CN108450333B

CN108450333B - Induction method of lilium tigrinum callus

Info

Publication number: CN108450333B
Application number: CN201810343821.3A
Authority: CN
Inventors: 符勇耀; 杨利平; 秦密; 高海洪
Original assignee: Yangtze Normal University
Current assignee: Yangtze Normal University
Priority date: 2018-04-17
Filing date: 2018-04-17
Publication date: 2020-02-07
Anticipated expiration: 2038-04-17
Also published as: CN108450333A

Abstract

The invention discloses an induction method of lilium tigrinum callus, which comprises the following steps: inoculating the pretreated and sterilized bulblets or scales of the lilium tigrinum into an induction culture medium, wherein the illumination intensity is 700lx-900lx, and culturing for 20-30 days to obtain the calli of the lilium tigrinum, the induction culture medium is MS +1.00 mg/L6-BA +0.15mg/L NAA +0.20mg/L KT + 0.05-0.1 mg/L2, 4-D, and the pH value of the culture medium is 5.8-6.2. The levels of endogenous hormones in plants are adjusted together through the synergistic compatibility among the contents of the 6-BA, the NAA, the KT and the 2,4-D components and certain illumination, so that the lilium tigrinum is induced to generate callus, and the inductivity reaches 71%. The method can effectively induce the formation of the callus of the lilium tigrinum within 20 days, overcomes the problem that the callus is difficult to induce, and lays a foundation for asexual propagation, disease-resistant breeding, molecular biology research and the like of the lilium tigrinum.

Description

Induction method of lilium tigrinum callus

Technical Field

The invention belongs to the technical field of lily tissue culture, and particularly relates to an induction method of lilium tigrinum and lily callus.

Background

Lilium tigrinum (Lilium lancifolium Thunb.) is the oldest cultivar of Liliaceae (Lilium) Lilium (Lilium), and although the origin is not detailed, the distribution is extremely wide. Wild lilium tigrinum is mostly distributed in east asia, japan and korea. The distribution is extremely wide in China, the region from the noble plateau of the cloud in the southwest to the Changbai mountain in the northeast has the trail of the region, and the main distribution regions are the regions of Tibet, Qinghai, Gansu, Shanxi, Jiangsu, Zhejiang, Anhui, Hebei, Shandong, Jilin and the like. The lilium brownii is a good plant integrating medication, eating and appreciation. The medicinal value of the lilium brownii is reflected in the efficacies of clearing lung and relieving cough, clearing heart and tranquilizing and the like, and the lilium brownii is widely cultivated in the middle and lower reaches of Yangtze river and is a main source of the lilium brownii medicinal material. The lilium brownii (Yixing lily) is called three edible lily varieties of China, namely the Lanzhou lily and the Longya lily, wherein 8 kinds of essential amino acid contents of human bodies in the lilium brownii are 7.05 percent higher than that of the Lanzhou lily, and the total amino acid content of the lilium brownii is 11.24 percent higher than that of the Lanzhou lily; in the mineral elements, phosphorus and potassium are removed, and the contents of calcium, iron, copper, manganese, zinc, magnesium and selenium in the lilium brownii are higher than those of the lilium brownii; in the basic nutrient components, except starch, pectin and reducing sugar, the contents of protein, fat, crude fiber, Vc and total phospholipid in the lilium tigrinum are all higher than that of the lilium davidii. Therefore, the potential value of lilium tigrinum in low fat, high nutrition and good health care function is worthy of development. The lilium tigrinum has the advantages of large lily, rich color, low temperature and drought resistance, convenient pearl bud seeding and propagation and other excellent properties, can be directly applied to urban landscaping, family yards, pot plants and the like in gardens as a ground cover, and is one of bulbous flowers commonly applied to gardens. Therefore, the lilium brownii has good market value.

The disease resistance of lilium tigrinum is poor, wherein the diseases are commonly gray mold (Botrytis vertictica), leaf tip blight (Phoma lilii), anthracnose (Colletotrichum lili) and the like, and the common viruses comprise more than 20 viruses such as Cucumber Mosaic Virus (CMV), Lily Symptomless Virus (LSV), lily mottle virus (LMoV) and the like. The disease can lead to withering of the lilium tigrinum plants, the ornamental value is reduced, the quality of the bulbs is influenced, and the medicinal value is greatly reduced. Improvements to the above problems can be achieved by breeding methods. Common plant disease-resistant breeding means comprise systematic breeding, distant hybridization, biotechnology breeding and the like, and disease-resistant in-vitro selection and somatic clone variation combining biotechnology are effective means which are tried by researchers in recent years and progress is achieved. The method for disease-resistant breeding by somatic cell clone variation is characterized by that it does not adopt in vitro selection, and directly adopts the plant regenerated by tissue culture or transplanted seedling to make disease-resistant detection, so that it can greatly shorten breeding period compared with screening disease-resistant plant in field. Among them, the dedifferentiation and redifferentiation are easy to generate variation, so the somaclonal variation is usually cultured by using Callus (calli), which means that a plant grows new cells or tissues in a place with a wound after being subjected to self-repair after being wounded. Three stages, a priming stage, a dividing stage and a maturation stage, are passed from the explant to the formation of callus. The callus induction ability and the formation part of different plants are different; the ability to induce callus also varies from part to part of the same plant. There have been some reports on the tissue culture of lilium tigrinum, such as Guo seashore and Lei Jia Jun (2006), Masu Yun (2012), Yangli peace and Song Xiao hong (2013); however, all studies showed that the induction efficiency of Lilium tigrinum callus is extremely low, and it is particularly difficult to induce a large amount of callus (clump). In addition, the induction culture medium utilizing the existing common varieties of wild lily, Lanzhou lily and the like is not suitable for the lilium brownii, and the reason may be that the lilium brownii lily is a triploid variety, so that the explant cannot be effectively induced to generate callus, and most of the lilium brownii can only generate adventitious buds. Therefore, the method is very necessary for the directional culture of the lilium tigrinum callus and has important scientific significance.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a method for inducing lilium tigrinum callus, which mainly solves the problems that lilium tigrinum explant callus is difficult to induce and has high pollution rate.

In order to achieve the purpose, the invention adopts the following technical scheme: the induction method of the lilium tigrinum callus comprises the following steps:

inoculating the pretreated and sterilized scale or bulbil of Lilium tigrinum into an induction culture medium for primary culture at the illumination intensity of 700lx-900lx for 20-30 days to obtain the callus and sterile adventitious bud of Lilium tigrinum; the induction culture medium is MS +1.00 mg/L6-BA +0.15mg/L NAA +0.20mg/L KT + 0.05-0.1 mg/L2-4, and the pH value of the culture medium is 5.8-6.2.

Preferably, the scale is a middle layer or an outer layer of the bulb.

Preferably, the sterile adventitious bud base swelling part (hereinafter, referred to as sterile bulblet) can be transferred to the induction culture medium for continuous culture, the illumination intensity is 700lx-900lx, and the induction culture is carried out for 20-30 days, so as to obtain the calli of the lilium tigrinum.

Preferably, the conditions of the induction culture are that the culture temperature is 25 +/-2 ℃, the humidity is 60% -70%, and the illumination time is 12-14 h/d.

Preferably, the pretreatment is to soak the bulblets or scales of the lily with a detergent solution for 5-10min and then wash the bulblets or scales with running tap water for more than 2 h; the sterilization is that the pretreated bulblets or scales of the lilium tigrinum are placed on a super-clean workbench, ethanol water solution with the volume concentration of 70-75% is used for treating for 20-30 s, then sterile water is used for cleaning for at least 2 times, mercuric chloride solution with the mass concentration of 0.1-0.2% is used for sterilization for 10-25 min, and finally sterile water is used for washing for at least 4 times.

Preferably, the detergent is soap, washing powder or soapberry saponin.

Compared with the prior art, the invention has the following beneficial effects:

1. the MS culture medium is used as a basic culture medium, and exogenous plant hormones and growth regulating substances are supplemented to provide energy for the generation of lilium tigrinum callus, so that the growth of the lilium tigrinum callus can be accelerated, the requirement of lilium tigrinum on cell dedifferentiation in nutrition and physiology can be met, particularly, the endogenous hormone level of the lilium tigrinum can be jointly regulated through the synergistic compatibility of the 6-BA, NAA, KT, 2,4-D component content and certain weak light, so that the lilium tigrinum can be induced to generate callus, a large amount of callus can be obtained within 20 days, the induction rate reaches 71%, the technical problem that the callus is difficult to generate in the lilium tigrinum tissue culture process is solved, and the invention lays a foundation for asexual propagation, disease-resistant breeding and molecular biology research of the lilium tigrinum.

2. The explant adopted by the invention is the middle and outer layer scales of the bulbil and the bulb or the sterile bulblet generated after primary culture, and has the characteristics of easily obtained materials, simple and convenient operation and rapid propagation, wherein the pollution rate of the sterile bulblet subjected to primary culture in the induction culture process is far lower than that of other explants, only 2.5 percent, and the callus induction rate reaches 35 percent, thereby solving the technical problems of high pollution rate and difficult callus generation in the tissue culture process of the lilium tigrinum.

Drawings

FIG. 1 shows the morphological changes of the induced Lilium tigrinum bulbil;

FIG. A shows the pearl bud scale 2-3 days later, B shows the pearl bud scale 10 days later, C shows the pearl bud scale 2 weeks later, D shows the callus produced by the pearl bud scale 20-30 days later, and E shows the callus produced by the pearl bud scale beginning to die after 30 days later;

FIG. 2 shows callus tissue generated by inducing sterile bulblets under different lighting conditions;

panel A shows callus induced under the condition of 0-300lx illumination intensity; b is callus induced under the condition of the illumination intensity of 700-; c is callus induced under the condition of 1500-2000lx illumination.

Detailed Description

The present invention will be described in further detail with reference to the following specific embodiments and the accompanying drawings.

1. The instrument equipment comprises:

steam pressure sterilization pot, dry heat sterilization cabinet, filtration sterilization device, spray sterilizer, ultraviolet lamp, electronic balance, superclean bench, refrigerator, balance, induction cooker, culture flask, stainless steel inoculation dish, operation inoculation scissors, straight tweezers, gun type inoculation tweezers, scalpel handle and the like.

2. Preparing a reagent:

(1) 75% alcohol: 75ml of 95% alcohol is added with water to reach 95 ml.

(2) 0.1% (0.2%) mercuric chloride solution: 1 g (2 g) of HgCl was weighed₂Distilled water was added thereto to 1000 ml.

(3)1mol/LHCL solution: 8.25ml of concentrated hydrochloric acid is taken and added with distilled water to reach the constant volume of 100 ml.

(4)1mol/LNaOH solution: weigh 8g NaOH, and make up to 200ml with distilled water.

3. Preparing a culture medium:

minimal medium for tissue culture (MS):

NH per liter of medium₄NO₃1650mg，KNO₃1900mg，CaCl₂·2H₂O 440mg，MgSO₄·7H₂O370mg，KH₂PO₄170mg，KI 0.83mg，H₂BO₃6.2mg，MnSO₄·4H₂0 22.3mg，ZnSO₄·7H₂O 8.6mg，Na₂MoO₄·5H₂O 0.25mg，CuSO₄·5H₂O 0.025mg，CoCl₂·6H₂O 0.025mg，FeSO₄·7H₂O 27.8mg，Na₂-EDTA·2H₂O37.3 mg, inositol 100mg, nicotinic acid 0.5mg, glycine 2mg, pyridoxine hydrochloride (vitamin B6)0.5mg, thiamine hydrochloride (vitamin B1)0.4mg and sucrose 30 g.

And (3) pH adjustment: adding 1mol of HC1 or NaOH aqueous solution dropwise to adjust the pH value of the culture medium to 5.8-6.2.

Subpackaging the prepared culture medium into culture bottles, sterilizing the subpackaged culture medium at the high temperature of 121 ℃ by using a steam pressure sterilization pot, and storing the culture medium in a sterile environment for later use.

4. Plant growth hormone:

auxin type: naphthylacetic acid (1-Naphthylacetic acid, NAA), 2,4-Dichlorophenoxyacetic acid (2, 4-dichlorphenoxyacetic acid, 2, 4-D);

cytokinins: 6-Benzylaminopurine (6-Benzylaminopurine, 6-BA), 6-glycosylaminopurine (6-Furfurylaminopurine, KT).

5. And (3) observing and counting the callus tissues:

observing the material every 2-3 days to see the growth and development conditions of the material, such as the change of the color, the size and the like of the material, and counting the pollution rate, the budding rate and the callus induction rate of the material at 20 days.

Example 1

1) Pretreatment of lilium tigrinum bulbil

Cleaning dirt on the surfaces of the bulblets of the lilium tigrinum in tap water, picking out the bulblets with mechanical damage and plant diseases and insect pests on the surfaces, soaking the healthy bulblets in clear soap water for 7-8min, and washing the healthy bulblets with running tap water for more than 2 h.

2) Sterilization of lilium tigrinum bulbil

Poking the bulbil into a plurality of bulbil scales with uniform size by using a dissecting needle on an aseptic operation table, firstly cleaning and disinfecting for 30s by using 75% ethanol, cleaning for 2-3 times by using high-temperature sterilized water, pouring out, then adding 0.20% mercuric chloride solution (the mercuric chloride solution needs to submerge the bulbil scales), covering a container cover, putting on a shaking table, shaking for 10-15min, fully contacting the mercuric chloride solution with the bulbil scales to ensure thorough disinfection, then pouring the mercuric chloride solution on the aseptic operation table, adding high-temperature sterilized water, and cleaning for 4-6 times.

3) Induction of lilium tigrinum bulblet

The pearl bud scales after pretreatment and sterilization are respectively inoculated into induction culture media with different concentrations of 2,4-D (0.01, 0.05, 0.1 and 0.5mg/L), 5 pearl bud scales are respectively placed on each bottle of culture media, and 10 bottles of culture media are respectively placed in each concentration gradient. The induction culture conditions comprise that the culture temperature is 25 +/-2 ℃, the culture is 20 days, the humidity is 60% -70%, and the illumination condition is 1500lx-2000lx and 12-14 h/d.

Induction medium composition: MS +6-BA 1.00mg/L + NAA0.15mg/L + KT 0.20mg/L +2-4, D (0.01, 0.05, 0.1 or 0.5 mg/L).

The material is observed every 2-3 days to see the growth and development conditions, such as the change of color, size and the like of the material. As shown in FIG. 1, the appearance of the bud scales became brown 2-3 days after inoculation (FIG. A). After 1 week, the bulbar bud scale is enlarged, after 10 days, the raised cell mass or bud point appears gradually at the periphery of the bulbar bud scale (figure B), after 2 weeks, the callus and adventitious buds appear obviously (figure C), and after 20-30 days, the callus appears most (figure D), but after 30 days, the callus begins to die slowly, the amount of the adventitious buds begins to increase, the roots also begin to appear, which indicates that the callus begins to differentiate (figure E).

And counting the pollution rate, the germination rate and the callus induction rate of the material at 20 days. As shown in table 1.

TABLE 1 conditions for callus culture of Lily bulbifera by induction of Lilium tigrinum bud scales (Primary culture)

Example 2

1) Pretreatment of Lilium tigrinum and Bulbus Lilii scales

Cleaning soil and impurities on the surface of the lilium tigrinum seed ball under tap water, peeling off scales damaged by diseases and insect pests or damaged by other pests on the periphery of the bulb, taking the scales without mechanical damage, diseases and insect pests and health on the middle layer, soaking the scales with soapberry saponin solution with certain concentration for 5min, and then washing with flowing tap water for more than 2h for later use.

2) Sterilization of lilium tigrinum scales

Primarily disinfecting the cleaned scales with 70% ethanol for 30s on an aseptic operation table, cleaning with high-temperature sterilized water for 2-3 times, adding 0.10% mercuric chloride solution, soaking, covering the container, placing the container on a shaking table, shaking for 20-24min to make the scales fully contact with the mercuric chloride solution, taking out, pouring the mercuric chloride solution on the aseptic operation table, and cleaning with high-temperature sterilized water for 4-6 times.

3) Induction of Lilium tigrinum scales

The scales after pretreatment and sterilization are horizontally arrangedCutting into 1cm²The left and right small pieces were inoculated on induction media containing 2,4-D (0.01, 0.05, 0.1, 0.5mg/L) of different concentrations, 5 pieces of scale were placed on each bottle of culture medium, and 10 bottles of culture medium were placed on each concentration of culture medium. The induction culture conditions comprise that the culture temperature is 25 +/-2 ℃, the culture is 20 days, the humidity is 60% -70%, and the illumination condition is 1500lx-2000lx and 12-14 h/d.

And counting the pollution rate, the germination rate and the callus induction rate of the material at 20 days. As shown in table 2.

TABLE 2 Induction of Lilium tigrinum bulb Scale (Primary culture) callus culture

Example 3

1) Pretreatment of lilium tigrinum bulb dish

Cleaning soil and impurities on the surface of the lilac seed ball under tap water, cutting the bulb disc from the base part of the bulb by using a knife, taking a healthy base disc which is free of plant diseases and insect pests and is mechanically damaged, soaking the base disc in a soap water solution for 10min, cleaning the base disc by using clean water, and then, allowing the base disc to be cleaned under clean flowing water for more than 3 h.

2) Sterilization of lilium tigrinum bulb plates

The clean bulb dish is firstly disinfected by 75% ethanol for 1min on a sterile operating platform, washed by high-temperature sterilizing water for 2-3 times, then soaked by 0.20% mercury bichloride for 10-15min, and placed on a shaking table to shake continuously during the soaking process, so that the bulb base dish is fully soaked by the mercury bichloride to ensure that the disinfection is more thorough, and finally the disinfectant is poured out and then washed by the high-temperature sterilizing water for 4-6 times.

3) Induction of lilium tigrinum bulb

Cutting the pretreated and sterilized bulbil into 1cm²The left and right small pieces were inoculated into induction medium containing 2,4-D (0.01, 0.05, 0.1, 0.5mg/L) at different concentrations, and 5 bulbs were placed on each flask of the mediumAnd a basal disc, wherein 10 bottles of culture medium are placed in each concentration gradient. The induction culture conditions comprise that the culture temperature is 25 +/-2 ℃, the culture is 20 days, the humidity is 60% -70%, and the illumination condition is 1500lx-2000lx and 12-14 h/d.

And counting the pollution rate, the germination rate and the callus induction rate of the material at 20 days. As shown in table 3.

TABLE 3 conditions for inducing calli of Pandanum convolvulus

Example 4

Randomly selecting 200 sterile bulblets generated in example 1 or 2 after culturing for 20 days in each group of induction culture medium with the same 2,4-D concentration, respectively inoculating the sterile bulblets on the induction culture medium with the corresponding 2,4-D concentration, and performing induction culture under the following conditions: the culture temperature is 25 +/-2 ℃, the humidity is 60-70%, the illumination condition is 1500lx-2000lx, and the induction culture is carried out for 20-30 days at 12-14 h/d.

And counting the pollution rate, the germination rate and the callus induction rate of the material at 20 days. As shown in table 4.

TABLE 4 sterile bulblet callus culture conditions for the induction of Toddalia chinensis

As can be seen from tables 1-4, the contamination rate is the highest when the explant is the Pandanum convolvulus bulb disc, and the contamination rate reaches more than 80%. When the explant is a sterile bulblet, the pollution rate is far lower than that of other explants and is only 2.5%, and the callus induction rate is not large compared with that when the explant is a bulblet or a flake, so that the problem of high callus induction pollution rate can be effectively solved. When the concentration of 2,4-D in the induction culture medium is 0.1mg/L, the germination rate is highest, the bulbil can reach 86%, and the aseptic bulblet can reach 87.5%. When the concentration of 2,4-D in the induction culture medium is 0.05mg/L, the callus rate is the highest, the bulbil can reach 38 percent, but the pollution rate is 14 percent; the aseptic bulblet can reach 35%, and the pollution rate is only 5%. Therefore, the induction culture medium is MS +6-BA 1.00mg/L + NAA0.15mg/L + KT 0.20mg/L +2-4, D0.05mg/L is beneficial to the induction of the lilium tigrinum callus, and has larger difference with the phytohormone in the existing induction culture medium of the lilium tigrinum callus, the reason may be that the endogenous hormone levels of different varieties of lilium brownii are different, and therefore, the exogenous growth regulator required in the in vitro culture is different. Since exogenous hormones act mainly by affecting plant endogenous hormones, the action effect of exogenous hormones is closely related to the kinds and levels of endogenous hormones in explants and callus tissues. In addition, after the sterile adventitious bud is transferred to a fresh induction medium for continuous culture, the adventitious bud can be limited from differentiating, and the callus (block) can be induced to form.

Example 5

The experimental procedure is the same as that of example 4, wherein the illumination intensity is 0-300lx, 700-900lx and 1500-2000lx, the illumination time is 12-14 h/d, and the induction medium is MS +6-BA 1.00mg/L + NAA0.15mg/L + KT 0.20mg/L +2-4, and D0.05mg/L.

Inoculating sterile bulblet into fresh induction culture medium for further culture, observing material at 20 days, inducing to form callus, and limiting differentiation of adventitious bud. As shown in FIG. 2, the calli formed under the illumination condition of 0-300lx had uneven sizes and were almost white and transparent (FIG. A). Under the illumination condition of 700-; while the callus formed under the illumination conditions of 1500-.

And counting the pollution rate, the germination rate and the callus induction rate of the material at 20 days. As shown in table 5.

TABLE 5 conditions of Lilium tigrinum callus under different illumination intensities

As shown in Table 5, when the illumination intensity is 700lx-900lx, the callus rate is the highest, and the 20-day sterile bulblet callus induction rate reaches 71%, so that the problem that the callus generation efficiency of the lilium tigrinum induced by the existing induction culture medium is extremely low is solved. The callus rate is improved by 2.38 times under the dark condition and 1.22 times under the stronger light condition, which is not consistent with the general thought that the dark condition has the promotion effect on the lily callus. The method is characterized in that certain illumination can adjust the endogenous hormone level in lilium tigrinum so as to induce and generate callus, while different varieties of lilium have different endogenous hormone levels so as to meet different requirements on illumination intensity.

Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.

Claims

1. The induction method of the lilium tigrinum callus is characterized by comprising the following steps:

inoculating the pretreated and sterilized scale or bulbil of Lilium tigrinum into an induction culture medium for primary culture at the illumination intensity of 700lx-900lx for 20-30 days to obtain the callus and sterile adventitious bud of Lilium tigrinum; transferring the expanded basal part of the sterile adventitious bud into the induction culture medium for continuous culture at the illumination intensity of 700lx-900lx for 20-30 days to obtain the callus of the lilium tigrinum;

the induction culture medium is MS +1.00 mg/L6-BA +0.15mg/L NAA +0.20mg/L KT + 0.05-0.1 mg/L2-4, and the pH value of the culture medium is 5.8-6.2.

2. The method for inducing lilium tigrinum callus according to claim 1, wherein the scale is the middle or outer scale of the bulb.

3. The induction method of calli of Lilium tigrinum L according to claim 1, wherein the induction medium is MS +1.00 mg/L6-BA +0.15mg/L NAA +0.20mg/L KT +0.05 mg/L2-4, and the pH of the medium is 5.8-6.2.

4. The induction method of lilium tigrinum callus according to claim 1 or 3, wherein the induction culture conditions are a culture temperature of 25 ± 2 ℃, a humidity of 60% to 70%, and an illumination time of 12 to 14 h/d.

5. The induction method of Lilium tigrinum callus as claimed in claim 1, wherein the pretreatment is to soak the bulbil or scale of Lilium tigrinum in detergent solution for 5-10min, and then to wash with running tap water for more than 2 h; the sterilization is that the pretreated bulblets or scales of the lilium tigrinum are placed on a super-clean workbench, ethanol water solution with the volume concentration of 70-75% is used for treating for 20-30 s, then sterile water is used for cleaning for at least 2 times, mercuric chloride solution with the mass concentration of 0.1-0.2% is used for sterilization for 10-25 min, and finally sterile water is used for washing for at least 4 times.

6. The method for inducing lilium tigrinum callus according to claim 5, wherein the detergent is soap, washing powder or soapberry saponin.