48 h; centrifuge the cultured bacterial liquid at 6000 rpm for 5 min to remove the supernatant, resuspend it with a sterile 10 mmol / L MgCl2 solution, and adjust the OD 600 value to 1.0; add 10 mmol / L 2-(N-morpholino)ethanesulfonic acid (MES) and 200 μmol / L acetosyringone (AS) to the bacterial liquid, and let it stand in the dark at room temperature for 3 h, which is the infection solution.

[0092] (4) Infection with Agrobacterium rhizogenes infection solution

[0093] Soak the avocado scions in the Agrobacterium rhizogenes infection solution, and then transfer them to a vacuum pump to evacuate for 15 min, 20 min, or 25 min.

[0094] Insert the treated cutting materials into the high-temperature sterilized coconut coir, spray water several times a day, and wrap them with a plastic film to keep the substrate moist. After 2 days of dark treatment, transfer them to light culture for 30-45 days to induce rooting and obtain cutting plants.

[0095] After 45 days of culture, investigate the average rooting time, average number of roots, rooting rate, root biomass, and cutting survival rate of different treatments. Investigate 80 cuttings for each treatment, and the results are shown in Table 4.

[0096] Table 4 Effects of Different Treatments on the Root Growth of Avocado Cuttings

[0097]

[0098] As can be seen from Table 4, the average rooting time is the shortest under the treatments of vacuum treatment for 20 min and 25 min, followed by 15 min; taking the rooting rate, average number of roots and root biomass as indicators, the treatment effects of vacuum treatment for 20 min and 25 min are the best, followed by 15 min. Thus, it can be seen that the vacuum treatment time will also affect the average rooting time, average number of roots, rooting rate and root biomass of avocado. Too long vacuum treatment will not improve the cutting rooting effect of avocado anymore. Vacuum treatment for 20 min is the most suitable vacuum treatment method for avocado.

[0099] Based on the above, it can be known that the technical route for inducing the rooting of avocado cuttings provided by the present invention is feasible, simple and highly efficient. Therefore, the present invention finally forms an optimal system for the rooting of avocado cuttings: using the current-year semi-lignified new shoots of the scions of 2- to 3-year-old avocado grafted plants as materials, using high-temperature sterilized coconut coir as the cutting substrate, soaking the cuttings in the Agrobacterium rhizogenes infection solution and infecting them by vacuum infiltration for 20 min, and then inserting the infected cuttings into the cutting substrate to induce the rooting of avocado cuttings. This method has established an Agrobacterium rhizogenes-induced cutting rooting system for avocado, using the current-year semi-lignified new shoots of the scions of 2- to 3-year-old avocado grafted plants as materials, ensuring the consistency of the genetic background of the materials, shortening the rooting time, improving the rooting rate and root growth potential, and at the same time reducing the requirements for experimental conditions.

[0100] The above-described embodiments are only descriptions of the preferred modes 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 method for inducing root growth in avocado cuttings using Agrobacterium rhizogenes, characterized in that, Comprising the following steps: Using the current-year semi-lignified new shoots of the scions of avocado grafted plants as cuttings, and subjecting the cuttings to vacuum infection with an Agrobacterium rhizogenes K599 infection solution to obtain treated cuttings; transferring the cuttings to coconut coir for cultivation; The scions of the avocado grafted plants are scions of 2- to 3-year-old avocado grafted plants.

2. The method according to claim 1, wherein The time of the vacuum infection is 15 min.

3. The method according to claim 1, wherein The preparation method of the Agrobacterium rhizogenes K599 infection solution comprises the following steps: Inoculating the activated Agrobacterium rhizogenes K599 into an LB liquid medium containing streptomycin for cultivation, followed by centrifugation and resuspension to obtain an Agrobacterium rhizogenes K599 bacterial solution; Mixing the Agrobacterium rhizogenes K599 bacterial solution, 2-morpholinoethanesulfonic acid, and acetosyringone to obtain the Agrobacterium rhizogenes K599 infection solution.

4. The method according to claim 3, wherein In the Agrobacterium rhizogenes K599 infection solution, the final concentration of 2-morpholinoethanesulfonic acid is 10 mmol / L, and the final concentration of acetosyringone is 200 μmol / L.

5. The method according to claim 3, characterized in that, In the LB liquid medium containing streptomycin, the final concentration of streptomycin is 50 mg / L.

6. Use of the method according to any one of claims 1-5 in the asexual propagation of avocado.

7. Use of the method according to any one of claims 1-5 in shortening the rooting time of avocado cuttings.

8. Use of the method according to any one of claims 1-5 in increasing the rooting rate of avocado cuttings.

9. Use of the method according to any one of claims 1-5 in increasing the number of roots of avocado cuttings.

10. Use of the method according to any one of claims 1-6 in increasing the root biomass of avocado cuttings.

Images