Method for promoting induction and rapid expansion of tetraploid Lanzhou lily tissue culture bulbs

Abstract

The invention discloses a method for promoting induction and rapid expansion of tetraploid Lanzhou lily tissue culture bulbs, which is carried out by adopting a two-step method and specifically comprises the following steps: (1) taking scales of tetraploid Lanzhou lily as explants, and inoculating the explants to a bulb induction culture medium after disinfection treatment to obtain sterile bulblets; and (2) inoculating the bulblet into a bulblet expansion culture medium to obtain a bulblet expanded tissue culture seedling. The induction of the tetraploid Lanzhou lily tissue culture bulblet can be effectively promoted, the diameter increase multiple and the weight increase multiple of the bulblet can be improved, and a foundation is laid for establishment of a tetraploid Lanzhou lily tissue culture seedling bulb efficient culture system.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant tissue culture, and particularly relates to a method for promoting the induction and rapid expansion of tissue-cultured bulbs of tetraploid Lilium davidii var. unicolor. Background Art

[0002] Lilies (Lilium spp.) are perennial bulbous flowering plants of the genus Lilium in the family Liliaceae. In China, as early as in Luo Yuan's Erya Yi in the Song Dynasty, it was described that: "The small ones are like garlic, and the large ones are like bowls. Dozens of pieces are stacked together, shaped like a white lotus flower, which means it is composed of a hundred pieces." That is to say, the bulbs of lilies are small like garlic and large like bowls, surrounded by white scale layers, just like a white lotus flower hugging tightly together, with a hundred pieces combined, thus getting the name "lily". Its underground bulb is composed of a bulb disc and a bulb body, which has the functions of producing daughter bulbs, generating roots, absorbing and storing nutrients, and supporting; the stem is grass green and erect without hair; the leaves are scattered, and the upper leaves are often smaller than the middle leaves, linear or lanceolate, with a flat leaf margin, no petiole and stipule; the flowers are trumpet-shaped, relatively large, fragrant, mostly white, yellow, pink, etc., solitary or clustered at the top of the stem, with the top bent but not curled; the capsule is oblong, with edges, and contains many seeds inside. The flowering and fruiting periods are from June to October. Lilium davidii var. unicolor is widely welcomed by consumers in the market because it contains rich nutrients and has certain medicinal value, and has become the largest medicinal and edible lily in China.

[0003] Inducing polyploid Lilium davidii var. unicolor can make the flowers and bulbs of the plant larger, the scales thicker, and the stress resistance stronger. By establishing a tissue culture rapid propagation and small bulb expansion system for tetraploid Lilium davidii var. unicolor through plant tissue culture technology, while maintaining the original excellent traits, the bulbs can be further expanded, which is of great significance for shortening the cultivation time of commercial lily bulbs and meeting the production practice of lily bulbs. At present, there have been studies on the induction of tetraploid Lilium davidii var. unicolor, but there are few reports on the further utilization of tetraploid germplasm. In this experiment, tetraploid Lilium davidii var. unicolor bulbs were used as experimental materials to explore the effects of various factors on the induction and expansion of tissue-cultured small bulbs, in order to construct an efficient cultivation system for tetraploid Lilium davidii var. unicolor bulbs and promote the production practice of tetraploid Lilium davidii var. unicolor germplasm resources. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an efficient cultivation method for promoting the induction and rapid expansion of tissue-cultured bulbs of tetraploid Lilium davidii var. unicolor. It is hoped that the induction rate of small bulbs and the expansion effect of small bulbs of tetraploid Lilium davidii var. unicolor under tissue culture conditions can be improved preferably, so as to solve the problem of the survival rate of tissue-cultured seedlings after transplantation and achieve the purpose of effectively utilizing tetraploid Lilium davidii var. unicolor germplasm resources.

[0005] The method for promoting the induction and rapid expansion of tissue-cultured bulbs of tetraploid Lilium davidii var. unicolor of the present invention is carried out by a two-step method, and specifically includes the following steps;

[0006] A method for promoting the induction and rapid swelling of tissue-cultured bulbs of tetraploid Lilium davidii var. unicolor, comprising the following steps:

[0007] (1) Take the outermost scales of healthy and plump tetraploid Lilium davidii var. unicolor bulbs as explants, after disinfection treatment, inoculate them on the bulb induction medium for culture to obtain aseptic small bulbs.

[0008] (2) Inoculate the aseptic small bulbs on the bulb swelling medium for culture to obtain tissue-cultured seedlings of tetraploid Lilium davidii var. unicolor with swollen bulbs.

[0009] In the method for promoting the induction and rapid swelling of tissue-cultured bulbs of tetraploid Lilium davidii var. unicolor of the present invention, the bulb induction medium is: solid MS medium containing 6-BA 1.0 mg / L + NAA 0.1 mg / L + gallnut extract 100 mg / L + sucrose 30 g / L + coconut water (CW) 5.00%.

[0010] In the method for promoting the induction and rapid swelling of tissue-cultured bulbs of tetraploid Lilium davidii var. unicolor of the present invention, the small bulb swelling medium is: MS solid medium containing 2,4-D 0.02 mg / L + maleic hydrazide 0.04 mg / L + sucrose 30 g / L + 5% earthworm cast extract.

[0011] In the method for promoting the induction and rapid swelling of tissue-cultured bulbs of tetraploid Lilium davidii var. unicolor of the present invention, the culture conditions are: the pH value of the medium is 5.8 - 6.0, the agar addition amount is 7 g / L, the temperature of the incubator is 24 ± 2 °C, the light intensity is 2400 Lx, and the light duration is 16 h / d.

[0012] In the method for promoting the induction and rapid swelling of tissue-cultured bulbs of tetraploid Lilium davidii var. unicolor of the present invention, the specific steps of the disinfection treatment of the explants are as follows:

[0013] Select the outermost scales of healthy and plump tetraploid Lilium davidii var. unicolor bulbs as explants, disinfect them with 75% ethanol for 30 s and soak them in 20% sodium hypochlorite for 15 min. After disinfection, rinse them with sterile water 5 times, and inoculate the scales with the concave surface up on the bulb induction medium. Inoculate 3 scales in each bottle to induce the production of small bulbs.

[0014] The difference between the method for promoting the induction and rapid swelling of tissue-cultured bulbs of tetraploid Lilium davidii var. unicolor of the present invention and the prior art lies in:

[0015] The present invention explores the methods for tissue-culturing and inducing small bulbs and swelling small bulbs of tetraploid Lilium davidii var. unicolor, which can effectively improve the induction rate of small bulbs and the swelling effect of small bulbs under the tissue-culturing conditions of tetraploid Lilium davidii var. unicolor, thereby solving the problem of the survival rate of transplanted tissue-cultured seedlings and achieving the purpose of effective utilization of the germplasm resources of tetraploid Lilium davidii var. unicolor. Brief Description of the Drawings

[0016] Figure 1 This is a potted seedling of tetraploid Lilium davidii var. unicolor Salisb. of the present invention

[0017] Figure 2 This is a schematic diagram of the cultivation process of tetraploid Lilium davidii var. unicolor Salisb. in the method of the present invention; wherein: the small bulbs (A) induced from the scales of tetraploid Lilium davidii var. unicolor Salisb.; after being transferred to the bulb swelling MS medium and cultured for 30 h, the small bulbs swell into (B); after 60 d, the small bulbs swell into (C), and after 90 d, the small bulbs swell into (D). Detailed Embodiment

[0018] The method for inducing and rapidly swelling tissue-cultured bulbs of tetraploid Lilium davidii var. unicolor Salisb. of the present invention will be further described below with reference to the accompanying drawings.

[0019] The present invention selects plump seeds of potted seedlings of tetraploid Lilium davidii var. unicolor Salisb. induced by colchicine, takes the outermost healthy scales without diseases and pests, rinses the soil on the surface of the scales with running water, and then transfers them into a super clean bench for disinfection treatment. The specific method is as follows: disinfect with 75% ethanol for 30 s and soak in 20% sodium hypochlorite for 15 min in sequence, rinse with sterile water 5 times after disinfection, place the selected scales in a culture dish containing filter paper after high-pressure steam sterilization, after the filter paper absorbs the moisture on the surface of the scales, use forceps to inoculate the scales with the concave surface upward into the bulb induction medium. Inoculate 3 scales in each bottle to induce the production of small bulbs.

[0020] The bulb induction medium used is: a solid MS medium containing 6-BA 1.0 mg / L + NAA 0.1 mg / L + 100 mg / L of gallnut extract + 30 g / L of sucrose + 5.00% of coconut water (CW). The culture temperature is 24 ± 2 °C, the light intensity is 2400 Lx, and the light duration is 16 h / d. When inoculating, place the disinfected scales in a culture dish containing filter paper after high-pressure steam sterilization. After the filter paper absorbs the moisture on the surface of the scales, use forceps sterilized by high temperature with an alcohol lamp to inoculate the scales with the concave surface upward into the bulb induction medium in sequence, and inoculate 3 scales in each bottle. Count the contamination rate after 10 d and count the induction rate after 45 d. The calculation formulas are as follows:

[0021] Contamination rate (%) = number of inoculated scales contaminated / total number of inoculated scales × 100%

[0022] Induction rate (%) = number of inoculated scales from which small bulbs are induced / total number of non-contaminated inoculated scales

[0023] The small bulb swelling medium used was: MS solid medium containing 2,4-D 0.02 mg / L + maleic hydrazide 0.04 mg / L + sucrose 30 g / L + 5% earthworm cast extract. The culture temperature was 24 ± 2 °C, the light intensity was 2400 Lx, and the light duration was 16 h / d. The induced small bulbs were taken out from the bulb induction medium with forceps sterilized by high-temperature of alcohol lamp, and after subculturing and cutting off the growing leaves in a sterile petri dish, the small bulbs were inoculated into the small bulb swelling medium. After 90 d, the diameter swelling multiple and weight swelling multiple were counted. The calculation formulas are as follows:

[0024] Note: The bulb diameter was measured with a vernier caliper sterilized by high-pressure steam, and the bulb weight was weighed with an electronic balance sterilized by ultraviolet light in a laminar flow hood;

[0025] Diameter swelling multiple = (bulb diameter after swelling test - bulb diameter before swelling test) / bulb diameter before swelling test

[0026] Weight swelling multiple = (bulb weight after swelling test - bulb weight before swelling test) / bulb weight before swelling test

[0027] Note: The preparation method of 5% earthworm cast extract was: The air-dried earthworm cast (cow dung treated by earthworms) and distilled water were soaked at a mass ratio of 1:20 for 48 h, and after centrifugation and filtration, 5% earthworm cast extract was prepared.

[0028] The small bulbs induced from the scales of tetraploid Lilium davidii var. unicolor were as Figure 2 shown. Swelling occurred at the base of the scales after 9 d; the rudiment of small bulbs appeared after 24 d, the small bulbs developed completely after 30 d, and almost no small bulbs occurred after 45 d. Compared with the medium supplemented with NAA, 6-BA alone or their combination, in the medium containing gallnut extract and coconut milk, the inoculated scale contamination rate was low and the induction rate was high, achieving the effect of promoting small bulb induction and saving a certain amount of time for the subsequent swelling of small bulbs.

[0029] The small bulbs cultured on the bulb induction medium were cut off the leaves and then inoculated into the bulb swelling medium. After culturing for 30 d, the small bulbs began to grow larger (B); after culturing for 60 d, the small bulbs further swelled (C); after culturing for 90 d, they swelled to (D).

[0030] The comparative test of the bulb induction medium compared with other traditional formulas was as follows:

[0031]

[0032] Except for the differences listed in the table, other test conditions were the same.

[0033] As can be seen from the above table, in terms of the pollution rate, the scale pollution rates of Comparative Examples 1-5 are between 15.97% and 19.10%, and there is no significant difference among the treatments. The pollution rate of the present invention is the lowest, at 9.03%, which is significantly lower than that of each comparative example treatment. In terms of the induction rate, the highest induction rate of the present invention is 98.30%, which is significantly higher than that of Comparative Example 1, Comparative Example 3, and Comparative Example 5. In the formula of the present invention, by adding the extract of Chinese gallnut and coconut juice to the culture medium, the scale pollution rate is the lowest and the induction rate is the highest, which is more conducive to the induction of the scales of tetraploid Lilium davidii var. unicolor.

[0034] The beneficial effects of the bulb induction culture medium of the present invention are as follows: The extract of Chinese gallnut is a natural product extracted from Chinese gallnut, mainly containing components such as high-concentration tannic acid and gallic acid, which has a significant inhibitory effect on the fungi and bacteria carried by the plant body. As a natural additive, coconut juice is rich in active substances such as amino acids, vitamins, minerals, and hormones required for plant growth, and plays an important role in promoting plant cell division and embryogenesis. After the explant is disinfected, the bacteria and fungi carried often cannot be completely removed, resulting in the pollution of the culture medium. By adding the extract of Chinese gallnut and coconut juice to the culture medium, while inhibiting the scale pollution rate, the induction rate of small bulbs is increased, thus promoting the induction of the scales of tetraploid Lilium davidii var. unicolor.

[0035] The comparison test of the small bulb swelling culture medium with other formulas is as follows:

[0036]

[0037] Except for the differences listed in the table, other test conditions are the same.

[0038] As can be seen from the above table, in terms of the diameter swelling multiple, there are significant differences between the present invention and Comparative Example 1, Comparative Example 2, and Comparative Example 3. The value of the diameter swelling multiple of the formula of the present invention is the highest at 2.31 times, and it is increased by 14.36% - 35.88% compared with Comparative Examples 1-3. There is no significant difference between Comparative Example 1 and Comparative Example 3. In terms of the weight swelling multiple, the value of the formula of the present invention is the highest at 9.07 times, which is significantly higher than that of Comparative Examples 1-3, and it is increased by 35.78% - 39.75% compared with Comparative Examples 1-3. There is no significant difference among Comparative Example 1, Comparative Example 2, and Comparative Example 3.

[0039] The beneficial effects of the bulb swelling culture medium of the present invention are as follows. Vermicompost, as a natural organic fertilizer, is rich in nutrients such as organic matter and humic acid. In addition, vermicompost is also rich in various hormones such as gibberellin, auxin, and cytokinin, which thus affect the growth, differentiation, and metabolic activities of plant cells; maleic hydrazide is a temporary plant growth inhibitor that increases the absorption of nutrients by the underground part of the plant by inhibiting the growth of the above-ground part. Adding the two together to the small bulb swelling culture medium can promote the growth and development of the bulbs and nutrient accumulation, thus significantly increasing the diameter and weight of the bulbs.

[0040] In the present invention, the scales of tetraploid Lilium davidii var. unicolor are taken as explants, and after disinfection treatment, they are inoculated into the bulb induction culture medium. After sterile small bulbs are induced in 45 days, the small bulbs are taken out, divided into plants in a sterile petri dish, inoculated into the small bulb swelling culture medium and cultured for 90 days. Finally, tissue culture seedlings of tetraploid Lilium davidii var. unicolor with swollen bulbs are obtained, and the swelling multiples of the diameter and weight reach 2.31 and 9.07 times respectively. The present invention does not require the proliferation and rooting processes of traditional lily tissue culture rapid propagation, is efficient and time-saving, and saves costs. It has established an efficient culture system for the bulbs of tetraploid Lilium davidii var. unicolor tissue culture seedlings, which plays a promoting role in the production practice of tetraploid germplasm resources of Lilium davidii var. unicolor.

Claims

1. A method for promoting the induction and rapid swelling of tissue-cultured bulbs of tetraploid Lilium davidii var. unicolor, characterized in that: It is carried out by a two-step method, which specifically includes the following steps: S1: Take the outermost scale of a healthy and plump tetraploid Lilium davidii var. unicolor Salisb. bulb as the explant. After disinfection treatment, inoculate it on a bulb induction medium for culture to induce small bulbs. The bulb induction medium is a solid MS medium containing 6-BA 1.0 mg / L, NAA 0.1 mg / L, gallnut extract 100 mg / L, sucrose 30 g / L, and coconut juice 5.00 wt%. S2: Inoculate the small bulbs in a small bulb swelling medium for culture to obtain tissue culture seedlings of tetraploid Lilium davidii var. unicolor Salisb. with swollen bulbs. The small bulb swelling medium is: an MS solid medium containing 2,4-D 0.02 mg / L, maleic hydrazide 0.04 mg / L, sucrose 30 g / L, and 5% earthworm cast extract.

2. The method for promoting the induction and rapid swelling of tissue-cultured bulbs of Lilium davidii var. unicolor Salisb. cv. "Lanzhou" tetraploid according to claim 1, characterized in that: The culture conditions are that the pH value of the medium is 5.8 - 6.0, the agar addition amount is 7 g / L, the temperature of the incubator is 24 ± 2 °C, the light intensity is 2400 Lx, and the light duration is 16 h / d.

3. The method for promoting the induction and rapid enlargement of tissue-cultured bulbs of tetraploid Lilium davidii var. unicolor Salisb. according to claim 1, wherein: The disinfection method for tetraploid Lilium davidii var. unicolor Salisb. bulbs is: disinfect with 75% ethanol for 30 s, soak in 20% sodium hypochlorite for 15 min. After disinfection, rinse with sterile water 5 times, and inoculate the scales with the concave surface up on the bulb induction medium.

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