A method for inducing adventitious roots from terminal buds of adult Masson pine trees

Abstract

The present invention belongs to the field of plant culture technology, and in particular to a method for inducing the growth of adventitious roots from the terminal buds of a Masson pine adult tree, the method comprising: (1) generating axillary buds by terminal bud induction culture; (2) generating adventitious roots by axillary bud induction culture; wherein, in step (1), a first culture medium is used for cultivation, and the first culture medium is a DCR culture medium containing 6-benzylaminopurine. The method does not need to be rejuvenated, saves steps and time compared with existing adult tree regeneration methods, and inducing axillary buds and breeding from the terminal buds, the bud induction rate and the single bud induction rate are high, and the growth is good; in addition, the adventitious root hairs obtained by the method are numerous, and the roots are thick and strong.

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. Characterized by rapid growth, high-quality wood, and strong resistance to adversity, it is an important afforestation and timber species in forestry production. However, traditional Masson pine propagation, primarily through seed propagation, is subject to significant genetic variation and segregation of offspring traits, making it difficult to meet the demand for high-quality germplasm resources in modern forestry.

[0003] Therefore, research on asexual propagation techniques for Masson pine, particularly inducing adventitious root growth from the terminal buds of mature trees, has important practical significance and application value. Due to the age effect of Masson pine, all known reports on regeneration of mature trees require the key step of rejuvenation, a process that requires at least six months 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. They 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 the growth of adventitious roots from the terminal buds of adult Masson pine trees. The method studies suitable methods for surface disinfection and sterilization of the 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, providing reliable theoretical support for the direct occurrence of terminal bud organs in adult Masson pine trees.

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

[0007] On the 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 removing the outer scales.

[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 naphthaleneacetic acid is 0.1 mg / L<naphthaleneacetic acid concentration<1 mg / L, and the concentration of indolebutyric acid is 0.1 mg / L<indolebutyric acid concentration<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 terminal bud induction culture generates axillary buds, and the first disinfection treatment comprises: 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 in 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 provided by the present invention for inducing the growth of adventitious roots from the terminal buds of adult Masson pine trees does not require rejuvenating treatment, thus saving steps and time compared to existing adult tree regeneration methods.

[0019] (2) The method for inducing the growth of adventitious roots from the terminal buds of adult Masson pine trees provided by the present invention has high bud induction rates and single bud induction rates in 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 produce a large number of adventitious roots that are thick and robust. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This study compares the sterilization treatment of terminal buds of adult Masson pine trees with and without the outer scales removed. A: Explants with the outer scales not removed; B: Explants with the outer scales not removed, sterilized, and inoculated on culture medium; C: Explants with the outer scales not removed, sterilized, and inoculated on culture medium for 2 months; D: Explants with the outer scales removed; E: Explants with the outer scales removed, sterilized, and inoculated on culture medium; F: Explants sterilized, inoculated, and incubated on culture medium for 2 months.

[0022] Figure 2 The figures are explants infected with bacteria again and explants that have been free of endophytes; A: explant infected with bacteria; B: explant infected with bacteria; C: explant treated with 0.2% PPM and grown normally for one month;

[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 state; B: 2mg / LTDZ induced axillary bud growth state; C: 2mg / LKT induced axillary bud growth state; D: 2mg / LZT induced axillary bud growth state;

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

[0026] The examples are provided to better illustrate the present invention, but are not intended to limit the present invention to the examples. Therefore, non-essential improvements and adjustments to the embodiments made by those skilled in the art based on the above-mentioned invention still fall within the scope of protection 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", "comprise" and the like 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] An 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; 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 (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 conditions are better than those 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 of the following conditions:

[0031] (a) In the first culture medium, the concentration of 6-benzylaminopurine is 0.25 mg / L to 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 to 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; 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;

[0034] (d) The terminal bud is the terminal bud after the outer scales are removed; specifically, the survival rate of the buds cultured 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 naphthaleneacetic acid and indolebutyric acid.

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

[0037] In some specific examples, the second culture medium further 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.

[0038] In some specific examples, a first disinfection treatment is performed before the apical bud induction culture generates axillary buds. 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 first disinfection treatment is followed by a second disinfection treatment, wherein the second disinfection treatment includes culturing the terminal 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 including: soaking the apical buds in a preservative.

[0043] In some specific examples, the third 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; the concentration of sucrose is 25g / L-35g / L, such as 27g / L, 29g / L, 32g / L or 34g / L.

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

[0045] In the following examples, 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, based on the actual growth of the explants, data on the sterilization effect of the terminal bud stem segment explant surface were collected 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; data on axillary bud induction and proliferation were recorded after 60 days of culture; and data on adventitious root induction were collected after 60 days of culture.

[0047] In the following example, DCR medium (without additives) is as follows: potassium dihydrogen phosphate 0.17 g, boric acid 6.2 mg, calcium chloride 0.085 g, zinc sulfate 8.6 mg, manganese sulfate 22.3 mg, potassium iodide 0.83 mg, sodium molybdate 0.25 mg, ferrous sulfate 27.8 mg, copper sulfate 0.25 mg, nickel chloride 0.025 mg, EDTA sodium salt 37.3 mg, vitamin B1 1.0 mg, cobalt chloride 0.025 mg, glycine 2.0 mg, vitamin B6 0.5 mg, inositol 200 mg, niacin 0.5 mg, glycine 2.0 mg, sucrose 30,000 mg, agar 6,000 mg, and magnesium sulfate 370 mg.

[0048] 1. Test Materials

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

[0050] 2. Disinfection

[0051] (1) 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 of outer scales ( Figure 1 D to Figure 1 F), the inoculated terminal buds were all about 0.5 cm long.

[0053] The first scheme retained the outer scales and included 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 Tween20 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 Tween20 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 Tween20 for 20 min (treatment ③).

[0054] The second scheme for removing outer scales includes three treatments: (1) soaking in 75% alcohol for 30 seconds, soaking in 40ml2% NaClO plus 1ml Tween20 for 25 minutes (treatment ④); (2) soaking in 500mg / L carbendazim solution for 5 minutes, soaking in 75% alcohol for 30 seconds, soaking in 40ml2% NaClO plus 1ml Tween20 for 25 minutes (treatment ⑤); (3) soaking in 500mg / L carbendazim solution for 10 minutes, soaking in 75% alcohol for 30 seconds, soaking in 40ml2% NaClO plus 1ml Tween20 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 minutes; 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 the results were 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: Values ​​are the mean ± standard error of three replicates, with 10 samples per group. For each variable, values ​​with the same superscript letter are not significantly different at the P>0.05 level, and 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 It can be seen 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 are completely contaminated.

[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 soaking 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] (2) 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 sterilization:

[0063] The first scheme: the explants of secondary bacterial or fungal infection were placed in Murashige and Skoog (MS) medium, 4% plant preservative mixture (PPM, Beijing Solebeau Technology Co., Ltd.) was added, and the culture was shaken and soaked for 4 hours without cleaning or removing the infected parts; the uninfected parts were inserted into DCR+6-BA0.025mg / L+IBA0.05mg / L medium without adding PPM (treatment ①), adding 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), and 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, with 2 replicates, for a total of 40 explants.

[0066] The explants treated with the above method 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 are roughly divided into two categories ( Figure 2 A, Figure 2 B). In addition, the contamination rate, browning rate and survival rate of the 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: Values ​​represent the mean ± standard error of two replicates, with 20 samples in each replicate. For each variable, values ​​with the same superscript letter are not significantly different at the P>0.05 level, and values ​​with different superscript letters are significantly different at the P<0.05 level.

[0070] As shown in Table 2 above, there were significant differences in contamination rates and survival rates among the different disinfection treatments, but no significant differences in browning rates (Table 2). Among all treatments, the highest contamination rate (90%) and the lowest survival rate (10%) were obtained when no PPM was added. Furthermore, when 0.25% PPM was added, the contamination rate was the lowest (25%) and the survival rate was the highest (75%). These results indicate that with increasing PPM concentration, the contamination rate of the pre-soaking treatments decreased while the survival rate increased. A comparative analysis revealed significant differences between the PPM-soaked treatments ⑤ and the unsoaked treatment ⑦. The contamination rate of treatment ⑦ was only 5% and the survival rate was 95%, while the contamination rate of treatment ⑤ was 35% and the survival rate was 65% (Table 2). This indicates that explants pre-soaked with PPM exhibited higher contamination rates and lower survival rates in the later stages 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 normally grown explants (uncontaminated explants) that had undergone primary and secondary surface disinfection. To evaluate the effect of 6-BA on axillary bud induction, explants with similar growth potential were placed on minimal medium (DCR was the minimal medium) 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 status of bud growth were recorded two months after inoculation. In addition, each medium was supplemented with 7 g / L agar, 30 g / L sucrose, and 0.1 mg / L IBA. Twenty explants were inoculated per treatment, with two replicates, for a total of 40 explants.

[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: 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 the P>0.05 level, and different superscript letters indicate a 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 buds of ① did not show robust growth; in contrast, ④ showed the highest number of individual buds and displayed a more vigorous growth pattern; significant differences were observed compared with the 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 the 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 was the basal culture media) supplemented with 2mg / L6-BA, 2mg / LTDZ, 2mg / LKT and 2mg / LZT, and the growth of the axillary buds was recorded. In addition, each culture medium was supplemented with 7g / L agar and 30g / L sucrose. 20 explants were inoculated for 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 ​​are the mean ± standard error of two replicates, each with 20 samples. For each variable, the same superscript letter indicates no significant difference at P>0.05.

[0082] Depend on Figure 4 As shown in Table 4, different cytokinins had no significant difference in the induction rate of axillary buds induced by needle bundles (Table 4), but had 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 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] Adventitious roots were induced using a two-factor completely randomized experimental design (Table 5). Single shoot tips with needle bundles were transferred to a minimal medium containing 1 / 4 DCR. Different concentrations of NAA and IBA were added to the rooting medium. In addition, 7 g / L agar and 15 g / L sucrose were also added to the rooting medium. Ten explants were inoculated per treatment, with three replicates, for a total of 30 explants. Rooting data were then 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 5As shown in Table 6, adventitious roots or fibrous roots were induced only in the five treatments (②, ④, ⑤, ⑥, and ⑧), and the growth of fibrous roots or lateral roots was different among the treatments. When NAA 0.5 mg / L was added, the number of rooted explants was significantly higher than that in the NAA 0.1 mg / L and NAA 1 mg / L treatments. Among them, the number of adventitious root explants induced in the NAA 0.5 mg / L + IBA 0.1 mg / 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 produce, and the higher the IBA concentration, the more roots each explant produces; the above results indicate 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 solutions of the present invention and are not limiting. 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 solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all 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 bud to generate axillary buds; (2) inducing the apical bud to generate adventitious roots; wherein, in step (1), a first culture medium is used for culture, and the first culture medium is DCR+0.25mg / L-2mg / L6-benzylaminopurine+0.05mg / L-0.15mg / L indolebutyric acid+5g / L-10g / L agar+25g / L-35g / L sucrose; the apical bud is the apical bud after removing the outer scales; in step (2), a second culture medium is used for culture, and the second culture medium is 1 / 4DCR culture medium+naphthaleneacetic acid+indolebutyric acid+7g / L agar+ 15 g / L sucrose, and the concentrations of naphthaleneacetic acid and indolebutyric acid are selected from any one of the following groups: (i) the concentration of naphthaleneacetic acid is 0.1 mg / L, and the concentration of indolebutyric acid is 0.5 mg / L; (ii) the concentration of naphthaleneacetic acid is 0.5 mg / L, and the concentration of indolebutyric acid is 0.1 mg / L; (iii) the concentration of naphthaleneacetic acid is 0.5 mg / L, and the concentration of indolebutyric acid is 0.5 mg / L; (iv) the concentration of naphthaleneacetic acid is 0.5 mg / L, and the concentration of indolebutyric acid is 1 mg / L; (v) the concentration of naphthaleneacetic acid is 1 mg / L, and the concentration of indolebutyric acid is 0.5 mg / L.

2. The method according to claim 1, characterized in that Before the terminal buds are induced and cultured to generate axillary buds, a first disinfection treatment is performed, which includes: soaking in a carbendazim solution.

3. The method according to claim 2, characterized in that The second disinfection treatment is performed after the first disinfection treatment, and the second disinfection treatment includes culturing the terminal bud using a third culture medium, and the third culture medium is DCR + 0.02mg / L-0.03mg / L 6-benzylaminopurine + 0.04mg / L-0.06mg / L indolebutyric acid + 0.05%-0.25% preservative + 5g / L-10g / L agar + 25g / L-35g / L sucrose.

4. The method according to claim 3, characterized in that The second disinfection treatment further includes: performing a pretreatment before culturing the terminal buds using the third culture medium, the pretreatment including: soaking the terminal buds in a preservative.

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