Tissue culture and rapid propagation method of lonicera caerulea

Abstract

The invention discloses a tissue culture and rapid propagation method of lonicera caerulea, and belongs to the technical field of tissue culture. The method comprises the following steps: S1, obtaining an explant; s2, carrying out callus culture; s3, carrying out induction culture; s4, rooting culture; s5, transplanting and domesticating tissue culture seedlings. According to the method, a tissue culture and rapid propagation system of the lonicera caerulea is successfully constructed by improving a culture medium formula and optimizing growth conditions, the propagation coefficient is increased, browning of the lonicera caerulea in the induction process is avoided, a reference basis is provided for a vegetative propagation technology of the lonicera caerulea, and a new reference way is provided for seedling resource shortage and breeding limitation of the lonicera caerulea.

Description

Technical Field

[0001] The present invention belongs to the technical field of tissue culture, and particularly relates to a method for rapid propagation of Lonicera caerulea by tissue culture. Background Art

[0002] Lonicera caerulea L., also known as mountain eggplant, black blind fruit, etc., is a perennial deciduous shrub of the genus Lonicera in the Caprifoliaceae family. It is a small berry unique to cold regions, with high nutritional and medicinal values. It is mainly distributed in the Greater and Lesser Khingan Mountains and Changbai Mountains in China. Currently, artificial cross-breeding is mainly used, and traditional propagation methods include seed propagation and cutting propagation. However, there are problems such as slow propagation speed, low survival rate, and large variability, which limit the development of excellent varieties. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a method for rapid propagation of Lonicera caerulea by tissue culture to solve the technical problems of low tissue culture propagation efficiency and low survival rate of Lonicera caerulea.

[0004] To achieve the above object, the technical solution adopted by the present invention is: to provide a method for rapid propagation of Lonicera caerulea by tissue culture, including the following steps:

[0005] S1. Obtaining explants: Inoculate the disinfected explants into the basal medium, and perform light culture for 10 - 14 h every day under a light intensity of 1900 - 2100 lx. The culture temperature is 24 - 26 °C, and the culture time is 7 - 14 d;

[0006] S2. Callus culture: Inoculate the explants obtained in S1 into the callus medium for callus culture. Perform light culture for 10 - 14 h every day under a light intensity of 1900 - 2100 lx. The culture temperature is 24 - 26 °C, and the culture time is 15 - 20 d;

[0007] S3. Induction culture: Inoculate the callus obtained in S2 into the induction medium for cluster bud induction culture, and perform dark culture at 21 - 23 °C for 19 - 21 d;

[0008] S4. Rooting culture: When the cluster buds obtained by induction culture grow to 2 - 3 cm, cut them from the base and transfer them to the rooting medium in sections for rooting culture to obtain tissue culture seedlings;

[0009] S5. Transplanting and acclimatizing the tissue culture seedlings.

[0010] Based on the above technical solution, the present invention can also be improved as follows:

[0011] Further, the disinfection treatment is as follows: Take shoot tips or axillary buds with a length of 2 - 3 cm, wash the surface attachments with clean water, disinfect them with 70 - 80% ethanol for 50 - 70 s in a laminar flow hood, then wash them 2 - 3 times with sterile water, and then disinfect them with 1.5 - 2.5% sodium hypochlorite solution for 2 min, and rinse them 5 - 7 times with sterile water.

[0012] Further, the basal medium is MS, N6, B5, H, White or WPM medium.

[0013] Further, the composition of the callus medium is: WPM + 6 - BA 2 mg·L -1 + 2,4 - D 1 mg·L -1 + sucrose 20 g / L.

[0014] Further, the composition of the induction medium is: WPM + thidiazuron (TDZ) 1 mg·L -1 + inositol 0.1 g·L -1 + sucrose 20 g / L.

[0015] Further, the composition of the rooting medium is: 1 / 2MS + indole - 3 - butyric acid (IBA) 1.0 mg·L -1 + sucrose 20 g / L.

[0016] Further, the rooting culture is carried out by first culturing in the dark at 20 - 25 °C for 3 - 5 d, and then culturing under a light intensity of 1900 - 2100 lx for 34 - 38 d until the main roots and lateral roots with a length > 7 cm grow out.

[0017] Further, S5 is specifically: Transplant the tissue - cultured seedlings into the sterile soil mixed by organic matter soil + vermiculite according to the mass ratio of 1 - 2:1 - 2, first culture in the dark for 2 - 4 d, then culture under light for 13 - 17 d, and then transplant the seedlings to the seedling shed and cover the soil for acclimation.

[0018] The beneficial effects of the present invention are as follows:

[0019] 1. The present invention can use different explants according to different culture seasons and can carry out artificial seedling cultivation throughout the year. Using NaClO 3 to sterilize the explants can ensure the disinfection effect while reducing the use of toxic reagents and reducing environmental pollution.

[0020] 2. By using the method of the present invention, tissue - cultured seedlings of Lonicera caerulea can be successfully obtained. By improving the medium formula and optimizing the growth conditions, a tissue - culture rapid propagation system of Lonicera caerulea is successfully constructed, increasing the multiplication coefficient, avoiding browning during the induction process of Lonicera caerulea, providing a reference basis for the asexual propagation technology of Lonicera caerulea, and providing a new reference way for the shortage of seedling resources and breeding limitations of Lonicera caerulea. Specific embodiments

[0021] The specific implementation manners of the present invention will be described below to facilitate those skilled in the art to understand the present invention. For those conditions not specified in the examples, they shall be carried out according to conventional conditions or the conditions recommended by the manufacturer. For those reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase. However, it should be clear that the present invention is not limited to the scope of the specific implementation manners. For those ordinary skilled in the art in the technical field, as long as various changes are within the spirit and scope of the present invention defined and determined by the appended claims, these changes are obvious, and all inventions and creations using the concept of the present invention are within the scope of protection.

[0022] The components of the culture medium used in the following examples are shown in Table 1.

[0023] Table 1 Components of the culture medium

[0024]

[0025]

[0026] Example

[0027] A tissue culture and rapid propagation method for Lonicera caerulea includes the following steps:

[0028] S1. Obtaining explants: In spring, the shoot tips of 2 - 3 cm can be directly collected as explants; during the dormant period, dormant branches (about 50 cm in length) can be collected. The bottom of the branches is immersed in water for 5 cm, and they are cultured under light for 12 h at 25 °C for 20 d. Finally, the axillary buds of 2 - 3 cm in water culture are taken as explants; the surface attachments are washed away with clean water, disinfected with 75% ethanol for 60 s in a super clean bench, then washed twice with sterile water, and then disinfected with 2% sodium hypochlorite solution for 2 min and rinsed 6 times with sterile water; the disinfected explants are inoculated into the basic medium, and cultured under light for 12 h every day at a light intensity of 2000 lx, the culture temperature is 25 °C, and the culture time is 10 d;

[0029] Lonicera caerulea is a perennial shrub (woody plant), and its metabolic pathways are different from those of herbs. Therefore, it is necessary to explore a suitable basic medium. By screening the common basic media MS, N6, B5, H, White, and WPM media, the results are shown in Table 2.

[0030] Table 2 Comparison of culture data of basic media

[0031]

[0032] The WPM medium is high in nitrate nitrogen, Ca, and K, and does not contain iodine and manganese (Table 1). It is suitable for the shoot tip culture of woody plants and can efficiently induce tissue culture seedlings of "Wulan". Moreover, (Table 2) it has a high multiplication coefficient, is not easily browned, and has a low cost, with remarkable effects, laying a foundation for the asexual propagation technology of blue honeysuckle.

[0033] S2. Callus culture;

[0034] The orthogonal design and variance analysis were used to study the effects of the types (6-BA and 2,4-D) and concentrations of hormones on the culture effect of "Wulan" callus. The results are shown in Table 3. Finally, the composition of the callus medium was determined as: WPM + 6-BA 2 mg·L -1 + 2,4-D 1 mg·L -1 + sucrose 20 g / L.

[0035] Table 3 Medium for inducing callus of "Wulan" tissue culture seedlings

[0036] Treatment group Medium components Proliferation coefficient M1 <![CDATA[WPM + 6 - BA (2 mg·L -1 ) + 2,4 - D (1 mg·L -1 ) + sucrose 20 g / L]]> 12±0.10b M2 <![CDATA[WPM + 6 - BA (2 mg·L -1 ) + 2,4 - D (2 mg·L -1 ) + sucrose 20 g / L]]> 10.0±0.53b M3 <![CDATA[WPM + 6 - BA(2mg·L -1 ) + 2,4 - D(3mg·L -1 ) + sucrose 20g / L]]> 9±0.17a M4 <![CDATA[WPM + TDZ (2 mg·L -1 ) + 2,4-D (1 mg·L -1 ) + sucrose 20 g / L]]> 1.6±0.69a M5 <![CDATA[WPM + TDZ (2 mg·L -1 ) + 2,4-D (2 mg·L -1 ) + Sucrose 20 g / L]]> 1.5±0.83b M6 <![CDATA[WPM + TDZ (2 mg·L -1 ) + 2,4-D (3 mg·L -1 ) + Sucrose 20 g / L]]> 3.4±0.28c

[0037] Note: Different letters indicate significant differences (P < 0.05). M1 - M3 are in one group, and M4 - M6 are in one group.

[0038] The explants obtained from S1 were inoculated into the callus medium (WPM + 6-BA 2 mg·L -1 + 2,4-D 1 mg·L -1 + sucrose 20 g / L) for callus culture. The light culture was carried out for 12 h every day under a light intensity of 2000 lx, the culture temperature was 25 °C, and the culture time was 18 d;

[0039] S3. Induction culture;

[0040] The orthogonal design and variance analysis were used to study the effects of the types (6-BA, TDZ, and inositol) and concentrations of hormones on the culture effect of "Wulan" tissue culture seedlings with multiple shoots. The results are shown in Table 4. Finally, the composition of the induction medium was determined as: WPM + TDZ 1 mg / L + inositol 0.1 g / L + sucrose 20 g / L, which is the best medium for inducing multiple shoots of "Wulan" tissue culture seedlings.

[0041] Table 4 Medium for inducing multiple shoots of "Wulan" tissue culture seedlings

[0042] Treatment group Medium components Proliferation coefficient W1 <![CDATA[WPM + TDZ (1 mg·L -1 ) + inositol (0.1 g·L -1 ) + sucrose 20 g / L]]> 1.2±0.00b W2 <![CDATA[WPM + TDZ (1 mg·L -1 ) + inositol (0.3 g·L -1 ) + sucrose 20 g / L]]> 1.0±0.33b W3 <![CDATA[WPM + TDZ (1 mg·L -1 ) + inositol (0.5 g·L -1 ) + sucrose 20 g / L]]> 5.6±0.67a W4 <![CDATA[WPM + 6 - BA (1 mg·L -1 ) + inositol (0.1 g·L -1 ) + sucrose 20 g / L]]> 12.6±0.67a W5 <![CDATA[WPM + 6 - BA (1 mg·L -1 ) + inositol (0.3 g·L -1 ) + sucrose 20 g / L]]> 7.5±0.33b W6 <![CDATA[WPM + 6 - BA (1 mg·L -1 ) + inositol (0.5 g·L -1 ) + sucrose 20 g / L]]> 4.4±0.58c

[0043] Note: Different letters indicate significant differences (P < 0.05). W1 - W3 are in one group, and W4 - W6 are in one group.

[0044] The callus obtained from S2 was inoculated into the induction medium (WPM + TDZ 1 mg·L -1+ Inositol 0.1 g·L -1 + Sucrose 20 g / L) for multiple shoot induction culture, and dark culture at 22 °C for 20 d;

[0045] S4. Rooting culture;

[0046] The types of hormones (IBA) and their concentrations affecting the rooting of "Wulan" tissue culture seedlings were studied by orthogonal design and variance analysis, and the results are shown in Table 5. Finally, the composition of the rooting medium was determined as: 1 / 2MS + IBA 1.0 mg / L, and sucrose 20 g / L was the best medium for inducing multiple shoots of "Wulan" tissue culture seedlings.

[0047] Table 5 Rooting medium for "Wulan" tissue culture seedlings

[0048] Treatment group Medium components Rooting rate (%) Number of roots (pieces) Root length (cm) N1 1 / 2MS + IBA (0.1 mg / L) + Sucrose 20 g / L 60.3±7.24b 5.0±0.00a 3.4±0.03b N2 1 / 2MS + IBA (0.5 mg / L) + Sucrose 20 g / L 45.2±1.67c 3.0±0.33b 1.2±0.03c N3 1 / 2MS + IBA (1.0 mg / L) + Sucrose 20 g / L 96.1±0.05a 5.0±0.00a 7.0±0.00a N4 1 / 3MS + IBA (0.1 mg / L) + Sucrose 40 g / L 33.6±2.98b 3.0±2.23b 3.6±0.03b N5 1 / 3MS + IBA (0.5 mg / L) + Sucrose 40 g / L 94.9±1.62a 4.0±0.58a 6.2±0.00a N6 1 / 3MS + IBA (1.0 mg / L) + Sucrose 40 g / L 46.3±4.41b 3.0±0.00b 2.1±0.07c

[0049] Note: Different letters indicate significant differences (P < 0.05). N1 - N3 are in one group, and N4 - N6 are in one group.

[0050] When the multiple shoots for induction culture grow to 2 - 3 cm, cut them from the base and transfer them to the rooting medium (1 / 2MS + IBA 1.0 mg·L -1 + Sucrose 20 g / L) for rooting culture. First, dark culture at 23 °C for 4 d, and then light culture at a light intensity of 2000 lx for 36 d until main roots and lateral roots with a length > 7 cm grow out to obtain tissue culture seedlings;

[0051] S5. Transplanting and acclimatization of tissue culture seedlings: Transplant the tissue culture seedlings into sterile soil (organic matter soil: vermiculite = 1:1, w:w, autoclaved), first dark culture for 3 d, then light culture for 15 d, and then transplant the seedlings to the seedling shed for soil covering and acclimatization. Organic matter soil can provide sufficient nutrients for tissue culture seedlings, and vermiculite can increase the air permeability of the soil, which is more conducive to the survival of tissue culture seedlings.

Claims

1. A method for rapid propagation of Lonicera caerulea by tissue culture, characterized in that: The following steps are involved: S1. Obtaining explants: inoculating the sterilized explants into a basal culture medium, and culturing them under a light intensity of 1900-2100 lx for 10-14 hours per day, a culture temperature of 24-26°C, and a culture time of 7-14 days; S2. Callus culture: inoculate the explant obtained in S1 into a callus culture medium for callus culture, and culture the callus under a light intensity of 1900-2100 lx for 10-14 hours per day, a culture temperature of 24-26°C, and a culture time of 15-20 days; S3, induction culture: the callus obtained in S2 is inoculated into the induction culture medium for cluster bud induction culture, and cultured in the dark at 21-23°C for 19-21 days; S4, rooting culture: when the induced buds grow to 2-3 cm, they are cut from the base and transferred to rooting medium in sections for rooting culture to obtain tissue culture seedlings; S5. Transplantation and domestication of tissue culture seedlings.

2. The method for tissue culture and rapid propagation of Lonicera caerulea according to claim 1, characterized in that: The disinfection treatment is as follows: take a stem tip or axillary bud with a length of 2-3 cm, wash off the surface attachments with clean water, disinfect with 70-80% ethanol for 50-70 seconds in a clean bench, then wash with sterile water for 2-3 times, then disinfect with 1.5-2.5% sodium hypochlorite solution for 2 minutes, and rinse with sterile water for 5-7 times.

3. The method for tissue culture and rapid propagation of Lonicera caerulea according to claim 1, characterized in that: The basic culture medium is MS, N6, B5, H, White or WPM culture medium.

4. The method for tissue culture and rapid propagation of Lonicera caerulea according to claim 1, characterized in that: The components of the callus culture medium are: WPM+6-BA2mg·L -1 +2,4-D 1mg·L -1 + sucrose 20g / L.

5. The method for tissue culture and rapid propagation of Lonicera caerulea according to claim 1, characterized in that: The components of the induction medium are: WPM + 1 mg·L thidiazuron -1 + inositol 0.1 g·L -1 + sucrose 20g / L.

6. The method for tissue culture and rapid propagation of Lonicera caerulea according to claim 1, characterized in that: The composition of the rooting medium is: 1 / 2MS + indolebutyric acid 1.0 mg·L -1 + sucrose 20g / L.

7. The method for tissue culture and rapid propagation of Lonicera caerulea according to claim 1, characterized in that: The rooting culture is first dark culture at 20-25° C. for 3-5 days, and then light culture at a light intensity of 1900-2100 lx for 34-38 days until a main root and lateral roots with a length greater than 7 cm are grown.

8. The method for tissue culture and rapid propagation of Lonicera caerulea according to claim 1, characterized in that: The S5 is specifically as follows: transplanting the tissue culture seedlings into sterile soil mixed with organic soil and vermiculite in a mass ratio of 1-2:1-2, first culturing in the dark for 2-4 days, then culturing in the light for 13-17 days, and then transplanting the seedlings to the seedling shed and covering with soil for acclimatization.

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