CN112931210B - Bulb proliferation culture method taking fritillaria paniculata bulb discs as explants - Google Patents
Bulb proliferation culture method taking fritillaria paniculata bulb discs as explants Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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Abstract
The invention discloses a method for bulb proliferation culture by taking a fritillaria paniculata bulb disc as an explant, which comprises the steps of explant selection and disinfection treatment, bulb disc regeneration bulblet culture and bulblet rapid growth; the explant adopted by the invention is a fritillaria obumati bulbodium disc which grows for two years and is cut off roots and scale leaves, and after disinfection treatment, MS + TDZ 1-3 mg.L is flatly inoculated in a way that the root surface of a biological student contacts a culture medium‑1+IAA0.5～1.5mg·L‑1+ 100-300 mg.L of cefazolin sodium‑1+ 5% -20% of coconut milk, and the culture medium for the rapid growth of the bulblet is B5+6-BA 1.0-2.0 mg.L‑1+NAA0.5～1.5mg·L‑1+2,4‑D2.0～4.0mg·L‑1(ii) a The method can rapidly produce a large amount of fritillaria paniculata bulbs, and has low culture cost and high reproduction rate.
Description
Technical Field
The invention relates to a rapid bulb proliferation culture method of fritillaria paniculata, in particular to a bulb proliferation culture method taking fritillaria paniculata bulb discs as explants.
Background
Fritillaria wabuensis S.Y.Tang et S.C.Yue is medicinal plant of Fritillaria of Liliaceae, and its dried bulb is Bulbus Fritillariae Cirrhosae as rare or endangered medicinal material; bulbus Fritillariae Cirrhosae is known as the first of Bulbus Fritillariae Cirrhosae in "Ben Cao gang mu Shi Yi", and was first reported and named in 1983 by obsidian and Yue Song Jian. The fritillaria obumati plant is usually 0.5-0.8 m long, the bulb is oblate, the diameter is 2-5 cm, the leaf strip is in a needle shape or nearly sickle shape, the length is 7-13 cm, the width is 0.9-2 cm, the tip is not curled, the flower is yellow green or yellow, the inner surface has purple spots or no spots, the capsule is 3-5 cm long, the diameter is 1.4-1.8 cm, the capsule has a ridge shape and a narrow wing, the width is 2mm or very narrow, and the fruit stem is upright. The flowering period is 6 months, and the fruit period is 7-8 months. Distributed in Sichuan Minjiang upstream mountain areas. Growing under shrubs with the elevation of 2500-3000 m.
Since 2010, the Fritillaria obu is formally loaded into one of the basic source plants of the Fritillaria cirrhosa in Chinese pharmacopoeia, and is gradually becoming the mainstream cultivated species of the Fritillaria cirrhosa. The dried bulbs of the fritillaria papyrifera are used as the medicine, have bitter, sweet and slightly cold properties, enter lung and heart channels, have the main drug effect components of alkaloids and saponins, have the effects of clearing heat, dispersing pathogenic accumulation, eliminating phlegm, relieving cough, relieving asthma, reducing blood pressure, regulating smooth muscle and resisting bacteria, and are mainly clinically used for symptoms such as lung heat dry cough, dry cough with little phlegm, yin deficiency over-strain cough, expectoration with bloody sputum and the like. At present, along with the research and development and production of a large number of fritillaria cirrhosa Chinese patent medicine products, the demand of fritillaria cirrhosa medicinal material basic source plants is increased year by year, so that wild resources are over developed, especially the wild fritillaria obushuensis resources are endangered, wild commodities are gradually extinct, and a new medicinal material rapid production technical method is urgently required to be searched.
The biological tissue culture technology has the advantages of short growth cycle and high reproduction rate, gets rid of the adverse effects of the changes of four seasons and day and night in the nature and the disastrous climate, has uniform conditions, is extremely favorable for the growth of plants, and is convenient for stably carrying out annual culture production, so the rapid reproduction by applying the tissue culture method is one of effective methods for solving the problem of shortage of the medicinal material resources of the bulbus fritillariae cirrhosae.
Disclosure of Invention
The invention aims to provide a method for bulb proliferation culture by taking a fritillaria paniculata bulb disc as an explant; the method has the advantages of short production cycle, low cost, high reproduction rate, etc., and can be used for mass production of Bulbus Fritillariae Orobanches bulb part for use as medicine in short time to relieve serious shortage of Bulbus Fritillariae Cirrhosae.
In order to achieve the above object, the solution adopted by the present invention comprises the following steps:
(1) selecting and disinfecting explants: selecting underground parts of a fritillaria paniculata plant which grows for two years, cutting off roots and scale leaves of the underground parts, washing the remained bulbil disc with mercuric chloride with the mass fraction of 0.05% for 1-3 min, washing the bulbil disc with sterile water for 2 times, and then sucking off water on the surface of the bulbil disc; then placing the mixture on an ultra-clean workbench, disinfecting the mixture for 30s by using 75% alcohol by volume, and washing the mixture for 2-5 times in sterile water; sterilizing with 0.1% mercuric chloride for 2-6 min, washing with sterile water for 3-5 times, and drying;
(2) bulb dish regeneration bulblet: the sterilized fritillaria obumati bulbil is flatly placed and inoculated into MS + TDZ 1-3 mg.L according to the way that the root surface of a biological student contacts with a culture medium-1+IAA 0.5～1.5mg·L-1+ 100-300 mg.L of cefazolin sodium-1+ 5% -20% of coconut milk in an induction culture medium for regenerating bulblet; culturing at the culture temperature of 10-15 ℃ in the dark for 15 days, and then culturing under the conditions of 5-10 hours of illumination per day and illumination intensity of 1000-2000 lx;
(3) rapid growth of bulblets: cutting off bulblets induced in the step (2), cutting off 2 bulblet plates which are taken as a group and provided with bases together, and inoculating the bulblets to B5+6-BA 1.0-2.0 mg.L-1+NAA0.5～1.5mg·L-1+2,4-D 2.0～4.0mg·L-1Culturing in the bulb rapid growth culture medium under the conditions that the culture temperature is 18-25 ℃, the illumination is 8-12 hours per day, and the illumination intensity is 1000-2000 lx;
the prepared pH value of all the culture media is 5.8-6.2, and the prepared pH value of sucrose is 30 g.L-15.5 to 6.5 g.L of agar-1。
The method adopts the bulb dish of the fritillaria obuma as the explant, and realizes the rapid culture method of the regenerated bulb; compared with the traditional cultivation method of the fritillaria papyrifera, the method greatly shortens the time, and overcomes the problems of long time, serious pest and disease damage, high production cost and the like in the traditional cultivation. In addition, the method can be implemented only by simple plant tissue culture equipment, has strong practicability, is not limited by seasons, and can be widely popularized in a large scale.
Detailed Description
The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.
Example 1
(1) Selecting and disinfecting explants: selecting an underground part of a fritillaria obueli plant which grows for two years, cutting off roots and scale leaves of the underground part, washing a reserved bulbus fritillariae pallidiflorae for 2min by using 0.05% of mercury bichloride, absorbing water on the surface of the bulbus fritillariae pallidiflorae after 2 times of aseptic water washing, placing the bulbus fritillariae pallidiflorae on a super clean workbench, disinfecting the bulbus fritillariae pallidiflorae for 30s by using 75% of alcohol by volume, disinfecting the bulbus fritillariae pallidiflorae for 3min by using 0.1% of mercury bichloride after 2 times of aseptic water washing, and absorbing water on sterilized filter paper after 5 times of aseptic water washing;
(2) bulb dish regeneration bulblet: the sterilized Fritillaria obueli bulb plate is flatly placed and connected into MS + TDZ2 mg.L according to the way that the root surface of a biological student contacts with the culture medium-1+IAA 1.0mg·L-1+ cefazolin sodium 200 mg. L-1+ culturing in inducing culture medium with 13% coconut juice and regenerated bulblet; culturing at 13 deg.C in dark for 15 days, illuminating for 8 hr per day under the illumination intensity of 1500lx for 50 days, and counting the bulblet inductivity of 94.3%, and the average number of induced bulblets is 7.86;
(3) rapid growth of bulblets: cutting off bulblets induced in the step (2), taking 2 bulblet discs which are taken as a group and are provided with bases, cutting off the bulblets together, and inoculating the bulblets into B5+ 6-BA1.5mg.L-1+NAA1.0mg·L-1+2,4-D 3.0mg·L-1In the culture medium for the rapid growth of the bulbs, the bulbs are cultured for 60 days at the temperature of 22 ℃ under the conditions of illumination for 10 hours per day and illumination intensity of 1500lx, and then the volume of the bulbs is increased by 5.73 times on average;
all the culture media have pH of 6 and sucrose content of 30 g.L-16 g.L agar-1。
Example 2
(1) Selecting and disinfecting explants: selecting underground part of a fritillaria paniculata plant which grows for two years, cutting off roots and scale leaves of the underground part, washing the remained bulbil disc with mercuric chloride with the mass fraction of 0.05% for 1min, washing with sterile water for 2 times, and then sucking off water on the surface of the bulbil disc; placing on a superclean bench, sterilizing with 75% alcohol for 30s, and washing with sterile water for 2 times; sterilizing with 0.1 wt% mercuric chloride for 2min, washing with sterile water for 3 times, and drying;
(2) bulb dish regeneration bulblet: the sterilized fritillaria obumati bulbil is flatly placed and inoculated into MS + TDZ1 mg.L according to the way that the root surface of a biological student contacts with the culture medium-1+IAA 0.5mg·L-1+ cefazolin sodium 100 mg.L-15% of coconut milk regenerated small scalesCulturing in an induction culture medium of the stem; culturing at 10 deg.C in dark for 15 days, then illuminating for 5 hr every day under illumination intensity of 1000lx, and counting bulblet inductivity of 82.7% after culturing for 50 days, and average number of induced bulblets is 5.91;
(3) rapid growth of bulblets: cutting bulblets induced in the step (2), cutting off 2 bulblet discs which are used as a group and are provided with bases together, and inoculating the cut bulblets into B5+ 6-BA1.0mg.L-1+NAA0.5mg·L-1+2,4-D 2.0mg·L-1In the culture medium for the rapid growth of the bulbs, the bulbs are cultured for 60 days at the temperature of 18 ℃ under the conditions of illumination for 8 hours per day and illumination intensity of 1000x, and then the volume of the bulbs is increased by 4.84 times on average;
all the culture media have pH of 5.8, sucrose of 30 g.L-1Agar 5.5 g. L-1。
Example 3
(1) Selecting and disinfecting explants: selecting underground part of a fritillaria paniculata plant which grows for two years, cutting off roots and scale leaves of the underground part, washing the remained bulbil disc with mercuric chloride with the mass fraction of 0.05% for 3min, washing with sterile water for 2 times, and then sucking off water on the surface of the bulbil disc; placing on a superclean bench, sterilizing with 75% alcohol for 30s, and washing with sterile water for 5 times; sterilizing with 0.1 wt% mercuric chloride for 6min, washing with sterile water for 5 times, and drying;
(2) bulb dish regeneration bulblet: placing sterilized Bulbus Fritillariae Orobanches into MS + TDZ3 mg.L-1+IAA 1.5mg·L-1+ cefazolin sodium 300 mg. L-1+ culturing in inducing culture medium with 20% coconut juice and regenerated bulblet; culturing at 15 deg.C in dark for 15 days, then culturing under the conditions of illumination for 10 hr per day and illumination intensity of 2000lx, and counting bulblet inductivity of 90.5% and average number of induced bulblets of 6.91 after culturing for 50 days;
(3) rapid growth of bulblets: cutting bulblets induced in the step (2), taking 2 bulblet discs which are taken as a group and are provided with bases, cutting off the bulblets together, and inoculating the bulblets into B5+ 6-BA2.0mg.L-1+NAA1.5mg·L-1+2,4-D 4.0mg·L-1In the culture medium for the rapid growth of the bulbs, the bulbs are cultured for 60 days at the temperature of 25 ℃ under the conditions of illumination for 12 hours every day and illumination intensity of 2000lx, and then the volume of the bulbs is increased by 5.27 times on average;
all the culture media have pH value of 5.8 and sucrose content of 30 g.L-1Agar 5.5 g. L-1。
Comparative example 1
In the step (2) of example 1, the sterilized fritillaria obumati bulb is inoculated into MS + TDZ 0 mg.L-1+IAA 1.0mg·L-1Culturing in 13% coconut milk culture medium; in other steps, as in example 1, the induction rate of bulblet was counted up to 24.23% after 50 days of culture, and the average number of induced bulblets was 2.21.
Comparative example 2
In the step (2) of example 1, the sterilized fritillaria obbusti bulb dish is inoculated with MS + TDZ 0.5 mg.L-1+IAA3.0mg·L-1+ 50 mg.L of cefazolin sodium-1Culturing in 1% coconut milk culture medium; in other steps, as in example 1, the bulblet induction rate was counted up to 31.63% after 50 days of culture, and the average number of induced bulblets was 3.26.
Comparative example 3
In step (3) of example 1, a small bulb having a bulb dish at the base thereof was inoculated with White +6-BA0.5 mg.L-1+NAA0.3mg·L-1+2,4-D 1.0mg·L-1The volume of the bulblet in the culture medium of (1) was increased by 2.47 times on average, as counted in example 1 after 60 days of culture.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (1)
1. A method for proliferation culture of bulbs by taking a fritillaria paniculata bulb disc as an explant is characterized by comprising the following steps:
(1) selecting and disinfecting explants: selecting an underground part of a fritillaria paniculata plant growing for two years, cutting off roots and scale leaves on the underground part, washing a reserved bulbil disc with 0.05% of mercury bichloride for 1-3 min, washing the bulbil disc with sterile water for 2 times, absorbing water on the surface of the bulbil disc, placing the bulbil disc on a super clean workbench, disinfecting the bulbil disc with 75% of alcohol for 30s, washing the bulbil disc with sterile water for 2-5 times, then disinfecting the bulbil disc with 0.1% of mercury bichloride for 2-6 min, washing the bulbil disc with sterile water for 3-5 times, and absorbing water on sterilized filter paper;
(2) bulb dish regeneration bulblet: the sterilized fritillaria obumati bulbil is flatly placed and inoculated into MS + TDZ 1-3 mg.L according to the mode that the root surface of a biological student contacts with a culture medium-1+IAA 0.5～1.5mg·L-1+ 100-300 mg.L of cefazolin sodium-1+ 5% -20% of coconut milk in an induction culture medium for regenerating bulblet; culturing at the culture temperature of 10-15 ℃ in the dark for 15 days, and then culturing under the conditions that the illumination is 5-10 hours per day and the illumination intensity is 1000-2000 lx;
(3) rapid growth of bulblet: cutting off bulblets induced in the step (2), cutting off 2 bulblet plates which are taken as a group and provided with bases together, and inoculating the bulblets to B5+6-BA 1.0-2.0 mg.L-1+NAA 0.5～1.5mg·L-1+2,4-D 2.0～4.0mg·L-1Culturing in the bulb rapid growth culture medium under the conditions that the culture temperature is 18-25 ℃, the illumination is 8-12 hours per day, and the illumination intensity is 1000-2000 lx;
the prepared pH values of all the culture media are 5.8-6.2, and the prepared pH values of sucrose are 30 g.L-15.5 to 6.5 g.L of agar-1。
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