A method for cultivating rooting of tissue culture stem segments of citrus rootstock

Abstract

The present invention discloses a method for cultivating rooting of citrus rootstock tissue culture stem segments, and belongs to the field of fruit tree propagation technology. The method comprises the following steps: (1) removing pectin and outer seed coat from citrus rootstock seeds, sterilizing and inoculating into MT basic culture medium, and cultivating to obtain sterile seedlings; (2) cutting the sterile seedlings into stem segments with nodes, inoculating into induction culture medium, and obtaining tissue culture young and tender sterile stem segments; (3) cutting off the tissue culture young and tender sterile stem segments, soaking the stem segments in ArMSU400 infection solution, and inserting the soaked stem segments into culture medium for rooting culture. The present invention improves the seedling breeding environment, and the entire operation is carried out in an indoor air-conditioned room, which greatly improves the working comfort, and each action can be standardized, and sterile rootstock seedling breeding can realize assembly line operation, laying the foundation for factory breeding.

Description

Technical Field

[0001] The invention relates to the technical field of fruit tree propagation, in particular to a rooting culture method for tissue cultured stem segments of citrus rootstocks. Background Art

[0002] Citrus is the world's largest fruit category and the largest cultivated fruit tree in southern my country, with an important economic status. Most citrus planting uses grafted seedlings, which consist of two parts: rootstock and scion. The length of rootstock breeding time and the consistency of its seedlings directly determine the cost and quality of grafted seedling breeding. Traditional citrus rootstock seedlings are mainly bred in the open air by seed sowing, which has a poor working environment, high labor input and high intensity. In addition, the seed supply is restricted by seasons and it is difficult to produce all year round. At the same time, because it is carried out in the open air, it is difficult to cultivate virus-free seedlings.

[0003] Based on the totipotency of cells, a large number of stem segments are proliferated through tissue culture, and then certain measures are taken to induce the formation of adventitious roots, so that seedlings can be propagated indoors and virus-free seedlings with good production consistency can be produced. The traditional method for stem segments obtained through tissue culture of citrus rootstocks is to add hormones to the culture medium to induce adventitious roots, and then harden the seedlings, clean them, and then plant them in the field. However, it is currently difficult for citrus tissue culture stem segments to take root. Not only are there few roots induced, but most of them are fleshy roots. Hardening is also required during transplantation, and the survival rate is extremely low and time-consuming.

[0004] After the plant stem segments are infected with Agrobacterium rhizogenes, a large number of new roots can be induced, which have the characteristics of fast growth rate and stable genetics. In the existing patents of Agrobacterium rhizogenes infection of citrus trifoliate orange and early golden sweet orange (ZL 201310242182.9 and ZL201310240707.5), the rooting effect of the stem segments of trifoliate orange and early golden sweet orange infected with Agrobacterium rhizogenes is good, but the hairy roots produced in the culture medium are a pathological state, which can be used for genetic transformation research; to become a real plant, adventitious buds need to grow on the hairy roots, and the regenerated buds elongate to produce adventitious roots in the rooting medium to produce early golden sweet orange plants, among which there are still problems such as low rooting ability in the rooting medium, the need for seedling hardening before planting, and survival rate. Summary of the invention

[0005] The purpose of the present invention is to provide a method for cultivating rooting of tissue cultured stem segments of citrus rootstocks to solve the problems of the above-mentioned prior art. The present invention provides a technology for inducing rooting of sterile stem segments obtained by tissue culture and forming aseptic seedlings of citrus rootstocks that can be directly used for grafting, laying the foundation for the factory-based breeding of citrus grafted seedlings.

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

[0007] One of the technical solutions of the present invention is a method for cultivating rooting of tissue cultured stem segments of citrus rootstock, comprising the following steps:

[0008] (1) Citrus rootstock seeds were removed of pectin and outer seed coat, sterilized, and inoculated into MT basic medium to obtain sterile seedlings;

[0009] (2) cutting the sterile seedlings into noded stem segments, inoculating them into an induction medium, and performing tissue culture to obtain semi-lignified sterile stem segments;

[0010] (3) cutting off the semi-lignified sterile stem segments, soaking the stem segments in ArMSU400 infection solution, and inserting the soaked stem segments into a culture medium for rooting culture.

[0011] The second technical solution of the present invention is a drug for promoting rooting of citrus seedlings, and the preparation method thereof comprises:

[0012] Mix the ArMSU400 bacteria with 1 / 2MSLIQ infection solution, resuspend the bacteria, and measure the OD 600 value, add 1 / 2MS to adjust OD 600 Value to 0.5~1;

[0013] The ArMSU400 bacterium is Agrobacterium rhizogenes ArMSU440, into which a recombinant strain with 35S:GFP is introduced;

[0014] The ingredients of the 1 / 2MSLIQ infection solution include: adding 2.2g of MS culture medium, 25g of sucrose, and 1mL of Vc per 1L, and adjusting the pH to 5.2.

[0015] The third technical solution of the present invention is the use of the drug in promoting the rooting of citrus plant cuttings.

[0016] Based on the above technical solution, the present invention has the following technical effects:

[0017] 1. The present invention solves the problem of difficulty in rooting tissue culture stem segments. After the plant is infected by Agrobacterium rhizogenes, the plant is induced to synthesize rooting-related hormones (such as auxin and cytokinin), which promotes the formation, growth and differentiation of the root system. However, after the plant stem segments are infected with Agrobacterium rhizogenes, they are traditionally cut into a culture medium, which will cause the plant to produce deformed roots (such as hairy roots or tumor roots). The present invention uses Agrobacterium rhizogenes to directly cut into a rooting matrix after infection, which not only promotes rooting, but also can improve the quality of the root system and the adaptability of the plant to the environment under certain circumstances.

[0018] 2. The present invention solves the problem of uneven quality of seedlings and easy virus carrying and transmission caused by seed sowing and cutting rooting. The citrus rootstock tissue culture stem segments are rooted, and all the stem segments are adventitious buds obtained by cutting the node stem segments obtained by sterile seedlings through culture medium and hormone induction. The sterile stem segments are separated and cut to form sterile stem segments. The quality of the obtained seedlings is controllable, and a large number of virus-free seedlings can be produced in batches in a short time.

[0019] 3. The present invention solves the problem that stock breeding is difficult to produce year-round. Through tissue culture technology, temperature, light, and nutrition can be fully controlled artificially and intelligently, and seedlings can be bred all year round without being limited by problems such as seed recalcitrance, polyembryony, large variation, and difficult storage. Moreover, through the preservation and proliferation of stem segments, a stem segment can produce multiple adventitious buds, and by adjusting the culture medium ratio, effective storage of seedlings can also be achieved.

[0020] 4. The present invention solves the problem that traditional tissue culture requires seedling hardening. The tissue culture stem segments are directly cut into the mixed matrix by infecting Agrobacterium rhizogenes to induce rooting. The appropriate moisture and air permeability in the matrix can promote the development of root hairs, enhance the plant's absorption of nutrients, and help the plant form an excellent root system. After rooting, it can be directly transplanted to a new culture matrix and container without seedling hardening, which is simple to operate, reduces manpower and resource waste, and greatly shortens the time for sterile rootstock seedlings to be directly used for grafting.

[0021] 5. The present invention improves the seedling breeding environment and lays the foundation for factory breeding of seedlings. The entire operation is carried out in an indoor air-conditioned room, which greatly improves the working comfort. Each action can be standardized, and sterile rootstock seedling breeding can achieve assembly line operation, laying the foundation for factory breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 The results show the effect of different hormone concentrations on inducing the production of young and sterile stem segments of bitter orange with nodes; among them, (a) represents different concentrations of 6-BA; (b) represents different concentrations of 6-BA and IBA; (c) represents different concentrations of 6-BA and GA3.

[0024] Figure 2 The whole technical process of rooting of trifoliate orange tissue culture stem segments; wherein, (a) represents the inoculation of trifoliate orange stem segments with nodes into the induction medium; (b) represents the germination of primary induced buds of trifoliate orange; (c) represents the production of buds by secondary proliferation of trifoliate orange; and (d) represents rooting induced by Agrobacterium rhizogenes.

[0025] Figure 3 Different OD values ​​for Agrobacterium rhizogenes 600 The induction effect of different values ​​and infection time on the rooting of young trifoliate orange stem segments in tissue culture; CK represents water treatment, O1 represents OD 600 =0.5, O2 represents OD 600 =1.0, T1 represents the infection time of 5 min, T2 represents the infection time of 15 min, O1T1 represents OD 600 =0.5 and the infection time was 5 min, and the other treatments were similar. (a) represents the number of root tips; (b) represents the number of bifurcations; (c) represents the average root length; and (d) represents the average root diameter.

[0026] Figure 4 Different OD values ​​for Agrobacterium rhizogenes 600 An example diagram showing how different infection values ​​and times promote rooting of young trifoliate orange stem segments in tissue culture.

[0027] Figure 5 The results show the effect of different hormone concentrations on inducing the production of young and sterile stem segments from the noded stem segments of Ziyang orange. Among them, (a) represents different concentrations of 6-BA; (b) represents different concentrations of 6-BA and IBA; (c) represents different concentrations of 6-BA and NAA.

[0028] Figure 6 Different OD values ​​for Agrobacterium rhizogenes 600 The rooting induction effect of different infection time and tissue culture young Ziyang orange stem segments; CK represents water treatment, O1, O2, O3 represent OD 600 =0.2, 0.5, 1.0, T1, T2, T3 represent the infection time of 5min, 15min, 30min, O1T1 represents OD 600 =0.2 and the infection time was 5 min, and the other treatments were similar. (a) represents the number of root tips; (b) represents the number of bifurcations; (c) represents the average root length; and (d) represents the average root diameter.

[0029] Figure 7 Different OD values ​​for Agrobacterium rhizogenes 600 An example diagram showing how different infection values ​​and times promote rooting of young, cultured Ziyang orange stem segments. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only 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 associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0036] The embodiment of the present invention provides a method for cultivating rooting of tissue culture stem segments of citrus rootstock, comprising the following steps:

[0037] (1) Citrus rootstock seeds were removed of pectin and outer seed coat, sterilized, and inoculated into MT basic medium to obtain sterile seedlings;

[0038] (2) cutting the sterile seedlings into noded stem segments, inoculating them into an induction medium, and performing tissue culture to obtain semi-lignified sterile stem segments;

[0039] (3) cutting off the semi-lignified sterile stem segments, soaking the stem segments in ArMSU400 infection solution, and inserting the soaked stem segments into a culture medium for rooting culture.

[0040] In some specific embodiments, the citrus rootstock seeds come from citrus fruits that naturally mature in the current year;

[0041] The method for removing pectin is as follows: mixing the seeds with 1 mol / L NaOH solution and stirring at room temperature for 15 min;

[0042] The disinfection method is: 30% sodium hypochlorite stirring disinfection for 20 minutes.

[0043] In some specific embodiments, the components of the MT minimal medium include: 4.43 g MT, 8 g agar, and 25 g sucrose;

[0044] The culture conditions for obtaining sterile seedlings include: culturing in the dark for 24-28 days and turning green for 7-14 days.

[0045] In some specific embodiments, when the cultured citrus seeds are trifoliate oranges, the induction medium includes 3 mg / L GA and 1 mg / L 6-BA;

[0046] When the cultured citrus seeds are orange, the induction medium includes 0.1 mg / L NAA and 3 mg / L 6-BA.

[0047] In some specific embodiments, the method of cutting the semi-lignified sterile stem segment is: cutting into a smooth incision along an oblique angle of 45°;

[0048] The preparation method of the ArMSU400 infection solution is as follows: the ArMSU400 bacterial cells are mixed with 1 / 2MSLIQ infection solution, the bacterial cells are resuspended, and the OD is measured. 600 value, add 1 / 2MS to adjust OD 600 Value to 0.5~1;

[0049] The ArMSU400 bacterium is Agrobacterium rhizogenes ArMSU440, into which a recombinant strain with 35S:GFP is introduced;

[0050] The ingredients of the 1 / 2MSLIQ infection solution include: adding 2.2 g of MS culture medium, 25 g of sucrose, and 1 mL of Vc per 1 L, and adjusting the pH to 5.2;

[0051] The stem segments were immersed in the ArMSU400 infection solution for 15 minutes.

[0052] In some specific embodiments, when the citrus seeds to be cultured are trifoliate oranges, the OD 600 Value to 0.5;

[0053] When the citrus seeds to be cultured are oranges, adjust the OD 600 value to 1.

[0054] In some specific embodiments, the culture matrix comprises: perlite and vermiculite mixed in a volume ratio of 2:1.

[0055] In some specific embodiments, the height of the cutting exceeds the cutting bevel;

[0056] The culture conditions of the rooting culture include: culturing in a culture room at 24° C. for 30 days and spraying water every 4 to 5 days.

[0057] The present invention also provides a drug for promoting rooting of citrus seedlings, and the preparation method thereof comprises:

[0058] Mix the ArMSU400 bacteria with 1 / 2MSLIQ infection solution, resuspend the bacteria, and measure the OD 600 value, add 1 / 2MS to adjust OD 600 Value to 0.5~1;

[0059] The ArMSU400 bacterium is Agrobacterium rhizogenes ArMSU440, into which a recombinant strain with 35S:GFP is introduced;

[0060] The ingredients of the 1 / 2MSLIQ infection solution include: adding 2.2g of MS culture medium, 25g of sucrose, and 1mL of Vc per 1L, and adjusting the pH to 5.2.

[0061] The embodiment of the present invention also provides the use of the drug in promoting the rooting of citrus plant cuttings.

[0062] Example 1

[0063] Effects of different hormone concentrations on inducing young and sterile stem segments of trifoliate orange with nodes:

[0064] In order to study whether there are differences in the induction of axillary buds of the internodes of the trifoliate orange with different hormones and different concentrations, three hormones with different proportions and different concentrations, 6-BA, 6-BA+IBA, and 6-BA+GA3, were selected to induce the germination of the internodes of the stem segments, and subculture was carried out. The specific experiments were as follows:

[0065] (1) Obtaining sterile seedlings

[0066] Collect the naturally matured citron fruits of the year, take out the seeds, wash them several times, remove the pectin, and rub the surface of the seeds with gauze until there is no excess fruit fluff and mucus. Put the seeds and 1mol / LNaOH solution into a beaker, stir at room temperature for 15 minutes, pour out the solution, rinse repeatedly with distilled water, and then immerse the seeds in water for 10 hours to soften the outer seed coat and facilitate peeling off the outer seed coat.

[0067] On the day of inoculation, remove the seed coat. In the clean bench, disinfect the seeds, add 30mL of sodium hypochlorite to a sterilized conical flask containing 70mL of sterile water, shake to mix, pour in the seeds that have been removed from the seed coat, stir continuously for 20 minutes, pour out the disinfectant, add sterile water and rinse repeatedly 6-8 times. Inoculate the disinfected seeds into MT basic medium (MT 4.43g, agar 8g, sucrose 25g), 2-3 seeds per bottle, culture in dark for 25 days, and turn green for 10 days.

[0068] (2) Inoculation of noded stem segments

[0069] The sterile seedlings were cut into 1 cm internoded stem segments, inoculated into induction medium supplemented with 6-BA, GA3, IBA and different proportions, and cultured in a conventional tissue culture room for 30 days to obtain 3-5 tissue culture young sterile stem segments for each stem segment.

[0070] Three comparison cases were set: (a) 6-BA was used alone at 0 mg / L, 0.5 mg / L, 1.0 mg / L, 1.5 mg / L, and 2.0 mg / L for a total of 5 concentrations; (b) 6-BA (0.5 mg / L, 1.0 mg / L, 2.0 mg / L) and IBA (0.05 mg / L, 0.1 mg / L, and 0.2 mg / L) for a total of 9 concentrations; (c) GA3 (2.0 mg / L, 3.0 mg / L) and 6-BA (0.5 mg / L, 1 mg / L) for a total of 4 concentrations. After 30 days of culture, the number of induced buds under different treatments was counted, and the proliferation times were calculated.

[0071] The results are as follows Figure 1 As shown in the results, after being induced by different hormones, the node stem segments of Poncirus trifoliata can form axillary buds within a certain period of time. Among them, the number of axillary buds produced by adding 6-BA alone is basically the same, and the proliferation multiples are all about 3; adding IBA on the basis of 6-BA can increase the proliferation multiples, but the effect is not obvious, and the proliferation multiples are about 4; adding GA3 on the basis of 6-BA can increase the proliferation multiples while increasing the internode height of the buds and producing new bud points, and the highest proliferation multiple is about 4.8; the above results show that using 3mg / L GA3+1mg / L 6-BA as the best hormone for the first-generation bud induction can not only increase the proliferation multiples, but also the new bud points brought by stem elongation are conducive to subculture and preservation.

[0072] Example 2

[0073] Effects of different cutting media on rooting of cuttings of trifoliate orange and sweet orange:

[0074] In order to study the effect of different substrates on the infection and rooting of trifoliate orange, the sterilized substrates were configured in different proportions to explore the effect of different substrates on the rooting of different varieties of stock, among which M1 was garden soil: vermiculite = 1:1, M2 was garden soil: vermiculite: coconut bran = 1:1:1, M3 was vermiculite: perlite = 2:1, M4 was vermiculite: substrate: perlite = 1:1:1, and M5 was substrate: coconut bran = 2:1. The cutting materials were collected in December 2023, sown in January, and grown in the substrate for 120 days. The cutting materials were cut off the base, leaving about 10 cm on the upper part, and the lower part was cut into a 45° bevel, and inserted vertically into the substrate.

[0075] 25 days after cutting, the root indicators were obtained through a root scanner. The seven root-related indicators of trifoliate orange and citrus orange were correlated through principal component analysis to obtain a correlation matrix. Two principal components were extracted respectively, and the cumulative contribution reached more than 90%. The comprehensive score can be calculated to obtain the best cutting medium.

[0076] Table 1 The effects of different ratios of substrates on rooting of cuttings of trifoliate orange and sweet orange were investigated by PCA method

[0077]

[0078] As shown in Table 1, through principal component analysis, the best cutting medium is M3, and M4 ranks second. Whether it is trifoliate orange or sweet orange, vermiculite:perlite=2:1 is the best cutting medium, which can produce more adventitious roots and secondary roots.

[0079] Example 3

[0080] Agrobacterium rhizogenes ArMSU440 with different OD 600 Effects of different values ​​and infection times on rooting induction of young trifoliate orange stem segments in tissue culture:

[0081] Agrobacterium rhizogenes is a Gram-negative soil bacterium of the genus Agrobacterium in the family Rhizobiaceae, which can infect most dicotyledons and a few monocotyledons as well as some gymnosperms. The ArMSU440 strain of Agrobacterium rhizogenes contains an agrobacterium-type Ri plasmid, has a wide host range (corn, tobacco, tea, Artemisia annua, etc.), and is also resistant to streptomycin.

[0082] (1) Agrobacterium rhizogenes transformation

[0083] The Agrobacterium rhizogenes used in the experiment was ArMSU440, purchased from Shanghai Weidi Biotechnology Co., Ltd. and stored in a -80° refrigerator. 35S:GFP was transformed into competent ArMSU440 by freeze-thaw method; then 700 μL of TY liquid medium without antibiotics was added and cultured at 28°C for 2 hours. The bacteria were collected by centrifugation at 6000 rpm for 1 minute, and about 100 μL of supernatant was taken and gently blown to resuspend the bacteria block and spread on the TY plate containing streptomycin, and then placed upside down in a 28°C incubator for 2-3 days.

[0084] A single colony was picked and, after verification, stored at -80°C for future use, which was the recombinant Agrobacterium rhizogenes ArMSU440.

[0085] (2) Activation of recombinant Agrobacterium rhizogenes ArMSU440

[0086] The recombinant Agrobacterium rhizogenes ArMSU440 was taken out from the -80°C refrigerator, inoculated on a TY plate containing streptomycin, and cultured at 28°C for 1 day until a single colony grew.

[0087] (3) Propagation of recombinant Agrobacterium rhizogenes ArMSU440

[0088] Pick a single colony, add 700 μL of TY liquid culture medium containing streptomycin to a 2 mL centrifuge tube, shake and culture at 28°C for 14 h, and observe the turbidity of the bacterial solution; aspirate all the bacterial solution, transfer it to a 50 mL conical flask of TY liquid culture medium containing streptomycin, shake and culture at 28°C for 10 h, and the expansion is completed.

[0089] (4) Preparation of infection fluid of recombinant Agrobacterium rhizogenes ArMSU440

[0090] Transfer all the bacterial liquid in the conical flask to a sterilized 50mL centrifuge tube; centrifuge at 8000rpm for 15min to collect the bacteria, discard the supernatant, add 20mL 1 / 2MSLIQ infection solution (add 2.2g MS medium, 25g sucrose, 1mL Vc per 1L, adjust pH to 5.2), pipette to resuspend the bacteria, and measure OD 600 value, add 1 / 2MS to adjust OD 600 The value was adjusted to 0.5 or 1.0, and the corresponding volume of streptomycin was added at a dilution of 2:1000 to improve the transformation efficiency.

[0091] (5) Infection of young sterile tissue cultured trifoliate orange stem segments

[0092] Pick out a young and tender stem segment of about 1 cm in length and in good growth condition (obtained in Example 1). Then, use a scalpel to cut it into a smooth incision at an angle of 45°. Soak the stem segment in the prepared ArMSU400 infection solution. The infection solution is set at different OD 600Values ​​(0.5, 1.0), two different time (5min, 15min) and a water control group were set. Then the treated stem segments were inserted into the culture matrix to ensure that the wound part of the seedling was covered by the culture matrix. The volume ratio of perlite and vermiculite in the culture matrix was 2:1, and both perlite and vermiculite were sterilized. Cover the seedlings with another layer of plastic wrap or cover the culture box, and spray water several times a day to keep the culture matrix moist in the early stage. The culture condition parameters are: culture at 25℃ for 20 days to induce the formation of capillary roots.

[0093] The results are as follows Figure 3 As shown. Different OD 600 The optimal OD value for promoting rooting of trifoliate orange by Agrobacterium rhizogenes was 600 =0.5, infection 15min, culture 30d the longest root length can reach 4cm, culture 45d can produce more secondary roots, Figure 4 It can be seen that the upper part of the plant also has obvious growth, the leaves become larger, the stems become thicker, and there is no need to harden the seedlings, and they can be transplanted directly out of the nursery.

[0094] Example 4

[0095] The effects of the technical solutions of Examples 1-3 were verified by using Citrus sinensis, and different hormone concentrations were found to induce the noded stem segments of Ziyang Citrus sinensis to produce young and tender sterile stem segments.

[0096] (1) Obtain sterile seedlings.

[0097] The naturally ripened orange fruits of the year were collected and the seeds were taken out. The other experimental methods were the same as those in Example 1.

[0098] (2) Inoculation of noded stem segments

[0099] The experimental method is the same as that in Case 1.

[0100] The difference from Example 1 is that 6-BA, 6-BA and IBA do not have a good effect on the induction of axillary buds of orange (see Figure 5 ). By improving the hormone ratio of the culture medium, a total of 6 concentrations of 6-BA (3 mg / L, 2.0 mg / L, 1.0 mg / L) and NAA (0.1 mg / L, 0.2 mg / L) were set.

[0101] pass Figure 5 It can be seen that through the treatment of 3mg / LNAA and 0.1mg / L6-BA, the proliferation multiples of the orange stem segments reached a maximum of 6 and a minimum of 3, and the buds grew vigorously and had good subsequent rooting.

[0102] Example 5

[0103] The effect of the technical scheme of Examples 1-3 was verified by using orange, and the root-generating Agrobacterium ArMSU440 at different OD600 The effect of different infection values ​​and different infection times on the rooting induction of tissue cultured young Ziyang orange stem segments.

[0104] (1) Activation of recombinant Agrobacterium rhizogenes ArMSU440

[0105] The experimental method is the same as that in Example 3.

[0106] (2) Propagation of recombinant Agrobacterium rhizogenes ArMSU440

[0107] The experimental method is the same as that in Example 3.

[0108] (3) Preparation of infection fluid of recombinant Agrobacterium rhizogenes ArMSU440

[0109] The experimental method is the same as in Example 3. The difference is that 1 / 2MS is added to adjust the OD 600 The values ​​are 0.2, 0.5, and 1.0, and the corresponding volume of streptomycin is added at a ratio of 2:1000 to improve the transformation efficiency.

[0110] (4) Infection of tissue culture stem segments

[0111] The experimental method is the same as in Example 3. The difference is that the orange stem segment is about 0.5 cm, and when cutting at a 45° angle, care should be taken to avoid too large an angle, which would cause the stems and leaves to become scattered. Due to the different materials and the results of the preliminary test, 10 treatment groups, 9 experimental groups, and 1 control group (clear water) were set for the orange infection. The experiment set up 3 treatment concentrations (OD 600 =0.2, 0.5, 1.0), 3 treatment times (5min, 15min, 30min), cross combination.

[0112] Depend on Figure 6 It can be seen that for tissue culture of orange stem segments, the best treatment combination is O3T2 (OD 600 =1.0, T2=15), the treatment effect is the best, and each index is significantly higher than other treatments. Figure 7 As can be seen from the examples, the roots induced by Agrobacterium rhizogenes have many branches, thick roots, lush stems and leaves, and there is no need for seedling hardening and they can be directly transplanted out of the nursery.

[0113] Verification was conducted on citrus fruits to determine whether ArMSU400 still had a good effect on the infection and rooting of citrus fruits stem segments, which fully confirmed the feasibility of the present invention.

[0114] In summary, by inoculating noded stem segments, 4-6 axillary buds can be proliferated in about 25 days, and more adventitious buds can be obtained by subculturing and multiplying them again. The buds obtained above can be divided and infected with Agrobacterium rhizogenes to achieve batch rooting of adventitious buds without tissue culture, which can not only improve the survival rate and high root quality; but also can greatly shorten the formation time of virus-free seedlings without hardening, and produce virus-free seedlings all year round. Among them, the best hormone for inducing axillary buds in tissue cultured trifoliate orange stem segments is 3mg / L GA+1mg / L6-BA, and the rooting infection concentration of cuttings is OD 600 =0.5, infection for 15 min; the best hormone for inducing axillary buds in orange is 3 mg / L6-BA+0.1 mg / LNAA, and the rooting infection concentration is OD 600 =1.0, infection 15min; therefore, the determination of hormone concentration and type as well as infection concentration shall be appropriately adjusted according to plant characteristics and economic factors; the present invention only takes 50d to complete the whole process from stem segment inoculation, rooting to sterile seedling transplanting, which greatly shortens the time of sterile seedling breeding.

[0115] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for cultivating rooting of citrus rootstock tissue culture stem segments, characterized in that: The following steps are involved: (1) removing pectin and outer seed coat from citrus rootstock seeds, sterilizing and inoculating them into MT basic medium, and culturing to obtain sterile seedlings; the citrus fruits are trifoliate orange and sweet orange; the components of the MT basic medium are: 4.43 g MT, 8 g agar, and 25 g sucrose; (2) Cutting the sterile seedlings into stem segments with nodes, inoculating them into an induction medium, and obtaining semi-lignified sterile stem segments by tissue culture; when the cultured citrus seeds are trifoliate orange, the hormone components in the induction medium are 3 mg / L GA and 1 mg / L 6-BA; when the cultured citrus seeds are citrus citrus, the hormone components in the induction medium are 0.1 mg / L NAA and 3 mg / L 6-BA; (3) cutting off the semi-lignified sterile stem segments, soaking the stem segments in ArMSU400 infection solution, and inserting the soaked stem segments into a culture medium for rooting culture; The preparation method of the ArMSU400 infection solution is as follows: the ArMSU400 bacterial cells are mixed with 1 / 2MSLIQ infection solution, the bacterial cells are resuspended, and the OD is measured. 600 value, add 1 / 2MS to adjust OD 600 Value to 0.5~1; The ArMSU400 is a recombinant strain of Agrobacterium rhizogenes ArMSU440, into which 35S:GFP is introduced; The composition of the 1 / 2MSLIQ infection solution is as follows: add 2.2 g of MS medium, 25 g of sucrose, and 1 mL of Vc per 1 L, and adjust the pH to 5.2; The stem segments were immersed in the ArMSU400 infection solution for 15 min; The culture matrix comprises: perlite and vermiculite mixed in a volume ratio of 2:

1.

2. The culture method according to claim 1, characterized in that The citrus rootstock seeds come from citrus fruits that naturally mature in that year; The method for removing pectin is as follows: mixing the seeds with a 1 mol / L NaOH solution and stirring at room temperature for 15 min; The disinfection method is: 30% sodium hypochlorite stirring disinfection for 20 minutes.

3. The culture method according to claim 1, characterized in that The culture conditions for obtaining sterile seedlings include: culturing in the dark for 24-28 days and turning green for 7-14 days.

4. The culture method according to claim 1, characterized in that The method for cutting the semi-lignified sterile stem segment is: cutting it into a smooth incision along an oblique angle of 45°.

5. The culture method according to claim 1, characterized in that The height of the cutting exceeds the oblique end of the cutting; The culture conditions of the rooting culture include: culturing in a culture room at 24° C. for 30 days and spraying water every 4 to 5 days.

6. The culture method according to claim 1, characterized in that When the citrus seeds to be cultured are trifoliate oranges, adjust the OD 600 value to 0.5; when the citrus seeds to be cultured are oranges, adjust the OD 600 value to 1.0.

Images