CN107996400B - Method for inducing adventitious buds of salvia miltiorrhiza by taking anthurium andraeanum inflorescence as explant - Google Patents
Method for inducing adventitious buds of salvia miltiorrhiza by taking anthurium andraeanum inflorescence as explant Download PDFInfo
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- CN107996400B CN107996400B CN201711235540.8A CN201711235540A CN107996400B CN 107996400 B CN107996400 B CN 107996400B CN 201711235540 A CN201711235540 A CN 201711235540A CN 107996400 B CN107996400 B CN 107996400B
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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
- A01H4/001—Culture apparatus for tissue culture
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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
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention discloses a method for inducing adventitious buds of anthurium andraeanum by taking anthurium andraeanum inflorescence as an explant, wherein unopened tender inflorescence extracted from anthurium andraeanum scape is selected as the explant, a soft brush is used for dipping a washing powder aqueous solution to lightly scrub surface dust, and the surface dust is disinfected and pretreated in a mixed disinfectant; transferring into super clean bench, sterilizing surface with 75% alcohol, soaking explant in mixed solution of mercuric chloride and tween for sterilization, washing with sterile water for 3-4 times to obtain sterile tender inflorescence, and inoculating in primary culture medium for inducing adventitious bud. The method solves the problems of short explant acquisition time, small amount, difficult disinfection and high browning rate in the tissue culture of the salvia miltiorrhiza bunge, can ensure that sufficient explants are obtained, can effectively reduce the pollution rate of the salvia miltiorrhiza bunge explant and improve the survival rate of the explant.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a method for inducing adventitious buds of a anthurium andraeanum inflorescence serving as an explant.
Background
Red root of herbaceous peony (Salvia prionitis Hance) has the effects of resisting bacteria, removing toxicity, clearing heat, removing dampness, resisting tuberculosis, resisting tumors and the like, and is a main raw material of a Chinese medicinal product, namely 'compound salvia tablet'. The sanguinarine extracted from Salvia miltiorrhiza Bunge is used as key intermediate for chemical modification to form diterpene quinone compound Shaerweixin with effect of inhibiting lung cancerThe anticancer medicine has the advantages of preventing the growth of cancer cells such as gastric cancer, ovarian cancer, liver cancer, colon cancer, cervical cancer and the like, blocking the dual functions of repairing and regenerating channels, having the characteristics of low toxicity, drug resistance, transfer resistance and the like, and being considered to be a novel antitumor medicine with independent intellectual property rights in China.
The germination rate of the salvia miltiorrhiza bunge seeds in a natural state is extremely low, in recent years, due to environmental changes, large acquisition of enterprises and large collection and excavation of mass of people in production areas, wild resources of the salvia miltiorrhiza bunge are exhausted, the clinical application of the salvia miltiorrhiza bunge is seriously influenced, and the tissue culture and rapid propagation of the salvia miltiorrhiza bunge are very urgent.
The sterilization of the explant and the acquisition of a sterile system are important links of tissue culture and in vitro rapid propagation, and have very important significance for large-scale production. The whole red-rooted salvia is molted, and when the disinfection treatment is carried out, the surface tension of the explant is increased, a disinfectant is not easy to permeate, so that the explant is not thoroughly disinfected, the pollution rate is high, and a sterile explant cannot be obtained; or the explant is excessively sterilized, so that the explant is browned and loses activity, and the survival rate of tissue culture of the explant is greatly reduced.
At present, there are only two reports on tissue culture of red-rooted salvia.
Huang-refined Tou (Huang-refined Tou, Xuyin Zeng, wall of Hu, fast reproduction of Renshen Redbetong in vitro) & communication of plant physiology (J)].1988, 34(5): 365.) discloses a method of using the stem nodes of the red rooted grass as explants, rinsing with tap water for 30s, soaking with 75% alcohol for 60 s, 0.1% HgCl2Sterilizing for 20min to obtain sterile external method for rapid propagation, but nothing is mentioned about the sterilizing effect of explants.
Study on tissue culture and rapid propagation of red-rooted salvia (J) of Guangxi plants].2006, 26(3): 282 + 285.) experiments prove that the stem nodes, stem tips and growing points of the red-rooted salvia before stem extraction are washed clean by tap water, then the dirt on the surface of the material is lightly wiped by gauze, the material is soaked in a solution of 800 times of thiophanate methyl for 20min, the solution is soaked in 75% alcohol for 1 min, and 0.1% HgCl is added2Treating for 5-10 min, washing with sterile water for 5-6 times, inoculating, and inoculatingThe contamination rates of the three explants are respectively as high as 46.7%, 60% and 85%, which indicates that the explant has poor disinfection effect.
It can be seen that the mangrove plant has not established a mature tissue culture system, the mangrove is a perennial herb, the current plant has only one growing point, generally does not sprout side buds, the basal leaves are close to the ground surface, there are short stems crossed in pairs, the high growth of the short stems is not obvious (Tanghui, Zhao Rui, Jiang Yuan, Cheng Zong. study of the biological characteristics of the mangrove plant [ J ] Chinese medicinal plant, 2008, No.29610: 1464-. The anthurium andraeanum florescence is 4-6 months, the inflorescence is of a umbrella-gathering type, the inflorescence quantity in the florescence is large, and a large quantity of unopened tender inflorescence can be obtained by taking materials from late ten days to middle ten days of 4 months, so that the requirement of in vitro sampling is met. At present, no report of adventitious bud induction by using the anthurium andraeanum inflorescence as an explant exists.
In view of the fact that the disinfection effect is not ideal and the explant with higher survival rate is difficult to obtain, the invention selects the unopened tender inflorescence extracted from the russian scape as the explant, provides a method for inducing the adventitious bud of the russian scape by using the inflorescence as the explant and provides reference for the rapid propagation of the russian scape.
The invention can reduce the browning rate of the explant, improve the survival rate of the explant and is also suitable for disinfecting the explant with villus.
Disclosure of Invention
In order to solve the technical problems that the whole body of the salvia miltiorrhiza bunge is hairy and difficult to disinfect, the invention provides a method for inducing adventitious buds of the salvia miltiorrhiza bunge by taking the inflorescence of the salvia miltiorrhiza bunge as an explant.
The technical scheme of the invention is as follows:
a method for inducing adventitious buds of a sanguinaria canadensis inflorescence by taking the sanguinea canadensis inflorescence as an explant comprises the following steps:
(1) preparing an explant: selecting unopened young inflorescences of the robust red-rooted grass scape before and after 11 am in sunny days, and cutting the inflorescences as explants with the length of about 0.5-1.0 cm by using a scalpel;
(2) pretreatment of explants: gently scrubbing the surface of the explant by dipping a washing powder aqueous solution with a soft brush, washing the explant by running water until no foam exists, soaking the explant in a mixed solution prepared from carbendazim and vitamin C for 25-30 min, washing the explant by running water for 30 min, and sucking off the surface water to obtain the pretreated explant;
(3) disinfection and inoculation of explants: soaking the pretreated explant in 75% alcohol for 10s in a super clean bench, then soaking the explant in mercuric chloride and Tween-80 mixed disinfectant for 7-11 min, slightly shaking the bottle at intervals during the soaking process to ensure that the explant is fully contacted with the disinfectant, then rinsing the explant with sterile water for 3-4 times, sucking surface water with sterile absorbent paper, and inoculating the explant to MS + TDZ 0.5mg/L +6-BA 0.5mg/L + NAA 0.2 mg/L for carrying out induction culture on the adventitious buds of the salvia miltiorrhiza bunge.
In the method, the carbendazim used in the mixed solution in the step (2) is 50% wettable powder with the concentration of 1g/L and the concentration of the vitamin C of 0.8 g/L.
In the method, the disinfectant in the step (3) is a mixed disinfectant, wherein the concentration of mercuric chloride is 0.1%, and the final concentration of tween-80 in volume ratio is 0.1%.
Compared with the existing disinfection method for the anthurium andraeanum explant, the disinfection method for the anthurium andraeanum inflorescence explant provided by the invention has the advantages that: at present, no report on a disinfection method of anthurium andraeanum inflorescence exists, and the inflorescence is used as an explant, so that a sterile explant with lower pollution rate and higher survival rate can be obtained. According to the experimental result, the pollution rate is lower than 30%, the survival rate is 63.64-71.74%, the average sprouting time of the adventitious buds is 10d, and then the adventitious buds are extracted from the inflorescence.
Drawings
FIG. 1 is a diagram showing the growth state of the explant of the red-rooted grass for 30d after being inoculated with the explant of the anthurium andraeanum in example 1;
FIG. 2 is a diagram showing the growth state of the explant of the anthurium andraeanum plant after 30 days of inoculation in example 2;
FIG. 3 is a diagram showing the growth state of the explant of the anthurium andraeanum plant of example 3 after being inoculated for 30 days;
FIG. 4 is a diagram showing the growth state of adventitious buds of sage induced in example 1;
FIG. 5 is a diagram showing the growth state of adventitious buds of sage induced in example 2;
FIG. 6 is a diagram showing the growth state of adventitious buds of R.sanguinea induced in example 3.
Detailed Description
The present invention will be further described with reference to the following examples, but the present invention is not limited thereto.
Example 1:
a method for inducing adventitious buds of a sanguinaria canadensis inflorescence by taking the sanguinea canadensis inflorescence as an explant comprises the following steps:
(1) preparing an explant: selecting unopened young inflorescence extracted from a strong red-rooted grass scape at noon in sunny weather, and cutting by using a scalpel to obtain an explant with the length of about 1.0 cm;
(2) pretreatment of explants: lightly scrubbing the surface of the explant by dipping a washing powder aqueous solution with a soft brush, washing the surface of the explant by running water until no foam exists, soaking the explant in a mixed solution prepared from 50% carbendazim and 0.8 g/L vitamin C for 25-30 min, washing the explant by running water for 30 min, and sucking the surface water to obtain the pretreated explant;
(3) disinfection and inoculation of explants: soaking the pretreated explant in 75% alcohol for 10s in a super clean bench, then transferring the explant into a mixed disinfectant with the concentration of 0.1% mercuric chloride and Tween-80 (the final concentration of Tween-80 is 0.1% by volume), soaking for 7 min, slightly shaking a bottle from time to time in the soaking process to ensure that the explant is fully contacted with the disinfectant, then rinsing the explant with sterile water for 4 times, absorbing surface water with sterile absorbent paper, and inoculating the explant into MS + TDZ 0.5mg/L +6-BA 0.5mg/L + NAA 0.2 mg/L to perform induction culture of the adventitious buds of the salvia miltiorrhiza bunge. Statistics is carried out 20 days after inoculation, the pollution rate is 27.45%, and the survival rate reaches 71.74%.
Example 1 growth status of sanguinaria officinalis inflorescence explants 30 days after inoculation, as shown in figure 1.
Example 1 growth status of adventitious buds of red rooted grass induced as shown in FIG. 4.
Example 2:
a method for inducing adventitious buds of a sanguinaria canadensis inflorescence by taking the sanguinea canadensis inflorescence as an explant comprises the following steps:
(1) preparing an explant: selecting unopened young inflorescence extracted from a strong red-rooted grass scape at noon in sunny weather, and cutting by using a scalpel to obtain an explant with the length of about 1.0 cm;
(2) pretreatment of explants: lightly scrubbing the surface of the explant by dipping a washing powder aqueous solution with a soft brush, washing the surface of the explant by running water until no foam exists, soaking the explant in a mixed solution prepared from 50% carbendazim and 0.8 g/L vitamin C for 25-30 min, washing the explant by running water for 30 min, and sucking the surface water to obtain the pretreated explant;
(3) disinfection and inoculation of explants: soaking the pretreated explant in 75% alcohol for 10s in a super clean bench, then transferring the explant into a mixed disinfectant with the concentration of 0.1% mercuric chloride and Tween-80 (the final concentration of Tween-80 is 0.1% by volume), soaking for 9 min, slightly shaking a bottle from time to time in the soaking process to ensure that the explant is fully contacted with the disinfectant, then rinsing with sterile water for 4 times, absorbing surface water with sterile absorbent paper, and inoculating the explant into MS + TDZ 0.5mg/L +6-BA 0.5mg/L + NAA 0.2 mg/L to perform induction culture of the adventitious buds of the salvia miltiorrhiza bunge. Statistics is carried out 20 days after inoculation, the pollution rate is 25.89%, and the survival rate is 69.49%.
Example 2 growth status of the induced adventitious bud of red rooted grass, as shown in fig. 2.
Example 2 growth status of the induced adventitious bud of red rooted grass, as shown in fig. 5.
Example 3:
a method for inducing adventitious buds of a sanguinaria canadensis inflorescence by taking the sanguinea canadensis inflorescence as an explant comprises the following steps:
(1) preparing an explant: selecting unopened young inflorescence extracted from a strong red-rooted grass scape at noon in sunny weather, and cutting by using a scalpel to obtain an explant with the length of about 1.0 cm;
(2) pretreatment of explants: lightly scrubbing the surface of the explant by dipping a washing powder aqueous solution with a soft brush, washing the surface of the explant by running water until no foam exists, soaking the explant in a mixed solution prepared from 50% carbendazim and 0.8 g/L vitamin C for 25-30 min, washing the explant by running water for 30 min, and sucking the surface water to obtain the pretreated explant;
(3) disinfection and inoculation of explants: soaking the pretreated explant in 75% alcohol for 10s in a super clean bench, then transferring the explant into a mixed disinfectant with the concentration of 0.1% mercuric chloride and Tween-80 (the final concentration of Tween-80 is 0.1% by volume), soaking for 11 min, slightly shaking a bottle from time to time in the soaking process to ensure that the explant is fully contacted with the disinfectant, then rinsing the explant with sterile water for 4 times, absorbing surface water with sterile absorbent paper, and inoculating the explant into MS + TDZ 0.5mg/L +6-BA 0.5mg/L + NAA 0.2 mg/L to perform induction culture of the adventitious buds of the salvia miltiorrhiza bunge. Statistics is carried out 20 days after inoculation, the pollution rate is 21.01 percent, and the survival rate is 63.64 percent.
Example 3 growth status of the induced adventitious bud of red rooted grass, as shown in fig. 3.
Example 3 growth status of the induced adventitious bud of red rooted grass, as shown in fig. 6.
The results of the rate of explant contamination and survival induced by examples 1-3 are as follows:
Claims (1)
1. a method for inducing adventitious buds of a sanguinaria canadensis inflorescence by taking the sanguinea canadensis inflorescence as an explant is characterized by comprising the following steps:
(1) preparing an explant: selecting unopened young inflorescences of a strong red-rooted grass scape before and after 11 am in sunny days, and cutting 0.5-1.0 cm in length by using a scalpel to serve as explants;
(2) pretreatment of explants: gently scrubbing the surface of the explant by dipping a washing powder aqueous solution with a soft brush, washing the explant by running water until no foam exists, soaking the explant in a mixed solution prepared from carbendazim and vitamin C for 25-30 min, washing the explant by running water for 30 min, and sucking off the surface water to obtain the pretreated explant;
the carbendazim used in the mixed solution is 50% wettable powder with the concentration of 1g/L and the concentration of vitamin C of 0.8 g/L;
(3) disinfection and inoculation of explants: soaking the pretreated explant in 75% alcohol for 10s in a super clean bench, then soaking in mercuric chloride and Tween-80 mixed disinfectant for 7-11 min, slightly shaking the bottle at times during the soaking process to ensure that the explant is fully contacted with the disinfectant, then rinsing with sterile water for 3-4 times, absorbing the surface water with sterile absorbent paper, and inoculating to MS + TDZ 0.5mg/L +6-BA 0.5mg/L + NAA 0.2 mg/L for culture to obtain the adventitious buds of the red-rooted salvia;
the disinfectant is a mixed disinfectant, wherein the concentration of mercuric chloride is 0.1%, and the final concentration of tween-80 in volume ratio is 0.1%.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5908771A (en) * | 1997-01-31 | 1999-06-01 | R. J. Reynolds Tobacco Company | Method for regeneration of salvia species |
| UA53637U (en) * | 2010-04-26 | 2010-10-11 | Наталья Алексеевна Егорова | METHOD FOR REGENERATING PLANTS OF CALLUS TISSUES OF Salvia sclarea L. in vitro |
| CN105393914A (en) * | 2014-09-12 | 2016-03-16 | 山东大学（威海） | Zostera marina isolated inflorescence callus induction method |
| CN106538382A (en) * | 2016-10-31 | 2017-03-29 | 江苏省中国科学院植物研究所 | A kind of method that eremochloa ophiuroides high-efficiency regeneration system is set up as explant with young fringe |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5908771A (en) * | 1997-01-31 | 1999-06-01 | R. J. Reynolds Tobacco Company | Method for regeneration of salvia species |
| UA53637U (en) * | 2010-04-26 | 2010-10-11 | Наталья Алексеевна Егорова | METHOD FOR REGENERATING PLANTS OF CALLUS TISSUES OF Salvia sclarea L. in vitro |
| CN105393914A (en) * | 2014-09-12 | 2016-03-16 | 山东大学（威海） | Zostera marina isolated inflorescence callus induction method |
| CN106538382A (en) * | 2016-10-31 | 2017-03-29 | 江苏省中国科学院植物研究所 | A kind of method that eremochloa ophiuroides high-efficiency regeneration system is set up as explant with young fringe |
Non-Patent Citations (2)
| Title |
|---|
| 红根草的离体快速繁殖;黄炼栋等;《植物生理学通讯》;19981031;第34卷(第5期);第365页 * |
| 红根草的组织培养与快速繁殖研究;唐凤鸾等;《广西植物》;20060531;第26卷(第3期);第282-285页 * |
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