Method for inducing growth of adventitious roots from terminal buds of adult masson pine trees

Abstract

The invention belongs to the technical field of plant culture, and particularly relates to a method for induced growth of adventitious roots from terminal buds of adult masson pine trees, which comprises the following steps: (1) performing induced culture on the terminal buds to generate axillary buds; (2) performing induction culture on the axillary buds to generate adventitious roots; wherein in the step (1), a first culture medium is used for culturing, and the first culture medium is a DCR culture medium containing 6-benayl aminopurine. According to the method, rejuvenation treatment is not needed, compared with an existing adult tree regeneration method, the working procedures and time are saved, in the process of inducing the axillary buds from the terminal buds and breeding, the bud induction rate and the single bud induction rate are high, and growth is good; in addition, the adventitious roots obtained through the method are large in number of root hairs and thick and strong in root hairs.

Description

Technical Field

[0001] The invention belongs to the technical field of plant cultivation, and in particular relates to a method for inducing the growth of adventitious roots from terminal buds of adult Masson pine trees. Background Art

[0002] Masson pine is an evergreen coniferous tree species widely distributed in southern my country. It has the characteristics of rapid growth, excellent material quality and strong resistance to adversity. It is an important afforestation and timber species in forestry production. However, the traditional Masson pine propagation method is mainly seed propagation, which has problems such as large genetic variation and separation of offspring traits, and it is difficult to meet the needs of modern forestry for high-quality germplasm resources.

[0003] Therefore, the research on asexual propagation technology of Masson pine, especially the asexual reproduction by inducing the growth of adventitious roots from the top buds of adult trees, has important practical significance and application value. Due to the age effect of Masson pine, the reports on the regeneration of adult trees all require the key step of rejuvenating to achieve regeneration. The rejuvenating process requires at least half a year of growth cycle for tree regeneration.

[0004] The top buds of adult Masson pine trees in spring are covered with brown scales when they first sprout, and are the youngest part of the entire tree. The top buds of adult Masson pine trees can be used as the starting point for plant regeneration, avoiding rejuvenation treatment and reducing the cycle. Summary of the invention

[0005] The present invention develops a method for inducing adventitious roots from terminal buds of adult Masson pine trees. The method studies suitable methods for surface disinfection and sterilization of terminal buds of adult Masson pine trees, screening of suitable culture media for inducing terminal buds and axillary buds, and screening of suitable culture media for inducing adventitious roots. The method compares and analyzes the effects of different exogenous disinfectant concentrations and durations on terminal bud growth, the effects of different cytokinins on axillary bud induction, and the effects of different auxins and their concentrations on adventitious root induction, so as to provide reliable theoretical support for the direct occurrence of terminal bud organs of adult Masson pine trees.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] On one hand, the present invention provides a method for inducing the growth of adventitious roots from terminal buds of adult Masson pine trees, comprising: (1) inducing and culturing the terminal buds to generate axillary buds; and (2) inducing and culturing the axillary buds to generate adventitious roots; wherein, in step (1), a first culture medium is used for culturing, and the first culture medium is a DCR culture medium containing 6-benzylaminopurine.

[0008] Preferably, one or more combinations of the following conditions are met in step (1): (a) in the first culture medium, the concentration of 6-benzylaminopurine is 0.25 mg / L-2 mg / L; (b) the first culture medium contains indolebutyric acid, and its concentration is 0.05 mg / L-0.15 mg / L; (c) the first culture medium contains agar and sucrose, the concentration of agar is 5 g / L-10 g / L, and the concentration of sucrose is 25 g / L-35 g / L; (d) the terminal bud is the terminal bud after the outer scales are removed.

[0009] Preferably, in step (2), a second culture medium is used for culturing, and the second culture medium is a DCR culture medium containing naphthylacetic acid and indolebutyric acid.

[0010] Preferably, the concentration of naphthylacetic acid is 0.1 mg / L<the concentration of naphthylacetic acid<1 mg / L, and the concentration of indolebutyric acid is 0.1 mg / L<the concentration of indolebutyric acid<1 mg / L.

[0011] Preferably, the second culture medium further comprises agar and sucrose, the concentration of agar is 5 g / L-10 g / L, and the concentration of sucrose is 25 g / L-35 g / L.

[0012] Preferably, a first disinfection treatment is performed before the above-mentioned apical bud induction culture to generate axillary buds, and the first disinfection treatment includes: soaking in a carbendazim solution.

[0013] Preferably, the first disinfection treatment is followed by a second disinfection treatment, wherein the second disinfection treatment comprises culturing the terminal buds using a third culture medium containing 6-benzylaminopurine, indolebutyric acid and a preservative.

[0014] Preferably, the concentration of 6-benzylaminopurine is 0.02 mg / L-0.03 mg / L; and / or the concentration of indolebutyric acid is 0.04 mg / L-0.06 mg / L; and / or the mass fraction of the preservative is 0.05%-0.25%.

[0015] Preferably, the second disinfection treatment further comprises: performing a pretreatment before culturing the terminal buds using the third culture medium, the pretreatment comprising: soaking the terminal buds using a preservative.

[0016] Preferably, the third culture medium further comprises agar and sucrose; the concentration of agar is 5 g / L-10 g / L, and the concentration of sucrose is 25 g / L-35 g / L.

[0017] The beneficial effects of the present invention include at least:

[0018] (1) The method for inducing the growth of adventitious roots from the terminal buds of adult Masson pine trees provided by the present invention does not require rejuvenation treatment, thus saving steps and time compared to existing adult tree regeneration methods.

[0019] (2) The method for inducing adventitious roots from the terminal buds of Masson pine adult trees provided by the present invention has high bud induction rates and single bud induction rates during the process of inducing axillary buds from the terminal buds and propagating them, and the buds grow well.

[0020] (3) The method for inducing adventitious roots from the terminal buds of adult Masson pine trees provided by the present invention can obtain a large number of adventitious roots that are thick and robust. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The disinfection and sterilization treatment of the top buds of adult Masson pine trees with and without peeling the outer layer of tender scales was compared; A: explants without peeling the outer layer of scales; B: explants without peeling the outer layer of scales, inoculated on the culture medium after disinfection; C: explants without peeling the outer layer of scales, inoculated on the culture medium after sterilization and cultured for 2 months; D: explants with the outer layer of scales removed; E: explants with the outer layer of scales peeled off, sterilized and inoculated on the culture medium; F: explants were disinfected and sterilized, inoculated on the culture medium and cultured for 2 months.

[0022] Figure 2 The explants are the explants infected by bacteria again and the explants that have eliminated endophytes; A: the explants infected by bacteria; B: the explants infected by bacteria; C: the explants that have grown normally for 1 month after being treated with 0.2% PPM;

[0023] Figure 3 The effects of different concentrations of 6-BA on axillary bud induction; A: 0.25 mg / L6-BA; B: 0.5 mg / L6-BA; C: 1 mg / L6-BA; D: 2 mg / L6-BA; E: 4 mg / L6-BA; F: 8 mg / L6-BA.

[0024] Figure 4 The induction of axillary buds by different cytokinins; A: 2mg / L6-BA induced axillary bud growth; B: 2mg / LTDZ induced axillary bud growth; C: 2mg / LKT induced axillary bud growth; D: 2mg / LZT induced axillary bud growth;

[0025] Figure 5 Adventitious roots induced by the combination of NAA and IBA; among them, A: 0.1mg / LNAA and 0.1mg / LIBA; B: 0.1mg / LNAA and 0.5mg / LIBA; C: 0.1mg / LNAA and 1mg / LIBA; D: 0.5mg / LNAA and 0.1mg / LIBA; E: 0.5mg / LNAA and 0.5mg / LIBA; F: 0.5mg / LNAA and 1mg / LIBA; G: 1mg / LNAA and 0.1mg / LIBA; H: 1mg / LNAA and 0.5mg / LIBA; I: 1mg / LNAA and 1mg / LIBA. DETAILED DESCRIPTION

[0026] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0027] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0028] The embodiment of the present invention provides a method for inducing the growth of adventitious roots from terminal buds of adult Masson pine trees, comprising: (1) inducing and culturing the terminal buds to generate axillary buds; (2) inducing and culturing the axillary buds to generate adventitious roots; wherein, in step (1), a first culture medium is used for culturing, and the first culture medium is a DCR culture medium containing 6-benzylaminopurine (6-BA).

[0029] It should be noted that the induction rate of axillary buds generated by apical bud induction culture using DCR medium containing 6-benzylaminopurine (6-BA) in the present invention is higher than that of other cytokinins, and the growth condition is better than that of other cytokinins, such as TDZ (diazadiazole), KT (6-furfurylaminopurine or 6-furfuryladenine) or ZT (zeatin).

[0030] In some specific examples, step (1) satisfies one or more combinations of the following conditions:

[0031] (a) In the first culture medium, the concentration of 6-benzylaminopurine is 0.25 mg / L-2 mg / L, such as 0.4 mg / L, 0.6 mg / L, 0.8 mg / L, 1 mg / L, 1.2 mg / L, 1.5 mg / L or 1.8 mg / L;

[0032] (b) the first culture medium contains indolebutyric acid at a concentration of 0.05 mg / L-0.15 mg / L, such as 0.07 mg / L, 0.09 mg / L, 0.11 mg / L or 0.13 mg / L;

[0033] (c) the first culture medium comprises agar and sucrose, wherein the concentration of agar is 5 g / L-10 g / L, such as 7 g / L, 8 g / L or 9 g / L, etc.; the concentration of sucrose is 25 g / L-35 g / L, such as 27 g / L, 29 g / L, 32 g / L or 34 g / L, etc.;

[0034] (d) The terminal bud is the terminal bud after the outer scales are removed; specifically, the survival rate of the culture after the outer scales are removed is higher than that without removing the outer scales.

[0035] In some specific examples, in step (2), a second culture medium is used for culturing, and the second culture medium is a DCR culture medium containing naphthylacetic acid and indolebutyric acid.

[0036] In some specific examples, the concentration of naphthylacetic acid is 0.1 mg / L<naphthylacetic acid concentration<1 mg / L, such as 0.3 mg / L, 0.5 mg / L or 0.7 mg / L, etc.; the concentration of indolebutyric acid is 0.1 mg / L<indolebutyric acid concentration<1 mg / L, such as 0.3 mg / L, 0.5 mg / L or 0.7 mg / L, etc.

[0037] In some specific examples, the second culture medium further comprises agar and sucrose, the concentration of agar is 5g / L-10g / L, such as 7g / L, 8g / L or 9g / L, etc.; the concentration of sucrose is 25g / L-35g / L, such as 27g / L, 29g / L, 32g / L or 34g / L, etc.

[0038] In some specific examples, a first disinfection treatment is performed before the apical bud induction culture generates axillary buds, and the first disinfection treatment includes: soaking in a carbendazim solution.

[0039] It should be noted that the first disinfection treatment may specifically include: soaking in 500 mg / L carbendazim solution for 10 minutes, and soaking in 75% alcohol for 30 seconds.

[0040] In some specific examples, the second disinfection treatment is performed after the first disinfection treatment, and the second disinfection treatment includes culturing the apical buds using a third culture medium containing 6-benzylaminopurine, indolebutyric acid and a preservative.

[0041] In some specific examples, the concentration of 6-benzylaminopurine is 0.02 mg / L-0.03 mg / L; and / or the concentration of indolebutyric acid is 0.04 mg / L-0.06 mg / L; and / or the mass fraction of the preservative is 0.05%-0.25%, such as 0.08%, 0.1%, 0.15%, 0.2% or 0.22%, etc.

[0042] In some specific examples, the second disinfection treatment further includes: performing a pretreatment before culturing the apical buds using the third culture medium, the pretreatment comprising: soaking the apical buds in a preservative.

[0043] In some specific examples, the third culture medium also contains agar and sucrose; the concentration of agar is 5g / L-10g / L, such as 7g / L, 8g / L or 9g / L, etc.; the concentration of sucrose is 25g / L-35g / L, such as 27g / L, 29g / L, 32g / L or 34g / L, etc.

[0044] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0045] In the following example, the culture conditions include: the culture is cultured in a light incubator (light intensity 3000 lx), the temperature is 25±2°C, and the photoperiod is 14 hours of light and 10 hours of darkness.

[0046] In the following example, according to the actual growth of the explants, the surface sterilization effect data of the terminal bud stem segment explants were counted after 30 days of culture; the sterilization effect of removing endophytes from the terminal bud stem segment explants was analyzed after 15 days of culture; the axillary bud induction and proliferation data were recorded after 60 days of culture; and the adventitious root induction data were counted after 60 days of culture.

[0047] In the following example, DCR medium (without additives) is as follows: 0.17 g potassium dihydrogen phosphate, 6.2 mg boric acid, 0.085 g calcium chloride, 8.6 mg zinc sulfate, 22.3 mg manganese sulfate, 0.83 mg potassium iodide, 0.25 mg sodium molybdate, 27.8 mg ferrous sulfate, 0.25 mg copper sulfate, 0.025 mg nickel chloride, 37.3 mg EDTA sodium salt, 1.0 mg vitamin B1, 0.025 mg cobalt chloride, 2.0 mg glycine, 0.5 mg vitamin B6, 200 mg inositol, 0.5 mg niacin, 2.0 mg glycine, 30000 mg sucrose, 6000 mg agar, and 370 mg magnesium sulfate.

[0048] 1. Test Materials

[0049] From March to April 2023, the terminal buds of excellent individual trees with a DBH of more than 25 cm were carefully collected from the mature forests of Masson pine forest farm in Duyun (see Figure 1 A) As explants, the forest farm is a pure Masson pine forest with a relatively simple forest structure, mainly composed of young forests, middle-aged forests, near-mature forests, mature forests and over-mature forests, which are half-sib families; then these samples are quickly stored in a refrigerator at 4°C for up to 10 days.

[0050] 2. Disinfection

[0051] (I) Primary surface disinfection

[0052] The collected explants were sterilized on the surface to remove surface fungi and bacteria; the two schemes were to retain the outer scales ( Figure 1 A to Figure 1 C) and peeling off the outer scales ( Figure 1 D to Figure 1 F), the inoculated terminal buds were all about 0.5 cm long.

[0053] The first scheme retains the outer scales and includes three treatments: (4) soaking in 500 mg / L carbendazim solution for 10 min, soaking in 75% alcohol for 30 s, and soaking in 40 ml 5% NaClO with a drop of Tween 20 for 15 min (treatment ①); (5) soaking in 500 mg / L carbendazim solution for 15 min, soaking in 75% alcohol for 30 s, and soaking in 40 ml 5% NaClO with 1 ml Tween 20 for 20 min (treatment ②); (6) soaking in anhydrous ethanol for 1 min, soaking in 75% alcohol for 30 s, and soaking in 40 ml 5% NaClO with 2 ml Tween 20 for 20 min (treatment ③).

[0054] The second scheme for removing the outer scales includes three treatments: (1) soaking in 75% alcohol for 30 seconds, and soaking in 40ml2%NaClO plus 1mlTween20 for 25 minutes (treatment ④); (2) soaking in 500mg / L carbendazim solution for 5 minutes, soaking in 75% alcohol for 30 seconds, and soaking in 40ml2%NaClO plus 1mlTween20 for 25 minutes (treatment ⑤); (3) soaking in 500mg / L carbendazim solution for 10 minutes, soaking in 75% alcohol for 30 seconds, and soaking in 40ml2%NaClO plus 1mlTween20 for 25 minutes (treatment ⑥).

[0055] In addition, in the above treatments ① to ⑥, after each immersion treatment, the plants were rinsed with sterile water 5 times, each time for 5 min; after immersion, each treatment group was cultured using culture medium (DCR as the basic culture medium, with 7 g / L agar and 30 g / L sucrose added, respectively); 10 explants were inoculated in each treatment, and repeated 3 times, that is, a total of 30 explants were inoculated in the first and second schemes, respectively.

[0056] The surface contamination rate, browning rate and survival rate of explants under different schemes and treatments are shown in Tables 1 and Figure 1 shown.

[0057] Table 1 Explant disinfection and sterilization methods and results Note: The values ​​are the mean ± standard error of 3 repeated experiments, with 10 samples in each group. For each variable, the same values ​​with superscript letters are not significantly different at the P>0.05 level, and the values ​​with different superscript letters are significantly different at the P<0.05 level.

[0058] From Table 1 and Figure 1 A to Figure 1 C shows that there are significant differences in the surface contamination rate and survival rate of treatments ①, ② and ③ in the first scheme; among them, the surface contamination rate of treatment ① is the lowest, while treatments ② and ③ show complete contamination.

[0059] From Table 1 and Figure 1 D to Figure 1 F It can be seen that there are significant differences in the surface contamination rate, survival rate and browning rate of ④, ⑤ and ⑥ in the second scheme, and as the immersion time of 500 mg / L carbendazim increases, the surface contamination rate gradually decreases and the survival rate gradually increases.

[0060] In addition, by comparing the treatment methods of the first and second options, it can be seen that the sterilization process of the regeneration of the apical bud organs of adult Masson pine trees preferably removes the outer scales, which can reduce the surface contamination rate and increase the survival rate; in addition, treating the explants with a carbendazim solution can significantly reduce the explant surface contamination rate and increase the explant survival rate.

[0061] (II) Secondary surface disinfection

[0062] The materials used for secondary sterilization are explants that have grown normally and are contaminated again after surface sterilization (explants after removing the outer scales). There are two options for removing bacteria:

[0063] The first scheme: place the explants of secondary bacterial or fungal infection in Murashige and Skoog (MS) medium, add 4% plant preservative mixture (PPM, Beijing Solebow Technology Co., Ltd.), shake and soak for 4 hours without cleaning or removing the infected parts; insert the uninfected parts into DCR+6-BA0.025mg / L+IBA0.05mg / L medium, do not add PPM (treatment ①), add 0.05% PPM (treatment ②), 0.1% PPM (treatment ③), 0.15% PPM (treatment ④), 0.2% PPM (treatment ⑤), and 0.25% PPM (treatment ⑥).

[0064] The second solution: directly take the explants infected with bacteria that have not been pre-soaked with PPM, remove the infected part, and insert them into DCR+6-BA0.025mg / L+IBA0.05mg / L+0.2%PPM (treatment ⑦), observe and record their contamination and growth conditions.

[0065] In addition, 7 g / L agar and 30 g / L sucrose were added to each culture medium in the first and second schemes, respectively; 20 explants were inoculated in each treatment, repeated twice, and a total of 40 explants were inoculated.

[0066] The explants treated with the above treatment were found to be infected again, which affected the induction of axillary buds and the formation of needles ( Figure 2 A. Figure 2 B and Figure 2 C) growth; the bacteria that appear during the culture process can be roughly divided into two categories ( Figure 2 A, Figure 2 B). In addition, the contamination rate, browning rate and survival rate of different treatment groups are shown in Table 2 below.

[0067] Table 2 Disinfection effect of PPM on surface bacteria

[0068] deal with Preprocessing method Contamination rate (%) Browning rate (%) Survival rate (%) ① yes 0.00PPM 90±0f 0±0a 10±0a ② yes 0.05% PPM 40±0cd 0±0a 60±0cd ③ yes 0.10% PPM 50±0de 0±0a 50±0bc ④ yes 0.15% PPM 55±7e 0±0a 45±7b ⑤ yes 0.20% PPM 35±7bc 0±0a 65±7de ⑥ yes 0.25% PPM 25±7b 0±0a 75±7e ⑦ no 0.2%PPM 5±7a 5±7a 90±0f

[0069] Note: The values ​​represent the mean ± standard error of two repeated experiments, with 20 samples in each repeat. For each variable, the values ​​with the same superscript letters are not significantly different at the P>0.05 level, and the values ​​with different superscript letters are significantly different at the P<0.05 level.

[0070] It can be seen from Table 2 above that there are significant differences in contamination rate and survival rate among different disinfection treatments, but there is no significant difference in browning rate (Table 2); among all treatments, when PPM was not added, the highest contamination rate (90%) and the lowest survival rate (10%) were obtained; in addition, when 0.25% PPM was added, the contamination rate was the lowest (25%) and the survival rate was the highest (75%). These results show that with the increase of PPM concentration, the contamination rate of pre-soaking treatment decreases and the survival rate increases; comparative analysis found that there is a significant difference between PPM ⑤ soaking treatment and non-soaking treatment ⑦; the contamination rate of treatment ⑦ is only 5%, and the survival rate is 95%, while the contamination rate of treatment ⑤ is 35%, and the survival rate is 65% (Table 2). The above shows that explants pre-soaked with PPM show a higher contamination rate and a lower survival rate in the later stage of continuous culture.

[0071] 3. Induction and propagation of axillary buds

[0072] (I) Effect of 6-BA concentration on adventitious bud induction

[0073] The materials used in the axillary bud induction test were explants that grew normally after one and two surface disinfection treatments (uncontaminated explants). In order to evaluate the effect of 6-BA on axillary bud induction, explants with basically the same growth potential were placed on basic culture media (DCR was the basic culture media) with 6-BA concentrations of 0.025 mg / L, 0.5 mg / L, 1 mg / L, 0.025 mg / L, 2 mg / L, 4 mg / L and 8 mg / L, and the number and state of bud growth 2 months after inoculation were recorded; in addition, each culture medium was also supplemented with 7 g / L agar, 30 g / L sucrose and 0.1 mg / L IBA; 20 explants were inoculated in each treatment, repeated twice, and a total of 40 explants were inoculated.

[0074] Axillary bud induction showed different growth characteristics under different concentrations of 6-BA ( Figure 3 ); In addition, the bud induction rate and single bud induction rate of different concentrations of 6-BA are shown in Table 3 below.

[0075] Table 3 Adventitious buds induced by different concentrations of 6-BA

[0076] deal with Hormone 1(6-BA) Hormone 2 (IBA) Bud induction rate (%) (%) Single bud induction rate ① 0.25 0.10 30.00±0.00d 3.50±0.71b ② 0.50 0.10 15.00±7.07ab 3.50±0.71b ③ 1.00 0.10 25.00±7.07bc 4.00±1.41b ④ 2.00 0.10 15.00±7.07ab 7.00±1.41c ⑤ 4.00 0.10 10.00±0.00a 1.00±0.00a ⑥ 8.00 0.10 10.00±0.00a 1.00±0.00a

[0077] Note: The values ​​represent the mean ± standard error of two repeated experiments, with 20 samples in each repeat. For each variable, the same superscript letters indicate no significant difference at the P>0.05 level, and different superscript letters indicate significant difference at the P<0.05 level.

[0078] As above Figure 3 As can be seen from Table 3, ① was observed to have a higher germination rate; however, the individual shoots of ① did not show robust growth; on the contrary, ④ showed the highest number of individual shoots and displayed a more vigorous growth pattern; significant differences were observed compared with other treatments that resulted in poor growth and malnutrition.

[0079] (II) Effects of different cytokinins on the induction of axillary buds in needle bundles

[0080] The materials used in the axillary bud proliferation test were newly formed needle bundles that grew normally after explants were treated with one and two surface disinfections. In order to evaluate the effect of cytokinins on axillary bud proliferation, the explants were placed in basal culture media (DCR is the basic culture media) supplemented with 2mg / L6-BA, 2mg / LTDZ, 2mg / LKT and 2mg / LZT, and the growth of axillary buds was recorded; in addition, each culture medium was supplemented with 7g / L agar and 30g / L sucrose; 20 explants were inoculated in each treatment, repeated twice, and a total of 40 explants were inoculated. The induction rate of axillary buds induced by different cytokinins is shown in Figure 2. Figure 4 And as shown in Table 4.

[0081] Table 4 Axillary buds induced by different types of cytokinins Note: Values ​​represent the mean ± standard error of two replicates, with 20 samples in each replicate. For each variable, the same superscript letter indicates no significant difference at P>0.05.

[0082] Depend on Figure 4 From Table 4, we can see that different cytokinins have no significant difference in the induction rate of axillary buds induced by needle bundles (Table 4), but have a greater effect on the growth of axillary buds and proto-needle bundles ( Figure 4 ); Adding 6-BA during induction can induce the most vigorous growth of axillary buds. Adding 6-BA can also prevent the browning and death of the original needle bundles. Figure 4 As shown in A; while TDZ( Figure 4 B) KT( Figure 4 C) and ZT( Figure 4 The addition of 6-BA showed the opposite effect. The original needle bundles began to turn brown and die, and the growth of new axillary buds was relatively poor. This shows that 6-BA is more suitable for the induction of axillary buds of Masson pine needles than TDZ, KT, and ZT.

[0083] 4. Adventitious root induction

[0084] A two-factor completely randomized experimental design was used to induce adventitious roots (Table 5), that is, a single shoot tip with a needle bundle was transferred to a basic medium containing 1 / 4DCR, and different concentrations of NAA and IBA were added to the rooting medium; in addition, 7 g / L agar and 15 g / L sucrose were added to the rooting medium; 10 explants were inoculated in each treatment, repeated 3 times, and a total of 30 explants were inoculated; then, the rooting data were observed and recorded.

[0085] Table 5 Two-factor completely randomized experimental design

[0086] deal with Hormone 1NAA (mg / L) Hormone 2IBA (mg / L) ① 0.10 0.10 ② 0.10 0.50 ③ 0.10 1.00 ④ 0.50 0.10 ⑤ 0.50 0.50 ⑥ 0.50 1.00 ⑦ 1.00 0.10 ⑧ 1.00 0.50 ⑨ 1.00 1.00

[0087] The results showed that different concentrations of NAA, IBA and the interaction between NAA and IBA (Table 6) showed significant differences in the induction of adventitious roots. Figure 5 and as shown in Table 6 below.

[0088] Table 6 Results of adventitious roots induced by two auxins

[0089]

[0090]

[0091] Depend on Figure 5From Table 6, we can see that adventitious roots or fibrous roots were induced only in the five treatments (②, ④, ⑤, ⑥, ⑧), and the growth of fibrous roots or lateral roots was different in each treatment. When NAA0.5mg / L was added, the number of rooted explants was significantly higher than that in NAA0.1mg / L and NAA1mg / L treatments. Among them, the number of adventitious root explants induced in the NAA0.5mg / L+IBA0.1mg / L treatment was the largest ( Figure 5 D); In the treatment of NAA0.1mg / L+IBA0.5mg / L, the number of roots of a single explant was the largest ( Figure 5 B); The above shows that the higher the NAA concentration, the more roots the explants will have, and the higher the IBA concentration, the more roots each explant will have; The above results show that appropriate addition of NAA and IBA is very important for the induction of adventitious roots.

[0092] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. A method for inducing adventitious roots from terminal buds of adult Masson pine trees, characterized in that: include: (1) inducing the apical buds to generate axillary buds; (2) inducing the axillary buds to generate adventitious roots; wherein, in step (1), a first culture medium is used for culture, and the first culture medium is a DCR culture medium containing 6-benzylaminopurine.

2. The method according to claim 1, characterized in that In step (1), one or more of the following conditions are met: (a) in the first culture medium, the concentration of 6-benzylaminopurine is 0.25 mg / L-2 mg / L; (b) the first culture medium contains indolebutyric acid at a concentration of 0.05 mg / L-0.15 mg / L; (c) the first culture medium comprises agar and sucrose, the concentration of agar is 5 g / L-10 g / L, and the concentration of sucrose is 25 g / L-35 g / L; (d) The terminal bud is the terminal bud after the outer scales are removed.

3. The method according to claim 1 or 2, characterized in that: In step (2), a second culture medium is used for culturing, and the second culture medium is a DCR culture medium containing naphthylacetic acid and indolebutyric acid.

4. The method according to claim 3, characterized in that The concentration of naphthylacetic acid is 0.1 mg / L<naphthylacetic acid concentration<1 mg / L, and the concentration of indolebutyric acid is 0.1 mg / L<indolebutyric acid concentration<1 mg / L.

5. The method according to claim 4, characterized in that The second culture medium further comprises agar and sucrose, wherein the concentration of agar is 5 g / L-10 g / L, and the concentration of sucrose is 25 g / L-35 g / L.

6. The method according to claim 1, 2, 4 or 5, characterized in that: Before the apical buds are induced to generate axillary buds, a first disinfection treatment is performed, and the first disinfection treatment includes: soaking in a carbendazim solution.

7. The method according to claim 6, characterized in that The second disinfection treatment is performed after the first disinfection treatment. The second disinfection treatment includes culturing the terminal buds using a third culture medium containing 6-benzylaminopurine, indolebutyric acid and a preservative.

8. The method according to claim 7, characterized in that The concentration of 6-benzylaminopurine is 0.02 mg / L-0.03 mg / L; and / or the concentration of indolebutyric acid is 0.04 mg / L-0.06 mg / L; and / or the mass fraction of the preservative is 0.05%-0.25%.

9. The method according to claim 7 or 8, characterized in that: The second disinfection treatment also includes: pre-treatment before culturing the apical buds using the third culture medium, the pre-treatment including: soaking the apical buds in a preservative.

10. The method according to claim 7 or 8, characterized in that: The third culture medium also contains agar and sucrose; the concentration of agar is 5g / L-10g / L, and the concentration of sucrose is 25g / L-35g / L.

Images