CN111165358B

CN111165358B - Method for inducing tetraploid plants by using uncaria adventitious buds

Info

Publication number: CN111165358B
Application number: CN202010176997.1A
Authority: CN
Inventors: 冷燕
Original assignee: Lincang Huikang Traditional Chinese Medicine Development Co ltd
Current assignee: Lincang Huikang Traditional Chinese Medicine Development Co ltd
Priority date: 2020-03-13
Filing date: 2020-03-13
Publication date: 2023-05-02
Anticipated expiration: 2040-03-13
Also published as: CN111165358A

Abstract

The invention relates to a polyploid culture method of traditional Chinese medicinal materials, in particular to a method for inducing tetraploid plants by using uncaria adventitious buds, which is realized by the steps of aseptic culture of adventitious buds, polyploid induction, chimera separation culture, tetraploid screening, tetraploid amplification and tetraploid rooting in sequence.

Description

Method for inducing tetraploid plants by using uncaria adventitious buds

Technical Field

The invention relates to a polyploid culture method of Chinese medicinal materials, belongs to the field of plant crop breeding and Chinese medicinal material planting, and in particular relates to a polyploid culture method of uncaria.

Background

Ramulus Uncariae cum UncisUncaria rhynchophylla(Miq.) Miq. Ex Havil.) is a perennial woody vine of Uncaria genus of Rubiaceae family, and is used as a drug with ramulus Uncariae cum Uncis, and has effects of calming endogenous wind, arresting convulsion, clearing heat, and suppressing liver, and can be used for treating diseases such as internal stirring of liver wind, convulsion, febrile convulsion, infantile convulsion, gestational eclampsia, headache, vertigo, etc. Modern medicine researches contain rhynchophylline, and has effects of lowering blood pressure, resisting blood platelet aggregation and resisting thrombosis, and can be used for treating part of cardiovascular and cerebrovascular diseases. At present, the traditional Chinese medicine preparation taking uncaria as the raw material medicine has more than 170 kinds and the annual demand of the medicinal materials is large. The wild is mainly distributed in regions such as Yunnan, guangdong, guangxi, guizhou, fujian, hunan, hubei, jiangxi and the like, and the Yunnan is produced in a forest or bush of a valley stream side with the altitude of 1300-190 m. At present, guizhou and Guangxi have been planted in a certain scale. In recent years, with the increase of development application and clinical acceptance of curative effect, demand quantity is increased year by year, price of uncaria medicinal materials is increased, and the current planting scale productivity can not meet market demands. The traditional propagation mode mainly has the defects of long propagation period, unstable germplasm characters and the like in seed propagation and cutting propagation, and the cutting propagation has the defects of low propagation rate, serious plant diseases and insect pests in the seedling and planting process and the like. Meanwhile, most of the uncaria in the drug standards of enterprises have uncaine content detection indexes, and the quality of the drug is uneven due to the fact that the uncaria has more varieties and even the difference among different varieties is larger.

Polyploidy is a natural plant that is commonly found in nature and has three or more chromosomes in the organism's own body cells, commonly found in higher plants, many of which undergo "polyploidization" and "diploidization" during the course of evolution. Because polyploid has the characteristics of large nutrition organ, enhanced stress resistance, improved growth rate, increased content of active ingredients and the like, the polyploid is an important mode of crop breeding, is also an important direction of medicinal plant breeding, and a plurality of important grain crops and economic crops are polyploids at present. Although polyploids exist widely in nature, the number is limited, and by artificial induction, the polyploids can be amplified in a large amount in a short period. The current mode of artificially inducing polyploidy mainly comprises chemical induction and physical induction, wherein the chemical induction is the most effective and widely applied method due to the advantages of simple operation, strong specificity and the like.

On one hand, the rhynchophylline contained in the uncaria is the most widely applied substance of the rhynchophylline content, the content of the rhynchophylline is the most important index for detecting whether the rhynchophylline reaches the standard or not by the country and enterprises, and meanwhile, the price and the value of the medicinal materials are larger as the content of the rhynchophylline is higher. On the other hand, the ramulus Uncariae cum Uncis is generally harvested by taking the ramulus Uncariae cum Uncis as a unit, and the yield per mu of ramulus Uncariae cum Uncis is only 40-80 kg according to the current planting parameters of the ramulus Uncariae cum Uncis, so that the yield is low, and the development of the ramulus Uncariae cum Uncis industry is restricted.

Disclosure of Invention

Aiming at the defects of the existing uncaria planting industry, the invention utilizes modern crop breeding and biotechnology to double the chromosomes of uncaria so as to obtain uncaria tetraploid, thereby increasing the yield per unit area and the medicinal value of uncaria.

The invention is realized by the following technical scheme:

1. culturing aseptic adventitious buds:

(1) Selecting strong and plant diseases and insect pests-free tender buds of uncaria, rinsing with saturated soap water solution for 10-15 min, then rinsing with tap water for 3-4 times until no soap solution remains, adding a proper amount of water, adding 2-3 drops of tween-80, shaking for 10-15 min, and rinsing with tap water for 50-60 min;

(2) Transferring the plant material into an ultra-clean workbench, adding 75% alcohol, shaking for 15-20 s, washing 3-4 times with sterile water, adding 0.1% mercuric chloride solution, shaking for 5-7 min, washing 6-8 times with sterile water, and sucking the surface moisture of the explant with sterile paper;

(3) And inoculating the sterilized young buds into an induction culture medium of 1/2MS+NAA 0.1~0.5mg/L+BA 3.0-5.0 mg/L, and culturing for 20-30 days in an environment with the illumination intensity of 1000-2000 lux and the illumination time of 10 hours per day at the temperature of 23-27 ℃.

2. Polyploid induction of adventitious buds:

cutting the induced adventitious buds into single buds, soaking the single buds in 0.1-0.15% colchicine solution containing 0.2% dimethyl sulfoxide, and placing the single buds in a non-illumination environment at 23-27 ℃ for induction for 24-48 hours.

3. Chimeric isolation culture:

the induced chimera is inoculated into a separation culture medium of MS+NAA 0.01-0.05 mg/L+KT 2.0-4.0 mg/L+BA 1.0-2.0 mg/L, and cultured for 20-30 days under the conditions of 23-27 ℃ and illumination intensity of 2000-3000 lux and illumination time of 10 hours per day.

4. Tetraploid screening: the cultured chimera is separated, plants with thicker leaves and thicker stems are used as screening objects, the leaves are taken as identification materials, and the identification is carried out by adopting a chromosome tabletting counting method, so that the plants with 2 pairs of chromosomes are screened.

5. Tetraploid amplification: cutting the selected tetraploid into 1-2 bud stem segments, inoculating the tetraploid into a proliferation culture medium of MS+NAA 0.01-0.05 mg/L+KT 2.0-4.0 mg/L+BA 1.0-2.0 mg/L, and culturing for 50-60 days in an environment with the temperature of 23-27 ℃ and the illumination intensity of 2000-3000 lux and the illumination time of 10 hours per day.

6. Rooting of tetraploid: cutting the tetraploid subjected to screening or amplification into stem segments with 2-3 buds, inoculating to rooting culture with 1/2WPM+NAA 0.5~2.0mg/L+IBA 0-2.0 mg/L+active carbon 0.5-1.0 g/L, and culturing for 25-35 days in an environment with the temperature of 23-27 ℃ and the illumination intensity of 3000-4000 lux and the illumination time of 12 hours per day.

The invention has the beneficial effects that:

the invention utilizes colchicine to carry out polyploid induction on the aseptic adventitious buds of uncaria, then screens and purifies and cultures the uncaria through a morphological identification method and a chromosome tabletting method to obtain tetraploid uncaria, the induction rate of the tetraploid uncaria is 18%, and a large number of tetraploid uncaria seedlings can be obtained after 90-180 days.

The tetraploid uncaria obviously thickens and thickens roots, stems and leaves in morphology, plants are obviously dwarfed after being planted, drought resistance and stress resistance of the plants are obviously improved, and daily management and picking of medicinal materials are facilitated.

After four years of planting, the medicinal hooks and hooked stems of the tetraploid uncaria disclosed by the invention are obviously increased in thickness, the hook forming rate in unit area is indirectly improved, and the mu yield of medicinal materials is improved by 50-100%.

After four years of planting, the tetraploid uncaria is subjected to uncarine detection, and the total alkali content of the uncaria is increased by about 0.12% compared with that of the original parent strain, so that the price and the medicinal value of medicinal materials are greatly increased.

Detailed Description

The invention will be further illustrated with reference to the following examples. Embodiments of the present invention include, but are not limited to, the following examples of implementation.

Example 1: 1. culturing aseptic adventitious buds:

(1) Selecting tender stems of healthy and strong uncaria with no plant diseases and insect pests, shearing the tender stems into stem segments with buds of 2-3 cm, rinsing the stem segments with saturated soap water solution for 10 times, then rinsing the stem segments with tap water for 3 times until no soap solution remains, adding proper amount of water, adding 2 drops of tween-80, shaking for 15min, and rinsing the stem segments with tap water for 60min;

(2) Transferring to an ultra-clean workbench, adding 75% alcohol, shaking for 15s, rinsing with sterile water for 3 times, adding 0.1% mercuric solution, shaking for 6min, rinsing with sterile water for 8 times, and sucking water on the surface of the explant with sterile paper;

(3) And (3) cutting the sterilized young buds, cutting wound parts, inoculating the cut parts into an induction culture medium of 1/2MS+NAA 0.1mg/L+BA3.0mg/L according to polarity, and culturing for 20 days under the environment of 23-27 ℃ and the illumination intensity of 1000-2000 lux for 10 hours in a single day, wherein the germination rate is 85.2%.

2. Polyploid induction of adventitious buds:

cutting the induced adventitious buds into single buds, soaking the single buds in 0.1% colchicine solution containing 0.2% dimethyl sulfoxide, and placing the single buds in a dark illumination environment at 23-27 ℃ for induction for 48 hours, wherein the survival rate of the adventitious buds is 52.4%, the polyploid induction rate is 20.8%, and the tetraploid induction rate is 18.7%.

3. Chimeric isolation culture:

absorbing surface induction liquid of the induced chimera by using sterile paper, flushing the surface induction liquid by using sterile water for 6-8 times, absorbing surface moisture, inoculating the surface induction liquid into a separation culture medium of MS+NAA0. mg/L+KT2. mg/L+BA 2. mg/L, and culturing for 20 days in an environment with the temperature of 23-27 ℃ and the illumination intensity of 2000-3000 lux and the illumination time of 10 hours per day.

4. Tetraploid screening:

the cultured chimera is separated, plants with thicker leaves and thicker stems are used as screening objects, the leaves are taken as identification materials, and the identification is carried out by adopting a chromosome tabletting counting method, so that the plants with 2 pairs of chromosomes are screened.

5. Tetraploid amplification

: cutting the selected tetraploid into 1-2 bud stem segments, inoculating the tetraploid into a proliferation culture medium of MS+NAA 0.05 mg/L+KT4.0 mg/L+BA 1.0mg/L, and culturing for 50 days at the temperature of 23-27 ℃ under the condition that the illumination intensity is 2000-3000 lux and the illumination time is 10 hours per day, wherein the proliferation coefficient is 4.5.

6. Rooting of tetraploid:

cutting the amplified tetraploid into stem segments with 2-3 buds, inoculating the stem segments on rooting culture of 1/2 WPM+NAA2.0mg/L+1.0g/L of activated carbon, and culturing for 30 days at the temperature of 23-27 ℃ under the illumination intensity of 3000-4000 lux in the environment of 12h of illumination time on a single day to obtain tetraploid uncaria seedlings, wherein the rooting rate is 96.7%.

Example 2: 1. culturing aseptic adventitious buds:

(1) Selecting strong and plant diseases and insect pests-free tender buds of uncaria, rinsing with saturated soap water solution for 15min, then rinsing with tap water for 3 times until no soap solution remains, adding a proper amount of water, adding 2 drops of tween-80, shaking for 10min, and rinsing with tap water for 60min;

(2) Transferring the plant material into an ultra-clean workbench, adding 75% alcohol, shaking for 20s, washing 3-4 times with sterile water, adding 0.1% mercuric chloride solution, shaking for 7min, washing 8 times with sterile water, and sucking the surface moisture of the explant with sterile paper;

(3) And (3) cutting the sterilized young buds, cutting wound parts, inoculating the cut parts into an induction culture medium of 1/2MS+NAA 0.5mg/L+BA5.0mg/L according to polarity, and culturing for 30 days in an environment with the illumination intensity of 1000-2000 lux and the illumination time of 10 hours on a single day at the temperature of 23-27 ℃.

2. Polyploid induction of adventitious buds:

cutting the induced adventitious buds into single buds, soaking the single buds in 0.15% colchicine solution containing 0.2% dimethyl sulfoxide, and placing the single buds in a dark illumination environment at 23-27 ℃ for induction for 36 hours, wherein the survival rate of the adventitious buds is 60.5%, the polyploid induction rate is 19.4%, and the tetraploid induction rate is 17.7%.

3. Chimeric isolation culture:

the induced chimera is sucked up with sterile paper to clean surface induction liquid, then is washed with sterile water for 8 times, is inoculated into a separation culture medium of MS+NAA 0.02 mg/L+KT 4.0 mg/L+BA 1.0mg/L, and is cultured for 20 days in an environment with the temperature of 23-27 ℃ and the illumination intensity of 2000-3000 lux and the illumination time of 10 hours per day after the surface water is sucked up.

4. Tetraploid screening:

5. Rooting of tetraploid:

cutting the selected tetraploid into stem segments with 2-3 buds, inoculating the stem segments on rooting culture of 1/2WPM+NAA 0.5 mg/L+IBA 2.0 mg/L+active carbon 1.0g/L, and culturing for 30 days at the temperature of 23-27 ℃ under the illumination intensity of 3000-4000 lux in the environment of 12 hours of illumination time per day to obtain tetraploid uncaria seedlings, wherein the rooting rate is 98.8%.

Claims

1. A method for inducing tetraploid plants by using uncaria adventitious buds is characterized by comprising the following steps:

1) Sterile culture of adventitious buds: selecting strong and healthy tender buds of uncaria without plant diseases and insect pests, sterilizing, inoculating to an adventitious bud induction culture medium, and culturing in a proper environment;

2) Polyploid induction: cutting the cultured aseptic adventitious bud into single bud, placing in polyploid induction liquid, and inducing under proper environment;

3) Chimeric isolation culture: washing the induced chimera, placing the washed chimera on a chimera separation culture medium, and culturing in a proper environment;

4) Tetraploid screening: after the chimera is separated and cultivated into a plant with stem leaves, selecting the chimera with fattened leaves and thickened stems according to a morphological identification method, taking young leaves as a plant unit, and carrying out chromosome identification;

5) Tetraploid amplification: cutting the selected pure tetraploid into single buds, inoculating the single buds to a proliferation culture medium, and culturing in a proper environment;

6) Tetraploid rooting: inoculating the obtained pure tetraploid seedling into rooting culture medium, and culturing in proper environment;

wherein:

the polyploid induction liquid in the step 2) is 0.1-0.15% colchicine solution, and the polyploid induction method is that the polyploid induction liquid is soaked for 24-48 hours at the temperature of 23-27 ℃ without illumination;

the culture medium for separating the chimera in the step 3) is MS+NAA 0.01-0.05 mg/L+KT 2.0-4.0 mg/L+BA 1.0-2.0 mg/L, the culture environment is at the temperature of 23-27 ℃, the illumination intensity is 2000-3000 lux, and the illumination time is 10 hours per day;

the proliferation culture medium in the step 5) is MS+NAA 0.01-0.05 mg/L+KT 2.0-4.0 mg/L+BA 1.0-2.0 mg/L, the culture environment is at the temperature of 23-27 ℃, the illumination intensity is 2000-3000 lux, and the illumination time per day is 10 hours.

2. The method for inducing tetraploid plants by using adventitious buds of uncaria according to claim 1, wherein the disinfection method in step 1) is sequentially carried out for 15-20 s by 75% alcohol, 5-7 min by 0.1% mercuric chloride solution, the adventitious bud induction culture medium is 1/2MS+NAA 0.1~0.5mg/L+BA 3.0-5.0 mg/L, the culture environment is at the temperature of 23-27 ℃, the illumination intensity is 1000-2000 lux, and the single day illumination time is 10h.

3. The method for inducing tetraploid plants by using adventitious buds of uncaria according to claim 1, wherein the rooting medium in the step 6) is 1/2WPM+NAA 0.5~2.0mg/L+IBA 0-2.0 mg/L+activated carbon 0.5-1.0 g/L, the culture environment is at the temperature of 23-27 ℃, the illumination intensity is 3000-4000 lux, and the single day illumination time is 12h.