Tissue culture method of citral cinnamomum bodinieri stem with axillary bud

Abstract

The invention discloses a tissue culture method of a citral type cinnamomum bodinieri stem with an axillary bud, and relates to the technical field of forest tree tissue culture. The tissue culture method comprises the following steps: selecting a current-year semi-lignified stem on a citral type cinnamomum bodinieri excellent single plant for disinfection treatment, and inoculating the stem on an axillary bud primary induction culture medium for axillary bud primary induction culture; after stem axillary buds in the axillary bud primary induction culture medium germinate, stem segments with axillary buds are soaked with a phycocyanobilin solution and then inoculated into the adventitious bud induction culture medium, red light irradiation treatment is conducted, conventional adventitious bud induction culture is conducted after red light irradiation, adventitious bud subculture multiplication culture, strong seedling culture and rooting induction culture are conducted, and the stem segments with the axillary buds are obtained. And finally, hardening seedlings and transplanting. According to the tissue culture method provided by the invention, the adventitious bud multiplication coefficient can reach more than 5, the rooting rate reaches up to 90.00%, the average rooting number also reaches 3.44, and the survival rate after two months is more than 85%. The invention provides support for better development and utilization of citral type Cinnamomum bodinieri, and has important significance for popularization and application of citral type Cinnamomum bodinieri.

Description

Technical Field

[0001] The present invention relates to the technical field of forest tissue culture, and particularly relates to a tissue culture method for the stem segments with axillary buds of Cinnamomum bodinieri var. citralum. Background Art

[0002] Cinnamomum bodinieri var. citralum refers to Cinnamomum bodinieri whose main component of leaf and branch essential oil is citral, belonging to the genus Cinnamomum of the Lauraceae family. Cinnamomum bodinieri var. citralum has a graceful tree shape, a large crown and dense shade, solid wood, luster and fragrance, and its branches and leaves can be used to extract essential oil. It is an important tree species for landscaping, timber and oil use. At present, the reproduction of Cinnamomum bodinieri var. citralum is mainly by seed reproduction, and there is less asexual reproduction of Cinnamomum bodinieri var. citralum. Studying the tissue culture method for the stem segments of Cinnamomum bodinieri var. citralum will provide a theoretical basis for the asexual reproduction of Cinnamomum bodinieri var. citralum, provide support for better development and utilization of Cinnamomum bodinieri var. citralum, and is of great significance for the popularization and application of Cinnamomum bodinieri var. citralum.

[0003] The prior art such as Patent No. CN110771505A discloses a tissue culture method for the stem segments of Cinnamomum bodinieri var. citralum. Specifically, in mid-May, the semi-lignified stem segment tissue of Cinnamomum bodinieri var. citralum is disinfected with 0.1% HgCl2 for 5 minutes, and the axillary bud germination rate of the stem segments is the highest, and the browning rate and contamination rate are relatively low. The suitable medium for the germination of the stem segments of Cinnamomum bodinieri is MS + 1.0 mg / L 6-BA + 0.05 mg / L IBA, and the germination rate is 60%. The suitable medium for proliferation is MS + 1.0 mg / L 6-BA + 0.2 mg / L IBA, the proliferation coefficient is 4.33, the plant height is 3.67 cm, and the ground diameter is 1.04 mm. Adventitious roots appear at the base after 11 days of rooting culture, and the suitable medium for rooting is 1 / 2MS + 1.5 mg / L IBA, the rooting rate is 75.00%, the root length is 5.06 cm, the number of roots is 3.50, and the root diameter is 0.82 mm. For the tissue culture seedlings with more than 3 roots, the survival rate of transplantation is more than 80%. In order to further optimize the tissue culture method for the stem segments of Cinnamomum bodinieri var. citralum and improve its transplantation survival rate, the present invention is thus proposed. Summary of the Invention

[0004] The purpose of the present invention is to propose a tissue culture method for the stem segments with axillary buds of Cinnamomum bodinieri var. citralum to overcome the above technical problems.

[0005] The present invention is realized through the following technical solutions. The present invention includes the following steps:

[0006] The present invention provides a tissue culture method for the stem segments with axillary buds of Cinnamomum bodinieri var. citralum, including the following steps:

[0007] (1) Pretreatment of explants

[0008] Select the current-year semi-lignified stem segments from the excellent individual plants of Cinnamomum bodinieri var. chenii with citral type. After trimming, cleaning, and disinfection treatments, divide them into small segments with axillary buds as explants;

[0009] (2) Initial induction culture of axillary buds

[0010] Inoculate the explants on the initial induction medium for axillary buds. The initial induction medium for axillary buds is: MS + 2.0 mg·L -1 6-benzylaminopurine + 0.05 - 0.1 mg·L -1 28-homobrassinolide + 0.2 - 1.0 mg·L -1 α-naphthaleneacetic acid;

[0011] (3) Induction culture of adventitious buds

[0012] After the axillary buds of the stem segments in the initial induction medium for axillary buds germinate, first dip the stem segments with axillary buds in the phycocyanin solution, then inoculate them into the adventitious bud induction medium, and perform red light irradiation treatment. After the red light irradiation treatment, perform conventional adventitious bud induction culture;

[0013] (4) Subculture proliferation culture and strong seedling culture of adventitious buds

[0014] Inoculate the adventitious bud clusters obtained by adventitious bud induction culture into the subculture proliferation medium for culture. Subculture at least 5 times to obtain cluster bud clusters. Divide the cluster bud clusters into bud clusters of uniform size and inoculate them into the strong seedling medium for culture to obtain tissue culture seedlings;

[0015] (6) Rooting induction culture, acclimatization and transplantation

[0016] Cut the tissue culture seedlings in the adventitious bud clusters obtained by strong seedling culture and put them into the rooting medium for culture to obtain rooted tissue culture seedlings, and then acclimatize and transplant the rooted tissue culture seedlings.

[0017] As a further optimized scheme of the present invention, in step (1), the disinfection treatment steps are: first treat the stem segments with 75% (v / v) alcohol for 45 s, then treat the stem segments with 0.1% (w / v) mercuric chloride reagent for 6 min, then treat the stem segments with 10% (w / v) hydrochloric acid solution for 1 min, and finally rinse the stem segments with sterile water 5 - 6 times.

[0018] As a further optimized scheme of the present invention, in step (3), the adventitious bud induction medium is MS + 0.5 - 1.5 mg·L -1 6-benzylaminopurine + 0.05 - 0.15 mg·L -1 α-naphthaleneacetic acid.

[0019] As a further optimization scheme of the present invention, the specific process of step (3) includes: after the axillary buds on the stem segments in the axillary bud initial induction medium germinate, remove the leaves on the stem segments, use a phycocyanin solution with a concentration of 0.01 mg / L as the dipping solution, evenly dip the stem segments with axillary buds in the dipping solution and then inoculate them into the adventitious bud induction medium. First, treat the stem segments with axillary buds with red light with a wavelength of 615 - 630 nm and a light intensity of 2500 Lux for 12 h, and then carry out the conventional adventitious bud induction culture.

[0020] As a further optimization scheme of the present invention, in step (3), the subculture proliferation medium is MS + 0.5 - 1.5 mg·L -1 6-benzylaminopurine + 0.05 - 0.15 mg·L -1 naphthaleneacetic acid + 0.10 - 3.0 mg·L -1 silver nitrate.

[0021] As a further optimization scheme of the present invention, in step (4), the seedling strengthening medium is MS + 0.5 mg·L -1 6-benzylaminopurine + 0.05 mg·L -1 naphthaleneacetic acid.

[0022] As a further optimization scheme of the present invention, in step (4), the rooting medium is 1 / 3MS + 0 - 1.0 mg·L -1 naphthaleneacetic acid + 0 - 1.0 mg·L -1 indolebutyric acid.

[0023] As a further optimization scheme of the present invention, in steps (2) to (4), the culture conditions for axillary bud initial induction culture, adventitious bud subculture proliferation culture, seedling strengthening culture and rooting induction culture are all: temperature 25 ± 2 °C, light intensity 2500 Lux, white light, light time 14 h / d.

[0024] As a further optimization scheme of the present invention, in step (7), at the time of transplanting, take out the rooted tissue culture seedlings, wash the culture medium on the roots with clear water, select the rooted tissue culture seedlings with good growth, having more than 3 branched roots and a root length of 1 - 2 cm, dip them in an 800 - 1000 - fold solution of 60% chlorothalonil wettable powder, and drain them to the seedling raising bed for transplanting.

[0025] As a further optimization scheme of the present invention, in step (7), the transplanting substrate uses peat soil and perlite with a mass ratio of 2:1, and the transplanting substrate is thoroughly watered with root - fixing water to keep it moist.

[0026] The present invention has the following advantages compared with the prior art:

[0027] In the present invention, the current-year semi-lignified stem branches of excellent individuals of Cinnamomum bodinieri var. chenii with citral type are selected. After trimming and disinfection, they are first inoculated into the initial induction culture for axillary bud induction. Through the component design of the initial induction culture of axillary buds, the germination rate is increased to 38.67% or more, and the germination time of axillary buds is shortened. When the axillary buds on the stem segments in the initial induction medium of axillary buds germinate to 2 - 3 cm, they are processed into stem segments with axillary buds and inoculated into the adventitious bud induction medium for adventitious bud induction culture. By optimizing the conditions for adventitious bud induction culture, the induction rate and quantity of adventitious buds are increased, and the growth state of adventitious buds is improved. Then, the obtained adventitious bud clusters are successively subjected to adventitious bud subculture proliferation culture, strong seedling culture, rooting induction culture, acclimatization and transplantation, completing the induction culture of the stem segments of Cinnamomum bodinieri var. chenii with citral type. In the culture method provided by the present invention, the adventitious bud proliferation coefficient can reach more than 5, the rooting rate is as high as 90.00%, the average number of roots also reaches 3.44, and the survival rate after two months is above 85%. The present invention provides support for better development and utilization of Cinnamomum bodinieri var. chenii with citral type, and has important significance for the popularization and application of Cinnamomum bodinieri var. chenii with citral type. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the collection and treatment of test materials provided by the present invention; in the figure, A is an excellent individual of Cinnamomum bodinieri var. chenii with citral type; B is the current-year semi-lignified branch; C is the stem segment with axillary buds without cleaning and sterilization; D is the stem segment with axillary buds after cleaning and sterilization;

[0029] Figure 2 Germination of axillary buds on stem segments under different combinations of growth regulators provided by the present invention; in the figure, A - I respectively correspond to treatments A - 1 to A - 9, and J corresponds to the blank control group;

[0030] Figure 3 Induction process of adventitious buds provided by the present invention; in the figure, A is the stem segment with axillary buds; B is the germination of axillary buds; C is the formation of adventitious buds; D is the growth of adventitious buds;

[0031] Figure 4 Effect of the composition and culture conditions of the adventitious bud subculture proliferation medium on adventitious bud subculture proliferation during the fifth subculture provided by the present invention; in the figure, A - I respectively correspond to treatments C - 1 to C - 9;

[0032] Figure 5 Effect of different subculture times on adventitious bud proliferation provided by the present invention;

[0033] Figure 6 Observation of adventitious buds in different subculture times provided by the present invention; in the figure, A is the growth of adventitious buds when the subculture times ≤ 3 times (subculture times ≤ 3 times); B is the growth of adventitious buds when 3 < subculture times ≤ 7 times;

[0034] Figure 7Growth of adventitious buds under different concentrations of AgNO₃ provided by the present invention; in the figure, A is 0 mg / L; B is 0.1 mg / L; C is 0.2 mg / L; D is 0.5 mg / L; E is the deformed tissue culture seedlings under the condition of high concentration (>1 mg / L) of AgNO₃;

[0035] Figure 8 Result of strong seedling culture of adventitious buds under treatment E-2 provided by the present invention; in the figure, A-B is before strong seedling; C-D is after strong seedling;

[0036] Figure 9 Adventitious root formation under different combinations of growth regulators provided by the present invention; in the figure, A-I respectively correspond to treatments F-1 to F-9;

[0037] Figure 10 Schematic diagram of transplanting regenerated plants of Cinnamomum bodinieri var. austrosinense with citral type; in the figure, A is 0 month after transplanting; B is 2 months after transplanting; C is 4 months after transplanting; D is 6 months after transplanting; E is 8 months after transplanting; F is 10 months after transplanting. Detailed implementation manners

[0038] The present invention will be further described in detail below with reference to the accompanying drawings. It is necessary to point out here that the following detailed implementation manners are only used to further illustrate the present invention and cannot be understood as limiting the protection scope of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention according to the above application content.

[0039] I. Reagents and materials

[0040] The following reagents and materials used are all commercially available products without special instructions.

[0041] The 28-HBL used in the present invention is 28-homobrassinolide, with the CAS number 74174-44-0; phycocyanin, with the CAS number 20298-86-6; 6-BA is 6-benzylaminopurine, with the CAS number 1214-39-7; NAA is naphthaleneacetic acid, with the CAS number 86-87-3; AgNO₃ is silver nitrate, with the CAS number 7761-88-8; IBA is indolebutyric acid, with the CAS number 133-32-4.

[0042] II. Methods

[0043] The following methods used are all conventional methods known to those skilled in the art without special instructions.

[0044] 1. Test materials

[0045] In September, on a sunny morning, select the current-year semi-lignified stem branches on excellent individual plants of Cinnamomum bodinieri var. lanceolatum with citral type as the initial test materials. After removing the leaves with scissors, cut them into small sections about 10 cm long. First, use a writing brush to dip the cleaning solution and brush the surface dirt, then wash off the cleaning solution with clean water, put them into an empty bottle, wrap the bottle mouth with gauze, place it under running water and rinse for about 2 h. Disinfect with 75% (v / v) alcohol for 45 s on a super clean bench, then treat with 0.1% (w / v) mercuric chloride reagent for 6 min. Pour out, add 10% (w / v) hydrochloric acid solution and shake for 1 min. Pour out, add sterile water and shake and rinse 5 - 6 times. Take out the disinfected materials with forceps and place them on the sterilized filter paper on the tray to absorb the moisture. Hold the stem segment with forceps in one hand and use a sterilized blade to cut off both ends of the stem segment and the petiole in the other hand, and divide them into small segments with 1 - 2 axillary buds as explants. The schematic diagram of the collection and treatment of test materials is as shown in Figure 1 shown.

[0046] 2. Initial induction culture of axillary buds

[0047] Inoculate the explants onto the initial induction medium for axillary buds. Use MS medium as the basic medium, and add 6-BA with a fixed concentration of 2.0 mg / L and different concentrations of 28-HBL (0, 0.05, 0.1 mg / L) and NAA (0.2, 0.5, 1.0 mg / L) respectively. Adopt a 3×3 completely randomized design and set a blank control (without adding growth regulators) to screen out the initial induction medium for axillary buds suitable for axillary bud induction. Inoculate 20 test tubes for each treatment, inoculate 1 stem segment in each test tube, and repeat 3 times. After culturing for 30 d, observe and record the number of buds sprouted and the growth status, and calculate the induction rate. The induction rate is calculated according to the following formula: Induction rate = (number of axillary buds germinated / number of inoculated explants) × 100%.

[0048] The culture conditions are: the temperature is controlled between 24℃ - 26℃, the light is 2500 Lux, white light, and the light time is 14 h / d.

[0049] Table 1. Effects of different combinations of growth regulators on the germination of axillary buds of stem segments

[0050]

[0051]

[0052] Note: Different lowercase letters in the same column indicate significant differences at the 0.05 level. The same applies to the following table.

[0053] As can be seen from Table 1, under the treatment conditions of all combinations of growth regulators, axillary buds can germinate, and the axillary bud germination rates among different treatments reach a significant difference level (p < 0.05). As can be seen from Table 1, the axillary bud germination rates under treatment A-3 and the blank control can reach 38.67% or more, which are significantly higher than other treatments.

[0054] Notably, for the axillary buds germinated under treatment A-3 and the blank control conditions, their leaves were in an undeveloped state, the axillary buds were short in length, and no secondary axillary buds were observed on the axillary buds. The axillary bud germination rates under treatment A-6 and treatment A-9 were at the lowest level, only 11.33%, which was significantly lower than other treatments. Under treatment A-4, the germination rate reached 32%, and the budding time was the earliest, at 6 days. The germinated axillary buds could reach 2.6 cm, and there were 4 secondary axillary buds on each germinated axillary bud on average ( Figure 2 ). Therefore, the axillary bud primary induction medium of treatment A-4 was selected for the axillary bud primary induction culture of explants.

[0055] 3. Adventitious bud induction culture

[0056] When the axillary buds on the stem segments in treatment A-4 germinated to 2 - 3 cm, the leaves were removed, and the stem branches with axillary buds were cut into stem segments with axillary buds of 0.5 cm in length and inoculated into the adventitious bud induction medium. The stem segments were flat on the surface of the medium. The culture conditions were a temperature of 24°C - 26°C, a light intensity of 2500 Lux, white light, and a light time of 14 h / d.

[0057] The adventitious bud induction medium was based on the MS medium, and different concentrations of 6-BA (0.5, 1.0, 1.5 mg / L) were randomly combined with NAA (0.05, 0.1, 0.15 mg / L) to screen out the best combination of plant growth regulators and their concentration ratios suitable for adventitious bud induction. 5 stem segments were inoculated into each culture bottle, 6 bottles were inoculated for each treatment, and repeated 3 times. Observation was carried out every 3 days, and the situation was recorded at the same time. After 30 days, the adventitious bud induction of the stem segments was counted. The adventitious bud induction rate was calculated according to the following formula: Adventitious bud induction rate = (Number of stem segments with induced adventitious buds / Number of inoculated stem segments) × 100%.

[0058] The results are shown in Table 2.

[0059] Table 2 Effects of different combinations of growth regulators on adventitious bud induction

[0060]

[0061]

[0062] The results showed that with the increase in the concentration of the growth regulator, the time for adventitious bud formation gradually shortened and finally remained unchanged, with the shortest time being 9 days. There were significant differences in the adventitious bud induction rate under different treatment conditions (p < 0.05). Except for treatment B-5, the adventitious bud induction rate increased with the increase in the concentration of the growth regulator, reaching a maximum of 66.66%. There were also significant differences in the number of adventitious buds produced under different treatment conditions (p < 0.05), with the range being 19.00 - 35.67. The treatment with the most adventitious buds was treatment B-4, with 35.67 adventitious buds. However, the lengths of the adventitious buds were uneven, and only some leaves were unfolded. There was no significant difference in the number of adventitious buds between treatment B-9 and treatment B-4, but the induction rate of treatment B-9 was the highest, and the growth status of its adventitious buds was consistent, with tall seedlings, thick stems, and unfolded leaves.

[0063] Furthermore, take the stem segments with axillary buds and use a phycocyanin solution with a concentration of 0.01 mg / L as the dipping solution. After evenly dipping the stem segments with axillary buds in the dipping solution, inoculate them into the adventitious bud induction medium (MS medium + 1.5 mg / L 6-BA + 0.15 mg / L NAA). The stem segments are flat against the surface of the medium. Under the temperature of 24°C - 26°C, first treat the stem segments with axillary buds with red light with a wavelength of 615 - 630 nm and a light intensity of 2500 Lux for 12 h, and then carry out conventional white light culture. The culture conditions are a temperature of 24°C - 26°C, a light intensity of 2500 Lux, and a light time of 14 h / d. Denote the above treatment as treatment B-10. The settings of this treatment are the same as above, that is, inoculate 5 stem segments in each culture bottle, a total of 6 bottles are inoculated, and repeat 3 times. Observe once every 3 days and record the situation at the same time. After 30 days, count the adventitious bud induction situation of the stem segments. The results showed that after dipping and red light treatment, the number of adventitious buds produced was 34.64, which was not significantly different from that of treatment B-9, but the induction rate was further increased compared with treatment B-9, being 73.33%, and the growth status of the adventitious buds was consistent, with tall seedlings, thick stems, and unfolded leaves ( Figure 3 ).

[0064] 4. Subculture and proliferation of adventitious buds

[0065] The adventitious bud clusters obtained from the treatment of B-10 were transferred to the subculture proliferation medium for multiple subculture proliferations. To explore the effects of the composition of different adventitious bud subculture proliferation media and culture conditions on the induction of adventitious buds from axillary bud-bearing stem segments. Using the MS medium as the basic medium, different concentrations of 6-BA (0.5, 1.0, 1.5 mg / L) and NAA (0.05, 0.1, 0.15 mg / L) were added, and a 3×3 completely randomized design was adopted. 5 adventitious bud clusters were inoculated into each culture bottle, 6 bottles were inoculated for each treatment, and the experiment was repeated 3 times. Observations were made every 3 days, and the situation was recorded simultaneously. After 30 days, the total number of adventitious buds was counted. The culture conditions were a temperature of 24°C - 26°C, a light intensity of 2500 Lux, white light, and a light duration of 14 h / d.

[0066] The results are shown in Table 3.

[0067] Table 3 Effects of different combinations of growth regulators on adventitious bud proliferation

[0068]

[0069]

[0070] Low concentrations of hormone conditions are not conducive to the proliferation of adventitious buds. Low concentrations of growth regulator conditions are not conducive to the proliferation of adventitious buds. Under the condition of the same concentration of NAA, as the concentration of 6-BA increases, the number of adventitious buds also increases. Except for the first subculture, the number of adventitious buds under the treatment of C-9 is the largest, and with the increase of the subculture times, the gap with other treatments becomes larger and larger. Under the treatment of C-9, after five subculture cultivations, the adventitious bud stems are thick and the leaf color is dark green, and the growth state is good ( Figure 4 ).

[0071] Furthermore, the treatment of C-9 was subcultured multiple times. From Figure 5 It can be seen that with the increase of the subculture times, the number of adventitious buds gradually increases, and starting from the 5th subculture, the number of adventitious buds exceeds 1500. The number of adventitious bud proliferations at the 7th subculture reaches 22011.48. With the increase of the base number, the number of adventitious buds shows a good exponential growth trend (R 2(>0.99). The proliferation coefficients of adventitious buds with different subculture times were studied, and it was found that the proliferation coefficients of adventitious buds varied greatly in the early stage, with a variation range of 1.11 - 3.96. Starting from the 6th subculture, the proliferation coefficients gradually stabilized and remained above 3.5. The adventitious bud clusters with different subculture times were observed, and it was found that adventitious buds mostly germinated at the part where the plant contacted the medium. In the early stage of subculture (the 1st - 3rd generation), adventitious buds mainly formed by direct occurrence, with only a small amount of callus appearing, and the number of bud primordia in the adventitious bud cluster was small. As the subculture continued to progress, in the later stage of subculture (after the 4th generation), the occurrence mode of adventitious buds became more diverse. In addition to direct occurrence, there was a large amount of callus in the adventitious bud cluster, and a large number of root primordia were contained in the callus. In addition, a small number of axillary buds were observed to germinate and form new branches. Figure 6 )

[0072] On the basis of the composition of the subculture proliferation medium given in treatment C - 9, AgNO3 was additionally added, and the concentration gradients of AgNO3 were set as 0, 0.1, 0.2, 0.5, 1, 2, 3 mg / L. After subculture for 7 times under the above subculture proliferation culture conditions, the effects of different concentrations of AgNO3 additives on the proliferation of adventitious buds were explored, and the results are shown in Table 4.

[0073] Table 4 Effects of AgNO3 on the proliferation of adventitious buds

[0074]

[0075] Under the treatment conditions of different concentrations of AgNO3, there were significant differences in the number of adventitious buds (p < 0.05), and AgNO3 could effectively promote the generation of adventitious buds. On the whole trend, the number of adventitious buds first increased and then decreased with the concentration of AgNO3, and high - concentration AgNO3 had an inhibitory effect on the growth of adventitious buds. When the concentration was 1 mg / L, the number of adventitious buds was the largest, up to 79 per bottle of medium on average, but the leaves of its adventitious buds were small and severely curled, with a brittle texture and poor growth state. The number of adventitious buds in the medium without adding AgNO3 was the least, only 44.67 per bottle of medium on average, and the growth state of adventitious buds was normal. Further research found that when the concentration of AgNO3 was higher than 0.5 mg / L, the growth state of adventitious buds began to show abnormalities, with curled leaves and even difficulty in normal leaf expansion, while low - concentration AgNO3 was more beneficial to the leaf expansion and leaf development of adventitious buds. Among them, under the treatment condition of 0.2 mg / L, the growth state of adventitious buds was the most normal, with not only good leaf expansion, but also a large number and large area of leaves. At the same time, there was no significant difference in the number of adventitious buds under this condition compared with the maximum value, but the proliferation coefficient was as high as 5.87, and the number of adventitious buds per bottle of medium was as high as 73.33. Figure 7 )

[0076] 5. Strengthening the seedlings of adventitious buds

[0077] The adventitious bud clusters subcultured and proliferated by D-3 were transferred to the strong seedling culture medium for strong seedling culture. Using MS as the basic medium, different concentrations of 6-BA (0.5, 1.0 mg / L) and NAA (0.05, 0.1, 0.15 mg / L) were randomly combined, and at the same time, the proliferation medium was used as the control to screen out the most suitable combination of plant growth regulators and concentration ratio for the strong seedling culture of Cinnamomum bodinieri Levl. adventitious bud clusters. 4 clusters were inoculated in each bottle, 5 bottles for each treatment, with 3 replicates. After 30 days, the number of effective buds (bud height > 2.5 cm) was counted. The conditions for strong seedling culture were temperature 24°C - 26°C, light intensity 2500 Lux, white light, and light time 14 h / d. The results are shown in Table 5.

[0078] Table 5 Effects of different growth regulator combinations on the strong seedlings of adventitious buds

[0079]

[0080] Before strong seedling culture, the adventitious bud clusters showed a dense and short state, with limited height growth, most of the leaves not unfolded, and even if unfolded, they were small and light green. The number of effective buds available for rooting was small (bud height > 2.5 cm), only 4 plants in each bottle, and the proportion of effective buds was only 7.34%. After strong seedling culture, the adventitious bud clusters changed significantly in morphology, with a significant increase in height, enhanced stem hardness, a significant increase in the number of unfolded leaves, and the leaves were wide, and the color changed from the original light green to dark green ( Figure 8 ). Further research found that there were significant differences in the number of effective buds under different treatment conditions (p < 0.05). Low concentrations of 6-BA promoted the height growth and leaf unfolding of tissue-cultured seedlings. Under the same condition of 6-BA, the number of effective buds in each bottle of medium increased with the decrease of NAA concentration. At the same time, the number of effective buds in each bottle of medium showed a trend of increasing first and then decreasing with the increase of NAA concentration, and 0.1 mg / L was the best concentration. In this study, the number of effective seedlings produced under treatment E-2 was the largest, and the proportion of effective buds in each bottle of medium was as high as 70.00% on average, showing a significant advantage compared with other treatment conditions, indicating that the strong seedling effect of this condition was the best. Therefore, the best medium formula for strong seedling culture was MS medium + 0.5 mg / L 6-BA + 0.1 mg / L NAA.

[0081] 6. Rooting

[0082] The tissue-cultured seedlings with a length of 3 - 4 cm were cut from the adventitious bud clusters strengthened by treatment E-2 and placed in the rooting medium. Using 1 / 3 MS medium as the basic medium, different ratios of NAA (0, 0.5, 1.0 mg / L) and IBA (0, 0.5,

[0083] 1.0 mg / L). Seven segments were inserted into each bottle, with five bottles for each treatment and replicated three times. After 30 days, the growth status was observed and the number of rooted plants was counted. The results are shown in Table 6.

[0084] Table 6 Effects of different combinations of growth regulators on adventitious root formation

[0085]

[0086] Under different treatment conditions, there were significant differences in the average rooting rate and the number of rooted plants of Cinnamomum bodinieri var. linaloolifera (p < 0.05). Among all treatments, treatment F-4 showed the best rooting effect, with an average rooting rate as high as 90.00% and an average number of rooted plants reaching 3.44; in sharp contrast, treatment F-3 had an average rooting rate of only 10.67% and an average number of rooted plants of only 1.77 (Table 6). Even without adding auxin growth regulators, there was still the phenomenon of rooting in the tissue-cultured seedlings of Cinnamomum bodinieri. Correlation analysis showed that the average rooting rate was negatively correlated with the starting time of rooting. The shorter the starting time of rooting, the higher the average rooting rate; at the same time, the rooting effect of single-type growth regulator treatment was significantly better than that of two combinations. Under the same concentration condition, the effect of adventitious root induction under the condition of NAA was better than that of IBA. The suitable medium for adventitious bud rooting of Cinnamomum bodinieri var. linaloolifera was 1 / 3MS + 0.5 mg / L NAA ( Figure 9 ).

[0087] 7. Acclimatization and transplantation

[0088] Thirty days after adventitious bud rooting of treatment F-2, the tissue-cultured seedlings with roots were taken out of the culture room with the bottles and acclimatized at room temperature for 3 - 5 days.

[0089] When transplanting, remove the cover and take out the rooted seedlings, and wash the root medium with clean water. Select seedlings with good growth, a plant height of about 2.5 cm, more than three branched roots, and a root length of 1 - 2 cm. After dipping them in 800 - 1000 times the liquid of 60% chlorothalonil wettable powder, drain them in a filter basin, cover them with a wet towel or wet newspaper, and then carry out transplantation. Using peat soil: perlite = 2:1 as the transplanting substrate, pour enough water to settle the roots, keep the substrate moist, cover it with a transparent plastic box to maintain the soil and air humidity, place the plug tray in the greenhouse. After two months, the survival rate was above 85%. Almost all the transplanted and survived seedlings could grow new leaves. The leaf color gradually changed from light green to dark green, the leaves were broad, the stem was thick and showed a reddish-brown color, and the growth was stronger. Figure 10 )

[0090] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent for the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention.

Claims

1. A tissue culture method for citral-type Cinnamomum camphora stem segments with axillary buds, characterized in that: The following steps are involved: (1) Explant pretreatment Select the semi-lignified stem segments of the current year from the superior individual plants of Cinnamomum camphora, which are pruned, cleaned and disinfected, and then divided into small segments with axillary buds as explants; (2) Initial induction culture of axillary buds The explants were inoculated on the axillary bud initial induction medium, which was MS + 2.0 mg·L -1 6-Benzylaminopurine + 0.05-0.1 mg·L -1 28-Homobrassinolide + 0.2-1.0mg·L -1 Naphthaleneacetic acid; (3) Adventitious bud induction culture After the axillary buds of the stem segments in the axillary bud initial induction medium germinate, the stem segments with axillary buds are first dipped in a phycocyanin solution, then inoculated into an adventitious bud induction medium, and irradiated with red light. After the red light irradiation, conventional adventitious bud induction culture is performed; (4) Adventitious bud subculture and seedling culture The adventitious bud masses obtained by adventitious bud induction culture are inoculated into a subculture proliferation medium for culture, and the subculture is repeated for at least 5 times to obtain clustered bud masses, and the clustered bud masses are divided into bud clusters of uniform size and inoculated into a seedling medium for culture to obtain tissue culture seedlings; (5) Rooting, hardening and transplanting The tissue culture seedlings in the adventitious bud clusters obtained through seedling cultivation are cut and placed in a rooting medium for cultivation to obtain rooted tissue culture seedlings, and then the rooted tissue culture seedlings are hardened and transplanted.

2. The tissue culture method of a citral-type Herba Cinnamomi stem segment with axillary buds according to claim 1, characterized in that: In step (1), the disinfection treatment step is: first treat the stem segment with 75% alcohol by volume for 45 seconds, then treat the stem segment with 0.1% mercuric chloride reagent by mass for 6 minutes, then treat the stem segment with 10% hydrochloric acid solution by mass for 1 minute, and finally rinse the stem segment with sterile water for 5-6 times.

3. The tissue culture method of a citral-type Herba Cinnamomi stem segment with axillary buds according to claim 1, characterized in that: In step (3), the adventitious bud induction medium is MS + 0.5-1.5 mg·L -1 6-Benzylaminopurine + 0.05-0.15 mg·L -1 Naphthaleneacetic acid.

4. The tissue culture method of a citral-type Herba Cinnamomi stem segment with axillary buds according to claim 1, characterized in that: The specific process of step (3) includes: after the axillary buds of the stem segments in the axillary bud initial induction culture medium germinate, the leaves on the stem segments are removed, a phycocyanin solution with a concentration of 0.01 mg / L is used as a dipping solution, the stem segments with axillary buds are evenly dipped in the dipping solution, and then inoculated into an adventitious bud induction culture medium, and the stem segments with axillary buds are first treated with red light with a wavelength of 615-630 nm and a light intensity of 2500 Lux for 12 hours, and then conventional adventitious bud induction culture is carried out.

5. The tissue culture method of a citral-type Herba Cinnamomi stem segment with axillary buds according to claim 1, characterized in that: In step (3), the secondary proliferation medium is MS + 0.5-1.5 mg·L -1 6-Benzylaminopurine + 0.05-0.15 mg·L -1 Naphthaleneacetic acid + 0.10-3.0mg·L -1 Silver nitrate.

6. The tissue culture method of a citral-type Herba Cinnamomi stem segment with axillary buds according to claim 1, characterized in that: In step (4), the seedling medium is MS + 0.5 mg·L -1 6-Benzylaminopurine + 0.05 mg·L -1 Naphthaleneacetic acid.

7. The tissue culture method of a citral-type Herba Cinnamomi stem segment with axillary buds according to claim 1, characterized in that: In step (4), the rooting medium is 1 / 3MS+0-1.0mg·L -1 Naphthaleneacetic acid + 0-1.0mg·L -1 Indolebutyric acid.

8. The tissue culture method of a citral-type Herba Cinnamomi stem segment with axillary buds according to claim 1, characterized in that: In steps (2) to (4), the culture conditions for the axillary bud initial induction culture, adventitious bud subculture proliferation culture, seedling culture and root induction culture are: temperature 25±2°C, light intensity of 2500Lux, white light, and lighting time of 14h / d.

9. The tissue culture method of a citral-type Herba Cinnamomi stem segment with axillary buds according to claim 1, characterized in that: In step (7), during the transplanting, the rooted tissue culture seedlings are taken out and the culture medium of the roots is washed with clean water. The rooted tissue culture seedlings with good growth, more than 3 branch roots and a root length of 1-2 cm are selected, and the seedlings are soaked with 800-1000 times diluted with 60% thiophanate-methyl wettable powder, and then drained to the nursery bed for transplanting.

10. The tissue culture method of a citral-type Herba Cinnamomi stem segment with axillary buds according to claim 1, characterized in that: In step (7), the transplanting medium is made of peat soil and perlite in a mass ratio of 2:1, and the transplanting medium is irrigated with rooting water to keep it moist.

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