Method for rapidly producing tissue culture seedlings of trichosanthes kirilowii

Abstract

The present invention belongs to the technical field of Trichosanthes kirilowii seedling production. Provided is a method for rapidly producing tissue culture seedlings of Trichosanthes kirilowii. The method for rapidly producing the tissue culture seedlings of Trichosanthes kirilowii comprises: S1, preparing a basic culture medium; S2, obtaining sterile seedlings; S3, performing proliferation inoculation of stem segments; S4, performing rooting inoculation of the stem segments; S5, culturing in a tissue culture room; and S6, processing test data. By means of producing Trichosanthes kirilowii seedlings by using indoor tissue culture, the problems in production of an imbalance in the male-to-female ratio and the reduction in yield when seeds are used for propagation and the problems of pests and diseases being spread and virus accumulation caused by using Trichosanthes kirilowii roots for propagation can be effectively solved.

Description

A method for rapidly producing Trichosanthes kirilowii tissue culture seedlings Technical Field

[0001] The invention relates to the technical field of trichosanthes kirilowii seedling production, and in particular to a method for rapidly producing trichosanthes kirilowii tissue culture seedlings. Background Art

[0002] Trichosanthes kirilowii is a plant of the Cucurbitaceae family, also known as Trichosanthes kirilowii, medicinal melon, hanging melon, wild gourd, etc. It is a perennial climbing herb and a perennial twining vine of the genus Trichosanthes of the Cucurbitaceae family. All its parts (fruit - whole Trichosanthes kirilowii, peel - Trichosanthes kirilowii peel, seeds - Trichosanthes kirilowii seeds, root tubers - Radix Trichosanthis) can be used as medicine. The whole Trichosanthes kirilowii has the effects of clearing heat and removing phlegm, relieving chest tightness and dispersing stagnation, and moistening and lubricating the intestines. It is used for lung heat cough, chest pain, constipation, etc.; Trichosanthes kirilowii peel has the effects of clearing heat and resolving phlegm, promoting qi and relieving chest tightness, and is used for phlegm heat cough, chest tightness and flank pain; Trichosanthes kirilowii seeds have the effects of moistening the lungs and resolving phlegm, moistening the intestines and relieving constipation, and are used for dry cough with sticky phlegm and dry intestinal constipation; Radix Trichosanthis kirilowii has the effects of clearing heat and purging fire, promoting body fluid and quenching thirst, and reducing swelling and discharging pus. It is used for fever, thirst, lung heat, etc.

[0003] Trichosanthes kirilowii is usually propagated by seed sowing and root tuber vegetative propagation. Seed propagation will cause a serious imbalance in the male-female ratio. There is no obvious difference between male and female plants during the seedling stage, making it difficult to distinguish, resulting in a large number of male plants being wasted, affecting yield. At the same time, seed propagation can easily lead to uneven seedlings, mixed germplasm, and variety degeneration. Root tuber vegetative propagation requires many underground tubers, and since the tubers are also medicinal materials, the reproduction coefficient is low. At the same time, root tuber propagation is prone to the spread of pests and diseases and the accumulation of viruses, especially root knot nematode disease and root rot, which can lead to serious deterioration of fruit yield and quality.

[0004] In response to the above problems, the inventors proposed a method for rapidly producing Trichosanthes kirilowii tissue culture seedlings to solve the above problems. Summary of the Invention

[0005] In order to solve the above problems, the present invention aims to provide a method for rapidly producing Trichosanthes kirilowii tissue culture seedlings.

[0006] To solve the above technical problems, the present invention adopts the following technical solution: a method for rapidly producing Trichosanthes kirilowii tissue culture seedlings, comprising the following steps:

[0007] S1. Preparation of basic culture medium: Use basic culture medium consisting of MS, 30 g / L sucrose, and 5 g / L agar. Adjust the pH to about 6.0 and dispense into tissue culture bottles. Sterilize in an autoclave at 121°C for 20 min and cool for later use.

[0008] S2. Obtaining sterile seedlings: Select healthy and healthy Trichosanthes kirilowii stem tips about 2 cm in diameter from the field. First, rinse the surface dust with tap water, then clean it with a detergent solution, and finally place it in a clean bench and disinfect it with different treatments of 75% alcohol and 0.1% mercuric chloride.

[0009] S3, stem segment proliferation inoculation: in the clean bench, cut the stem tip regeneration seedlings about 1 cm stem segments and inoculate them into the proliferation medium with different treatments;

[0010] S4, stem segment rooting inoculation: in the clean bench, cut the stem tip of the regenerated seedlings about 1 cm and inoculate them into the rooting medium with different treatments;

[0011] S5. Tissue culture room culture: After the stem segments have been multiplied and rooted, they are placed on a tissue culture rack for culture. The culture room is illuminated by fluorescent lamps and the culture temperature is controlled at 20-25°C. After 30 days of culture, the number of regenerated buds, plant height, rooting rate, root number, root length, and growth of regenerated seedlings are counted.

[0012] S6. Experimental data processing: Multiplication factor = total number of regenerated seedlings / total number of inoculated stem segments*100%; rooting rate = total number of rooted regenerated seedlings / total number of inoculated stem segments*100%. Experimental data were processed and analyzed using Excel and DPS software.

[0013] Preferably, in step S2, after disinfection with alcohol and mercuric chloride, the stem tip is washed 4-5 times with sterile water respectively, the disinfected stem tip is placed under a dissecting microscope to peel off the growth point, and the growth point of about 1 mm is cut and inoculated into the basic culture medium for culture.

[0014] Preferably, one cell is inoculated per bottle, 30 bottles are inoculated per treatment, and three replicates are performed. After culturing for 20 days, the contamination rate, mortality rate, and seedling rate are calculated.

[0015] Preferably, in step S3, 9 bottles are inoculated per treatment, 3 bottles are repeated for 3 times, and 4 stem segments are inoculated into each bottle.

[0016] Preferably, in step S4, 9 bottles are inoculated per treatment, 3 bottles are repeated for 3 times, and 4 stem segments are inoculated into each bottle.

[0017] Preferably, in step S5, the illumination intensity is 2500-3000 lx and the illumination time is 12 h / d.

[0018] Compared with existing technologies, the present invention has the beneficial effect of producing Trichosanthes kirilowii seedlings through indoor tissue culture, effectively solving the problems of seed propagation leading to an imbalanced male-female ratio and reduced yield, as well as the problem of using Trichosanthes kirilowii roots to propagate seedlings, which can lead to the spread of pests and diseases and the accumulation of viruses. Furthermore, tissue culture is not restricted by season, and tissue culture seedlings can be produced year-round, forming a factory-based seedling propagation and production system that ensures both quality and quantity to meet market demand for seedlings.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] FIG1 is a schematic diagram of the tissue culture operation structure of the present invention.

[0022] DETAILED DESCRIPTION

[0023] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0024] Embodiment: As shown in FIG1 , the present invention provides a method for rapidly producing Trichosanthes kirilowii tissue culture seedlings, comprising the following steps:

[0025] S1. Preparation of basic culture medium: Use basic culture medium consisting of MS, 30 g / L sucrose, and 5 g / L agar. Adjust the pH to about 6.0 and dispense into tissue culture bottles. Sterilize in an autoclave at 121°C for 20 min and cool for later use.

[0026] S2. Obtaining sterile seedlings: Select healthy, field-grown Trichosanthes kirilowii stem tips, approximately 2 cm in diameter. First, rinse the surface dust with tap water, then with a detergent solution. Finally, place the tips in a clean bench and disinfect them with different treatments of 75% alcohol and 0.1% mercuric chloride (see Table 1). After disinfection with alcohol and mercuric chloride, rinse them 4-5 times with sterile water. Place the disinfected tips under a dissecting microscope to remove the growing point. Cut the growing point, approximately 1 mm in diameter, and inoculate it into a basic culture medium for culture. Inoculate one seed per bottle, 30 bottles per treatment, and replicate three times. After 20 days of culture, calculate the contamination rate, mortality rate, and seedling rate.

[0027] S3. Stem segment proliferation inoculation: In a clean bench, cut stem segments of about 1 cm from the shoot tips of the regenerated seedlings and inoculate them into proliferation medium with different treatments (see Table 2). Inoculate 9 bottles per treatment, with 3 bottles as a replicate, 3 replicates, and 4 stem segments per bottle.

[0028] S4. Stem segment rooting inoculation: In a clean bench, cut the stem tip of the regenerated seedlings to about 1 cm and inoculate them into the rooting medium of different treatments (see Table 3). Each treatment was inoculated into 9 bottles, with 3 bottles as a replicate, 3 replicates, and 4 stem segments per bottle;

[0029] S5. Tissue culture room culture: After the stem segments have proliferated and rooted, they are placed on a tissue culture rack for culture. The culture room is illuminated by fluorescent lamps with a light intensity of 2500-3000 lx and a light duration of 12 h / d. The culture temperature is controlled at 20-25°C. After 30 days of culture, the number of regenerated buds, plant height, rooting rate, root number, root length, and growth of regenerated seedlings are counted.

[0030] S6. Experimental data processing: Multiplication factor = total number of regenerated seedlings / total number of inoculated stem segments*100%; rooting rate = total number of rooted regenerated seedlings / total number of inoculated stem segments*100%. Experimental data were processed and analyzed using Excel and DPS software.

[0031] Table 1 Effects of different disinfection times on in vitro culture of shoot tips

[0032]

[0033] Note: Different capital letters in the same column indicate significant differences at the 0.01 level.

[0034] As shown in Table 1, different disinfection time treatments had a very significant effect on the in vitro regeneration of shoot apex. When the 75% alcohol treatment time was constant, the shoot apex contamination rate gradually decreased and the mortality rate gradually increased with the extension of the 0.1% mercuric chloride treatment time. When the 0.1% mercuric chloride treatment time was constant, the shoot apex contamination rate and mortality rate did not change regularly with the extension of the 75% alcohol treatment time. The seedling rate of treatment T5 was the highest, at 83.8%, which was significantly different from the other treatments. Its contamination rate and mortality rate were relatively low, at 11.1% and 5.1%, respectively. Treatment T1 had the highest contamination rate and the lowest mortality rate, at 36.5% and 2.4%, respectively. Treatment T9 had the lowest contamination rate and the highest mortality rate, at 3.4% and 38.4%, respectively. Taking all factors into consideration, T5 (75% alcohol 40 s + 0.1% mercuric chloride 6 min) was the most suitable disinfection combination for shoot apex explants.

[0035] Table 2 Effects of different concentrations of 6-BA and NAA on proliferation

[0036]

[0037] As shown in Table 2, different concentrations of 6-BA and NAA combinations had an effect on the proliferation of regenerated seedlings. The proliferation times and plant height were significantly different from those of the control. When the 6-BA concentration was the same, the proliferation times first increased and then decreased with the increase of NAA concentration, and the plant height changed irregularly. High concentrations of NAA had a certain inhibitory effect on proliferation. When the NAA concentration was the same, the proliferation times first decreased and then increased with the increase of 6-BA concentration. High concentrations of 6-BA within the tested range promoted proliferation. The proliferation times and plant height of regenerated seedlings in treatment N8 were 5.42 and 2.12 cm, respectively. The stem nodes elongated normally and the leaves were light green. The proliferation times were significantly different from those of other treatments and the highest. The plant height was not significantly different from those of treatments N1, N2, N3, N4, N6, and N9. Therefore, the optimal hormone combination for proliferation of Trichosanthes kirilowii stem segments was 0.4 mg / L 6-BA and 0.06 mg / L NAA.

[0038] Table 3 Effects of different concentrations of NAA and IBA combinations on rooting

[0039]

[0040] As shown in Table 3, the addition of different concentrations of NAA and IBA can promote the rooting of Trichosanthes kirilowii to varying degrees, but the rooting rate did not reach 100%. Too low or too high concentrations inhibited the growth of regenerated roots. The overall rooting rate of NAA treatment was higher than that of IBA. Among them, A3 (NAA was 0.15 mg / L) had the highest rooting rate of 95.1%, but the growth quality of regenerated seedlings was weak. Some roots in A2 and A3 treatments became thicker and deformed, making them difficult to survive after transplantation. The regenerated roots in treatments with different concentrations of IBA grew normally, with more lateral roots and larger seedlings. Among them, B4 (IBA was 1.0 mg / L) had a relatively high rooting rate of 92.2%, with 5.3 roots, a root length of 7.6 cm, light green leaves, and strong seedlings. It was a more suitable rooting concentration for Trichosanthes kirilowii.

[0041] Through the above experimental method, it can be concluded that the explant disinfection combination is 75% alcohol for 40 seconds + 0.1% mercuric chloride for 6 minutes;

[0042] The most suitable hormone combination for stem segment proliferation was 0.4 mg / L 6-BA + 0.06 mg / L NAA;

[0043] The most suitable hormone combination for stem segment rooting was 1.0 mg / L IBA;

[0044] In the field, the stem tips of Trichosanthes kirilowii No. 9 and Trichosanthes kirilowii No. 20 varieties with healthy growth of about 2 cm were selected, and after disinfection, the growth points were peeled off and cultured to obtain regenerated seedlings. The regenerated seedlings were respectively inoculated into the above-mentioned culture medium with the most suitable combination of stem segment proliferation and rooting hormones, so that Trichosanthes kirilowii tissue culture seedlings could be quickly obtained.

[0045] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims, not the foregoing description, and all variations within the meaning and range of equivalents of the claims are intended to be included therein. Any reference sign in a claim should not be construed as limiting the claim to which it relates.

[0046] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A method for rapidly producing Trichosanthes kirilowii tissue culture seedlings, characterized in that: The method includes the following steps: S1. Preparation of basic culture medium: Use basic culture medium consisting of MS, 30 g / L sucrose, and 5 g / L agar. Adjust the pH to about 6.0 and dispense into tissue culture bottles. Sterilize in an autoclave at 121°C for 20 min and cool for later use. S2. Obtaining sterile seedlings: Select healthy and healthy Trichosanthes kirilowii stem tips about 2 cm in diameter from the field. First, rinse the surface dust with tap water, then clean it with a detergent solution, and finally place it in a clean bench and disinfect it with different treatments of 75% alcohol and 0.1% mercuric chloride. S3, stem segment proliferation inoculation: in the clean bench, cut the stem tip regeneration seedlings about 1 cm stem segments and inoculate them into the proliferation medium with different treatments; S4, stem segment rooting inoculation: in the clean bench, cut the stem tip of the regenerated seedlings about 1 cm and inoculate them into the rooting medium with different treatments; S5. Tissue culture room culture: After the stem segments have been multiplied and rooted, they are placed on a tissue culture rack for culture. The culture room is illuminated by fluorescent lamps and the culture temperature is controlled at 20-25°C. After 30 days of culture, the number of regenerated buds, plant height, rooting rate, root number, root length, and growth of regenerated seedlings are counted. S6. Experimental data processing: Multiplication factor = total number of regenerated seedlings / total number of inoculated stem segments*100%; rooting rate = total number of rooted regenerated seedlings / total number of inoculated stem segments*100%. Experimental data were processed and analyzed using Excel and DPS software.

2. A method for rapidly producing Trichosanthes kirilowii tissue culture seedlings according to claim 1, characterized in that: In step S2, the stem tip is disinfected with alcohol and mercuric chloride and then washed 4-5 times with sterile water respectively. The disinfected stem tip is placed under a dissecting microscope to peel off the growing point, and the growing point of about 1 mm is cut and inoculated into the basic culture medium for culture.

3. A method for rapidly producing Trichosanthes kirilowii tissue culture seedlings according to claim 2, characterized in that: One inoculation was performed in each bottle, 30 bottles were inoculated in each treatment, and three replicates were performed. After 20 days of culture, the contamination rate, mortality rate, and seedling rate were calculated.

4. A method for rapidly producing Trichosanthes kirilowii tissue culture seedlings according to claim 1, characterized in that: In step S3, 9 bottles were inoculated per treatment, 3 bottles were repeated for 3 times, and 4 stem segments were inoculated into each bottle.

5. A method for rapidly producing Trichosanthes kirilowii tissue culture seedlings according to claim 1, characterized in that: In step S4, 9 bottles were inoculated per treatment, 3 bottles were repeated for 3 times, and 4 stem segments were inoculated into each bottle.

6. A method for rapidly producing Trichosanthes kirilowii tissue culture seedlings according to claim 1, characterized in that: In step S5, the illumination intensity is 2500-3000 lx and the illumination time is 12 h / d.

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