CN112997881A - Sedum aizoon explant sterilization method, callus induction culture medium and induction method - Google Patents
Sedum aizoon explant sterilization method, callus induction culture medium and induction method Download PDFInfo
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- CN112997881A CN112997881A CN202110145862.3A CN202110145862A CN112997881A CN 112997881 A CN112997881 A CN 112997881A CN 202110145862 A CN202110145862 A CN 202110145862A CN 112997881 A CN112997881 A CN 112997881A
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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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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Abstract
The invention provides a sterilization method of an exophyte of a sedum plant, which comprises the following steps: obtaining an exophyte of the sedum; soaking the Crassulaceae plant explant with alcohol; using 0.08-0.12 w/w% of HgCl2Sterilizing the explant of the Crassulaceae plant with the aqueous solution for 2.8-3.2 min; washing the Crassulaceae plant explant with sterile water to obtain sterilized Crassulaceae plant explant. The invention also provides an induction culture medium for sedum plant callus induction, which comprises the following components: MS culture medium, 5-8g/L agar, 25-35g/L sucrose, 2.4-2.6 mg/L6-BA and 0.09-0.11mg/L NAA. The invention also provides a sedum callus induction method, which uses the induction culture medium to induce and culture the explants of sedum plants sterilized by the method to form sedum callus. The method has high callus induction rate.
Description
Technical Field
The invention belongs to the technical field of garden biology, relates to a sterilization method of a sedum aizoon explant, a callus induction culture medium and an induction method, and particularly relates to a sterilization method of a sedum aizoon explant, a formula of a callus induction culture medium and an induction method.
Background
Sedum plumbizicola (Sedum plumbizinicola) belongs to Crassulaceae, is found in new species in Zhejiang province, and is favored to grow in Pb-and Zn-rich ore areas. The perennial meat herbage has large biomass, strong fertility and the highest absorption concentration of 1241mg/kg, is an important repairing material for repairing cadmium-polluted farmland soil and has wide prospect.
An efficient sterilization method and a callus induction method for the rhodiola sacra explant are lacked, and the development of the work of breeding the variety of the rhodiola sacra and the like is restricted.
Disclosure of Invention
The invention aims to provide an explant sterilization scheme and a callus induction culture medium formula for tissue culture of sedum plumbizincicola, and a screening method for the tissue culture of sedum plumbizincicola comprises HgCl2Sterilizing for a certain time, and screening with sterile water, wherein the culture medium comprises callus induction culture medium.
The technical scheme for realizing the purpose of the invention is to ensure the survival rate of the explant by screening through an explant sterilization method and provide the explant with good growth for callus induction. The callus induction culture medium improves the induction rate of explants by screening 6-BA and NAA combinations with different concentrations, and provides excellent callus for callus proliferation.
The method is mainly characterized in that: the test scheme mainly takes MS as a basic culture medium, selects complete and good leaves from healthy field seedlings as explants, and ensures the survival rate of the explants by screening through a sterilization method. The sterilized explants are inoculated into callus induction culture media added with the combination of hormones 6-BA and NAA with different mixture ratio concentrations, so that the callus induction rate is improved, and good-growing callus is provided for later-stage experiments, thereby realizing the cultivation of tissue culture seedlings of the Sedum plumbizincicola with excellent characters. The test operation is simple and convenient, and is beneficial to screening out the culture medium formula most suitable for the callus induction of the rhodiola plumbata.
More specifically, in order to solve the problems in the prior art, the first aspect of the present invention provides a sterilization method for an explant of a sedum plant, comprising the following steps:
(i) obtaining an exophyte of the sedum;
(ii) soaking the Crassulaceae plant explant with alcohol;
(iii) using 0.08-0.12 w/w% (e.g., 0.09 w/w%, 0.10 w/w%, 0.11 w/w%) of HgCl2Sterilizing the Crassulaceae explant with water solution for 2.8-3.2min (such as 2.9min, 3.0min, 3.1 min);
(iv) washing the Crassulaceae plant explant by sterile water to obtain a sterilized Crassulaceae plant explant.
In some embodiments, in step (i), the sedum explant is washed clean and used after rinsing with running water.
In some embodiments, in step (i), the rinsing is a 0.4-0.6 w/w% aqueous NaClO solution surface disinfection for 4-6 min.
In some embodiments, in step (ii), the alcohol is 70-80% (e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%) alcohol aqueous solution by volume.
In some embodiments, in step (ii), the soaking time is 25-35s (e.g., 26s, 27s, 28s, 29s, 30s, 31s, 32s, 33s, 34 s).
In some embodiments, in step (ii), the soaking is performed under stirring conditions.
In some embodiments, in step (ii), after the soaking, the sedum explants are rinsed with sterile water for 1.8-2.2min (e.g., 1.9min, 2.0min, 2.1 min).
In some embodiments, in step (ii), the rinsing with sterile water is performed under stirring conditions.
In some embodiments, in step (iii), the sterilization is performed under stirring conditions.
In some embodiments, in step (iv), the crassulaceae explants are rinsed with sterile water for 2-6 times (e.g., 3 times, 4 times, 5 times) with each rinse time being 0.8-2.2min (e.g., 0.9min, 1.0min, 1.1min, 1.2min, 1.3min, 1.4min, 1.5min, 1.6min, 1.7min, 1.8min, 1.9min, 2.0min, 2.1 min).
In some embodiments, in step (iv), the crassulaceae explants are rinsed with sterile water for 4 rinses, the first rinse being 1.8-2.2min (e.g., 1.9min, 2.0min, 2.1 min); flushing for a second time for 0.8-1.2min (e.g., 0.9min, 1.0min, 1.1 min); washing for the third time for 1.8-2.2min (e.g., 1.9min, 2.0min, 2.1 min); the fourth rinsing step is performed for 1.8-2.2min (e.g., 1.9min, 2.0min, 2.1 min).
In some embodiments, in step (iv), the rinsing with sterile water is performed under stirring conditions.
In some embodiments, the sedum plant is sedum plumbizincicola.
In some embodiments, the sedum explant is a leaf of a sedum.
In a second aspect, the present invention provides an induction medium for callus induction of sedum plants, comprising: liquid basal culture medium and phytohormones 6-BA and NAA, wherein the concentration of the phytohormones in the induction culture medium is 2.4-2.6mg/L of 6-BA and 0.09-0.11mg/L of NAA.
In some embodiments, the concentration of the hormone in the induction medium is 2.5mg/L6-BA and 0.1mg/L NAA.
In some embodiments, the liquid basal medium is MS liquid medium.
In some embodiments, the MS liquid medium has a composition of:
1900mg/L potassium nitrate, 285.85mg/L calcium chloride, 1650mg/L ammonium nitrate, 180mg/L magnesium sulfate, 170mg/L monopotassium phosphate, 0.025mg/L CoCo-6H2O, 0.025mg/L bluestone 5H2O, 6.2mg/L boric acid, 16.9mg/L manganese sulfate, 0.25mg/L sodium molybdate 2H2O, 8.6mg/L Zinc sulfate 7H2O, 0.83mg/L potassium iodide, 27.8mg/LFeSO4·7H2O、37.3mg/L Na2-EDTA·2H2O, 2.0mg/L glycine, 100mg/L inositol, 0.1mg/L thiamine VB1, 0.50mg/L nicotinic acid, 0.5mg/L pyridoxine VB6, and the balance water.
In some embodiments, the induction medium further comprises: 5-8g/L agar and 25-35g/L sucrose.
In some embodiments, the induction medium further comprises: 7g/L agar and 30g/L sucrose.
In some embodiments, the MS liquid medium has a PH of 5.75 to 5.95.
In some embodiments, the MS liquid medium has a PH of 5.8.
In some embodiments, the sedum plant is sedum plumbizincicola.
In some embodiments, the induction medium is used to induce callus formation from leaves of the sedum plant.
The third aspect of the invention provides a sedum plant callus induction method, which comprises the following steps: inducing the explant of the sedum plant to form the sedum callus using the induction medium of the second aspect of the invention.
In some embodiments, the induction culture is performed after the explant of the sedum plant is sterilized by the sterilization method according to the first aspect of the present invention.
In some embodiments, the temperature of the induction culture is 25 ± 2 ℃.
In some embodiments, the temperature of the induction culture is 25 ℃.
In some embodiments, the light intensity of the induction culture is 1800-3200 LX.
In some embodiments, the light intensity of the induction culture is 2000-3000 LX.
In some embodiments, the induction culture is performed at a light time of 12-16h per day.
In some embodiments, the light exposure time for the induction culture is 14h per day.
In some embodiments, the induction culture is for culture days 23-27 d.
In some embodiments, the induction culture is for a culture day of 25 d.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in further detail below.
Example 1: sterilization of Sedum plumbizincicola explants
(1) Explant harvesting
The original germplasm is collected from Zhejiang province, the test material is the open-air flowerpot cultivated sedum plumbizincicola, the conventional cultivation method is adopted, and the flowerpot is placed in a laboratory for a week to adapt to the indoor environment. After one week, young leaves of plants with consistent growth vigor and good growth conditions are taken as explants to be tested.
(2) Explant sterilization method screening
And (2) disinfecting the surface of the explant obtained in the step (1) with 0.5 w/w% NaClO aqueous solution for 5min, washing the surface with running water for 1h, putting the explant into a clean bench, soaking the explant in 75% alcohol aqueous solution by volume fraction for 30s in the clean bench, and washing the explant with sterile water for 2 min. A sterile combinatorial screen was then performed: HgCl with the mass fraction of 0.1 percent is added2The aqueous solution and sterile water were combined in a cycle, the specific cycle combination design is shown in table 1, and after the combination protocol was completed, the explants were rinsed twice with sterile water, 2min each time. Finally, the excess water is sucked up by sterile filter paper, and then the mixture is cut into small pieces with the square of 1cm and inoculated to MS0Culturing in culture medium (containing sucrose 30g/L and agar 7g/L) at pH5.8 and light intensity 2000lx under illumination of 14 hr per day and temperature 25 deg.C for 30 days, and counting the survival rate of each treated culture (i.e., culture time)Percentage of non-dead explants compared to inoculated explants after 30 days of culture), 9 flasks per group, 3 explants per flask, for a total of 5 45 flasks. After 20d, the experimental results were counted, and the average values are shown in Table 2.
MS0The components of the culture medium are as follows:
30g/L of sucrose;
7g/L of agar;
macroelements:
KNO3 1900m g/L；
NH4NO3 1650mg/L；
KH2PO4 170mg/L；
MgSO4·7H2O 370mg/L；
CaCl2 285.85mg/L；
trace elements:
H3BO4 6.2mg/L；
MnSO4·H2O 16.9mg/L；
ZnSO4·7H2O 8.6mg/L；
KI 0.83mg/L；
Na2MoO4·2H2O 0.25mg/L；
CuSO4·5H2O 0.025mg/L；
CoCl2·6H2O 0.025mg/L；
organic matter:
2mg/L of glycine;
thiamine hydrochloride (VB1)0.1 mg/L;
pyridoxine hydrochloride (VB6)0.5 mg/L;
inositol 100 mg/L;
nicotinic acid (VB3)0.5 mg/L;
the balance of water.
TABLE 1 explant Sterilization treatment design
(3) Sterilization protocol screening results
TABLE 2 screening results
Through screening, the optimal sterilization method comprises the following steps: 75% alcohol 30s + sterile water 2min + 0.1% HgCl23min + sterile water 2min + sterile water 1min + sterile water 2min, the survival rate is 94.6%.
Compared with the continuous use of the sterile water, the separate and multiple use of the sterile water can play a better role in cleaning and removing the HgCl in the plant tissues2。
In the operation of this example, the glass rod was used for continuous stirring during the sterile water washing, alcohol treatment and mercuric chloride disinfection.
Example 2: sedum plumbizincicola callus induction
The composition of the MS liquid medium is as follows:
macroelements:
1900mg/L potassium nitrate;
285.85mg/L calcium chloride;
1650mg/L ammonium nitrate;
180mg/L magnesium sulfate;
170mg/L potassium dihydrogen phosphate;
trace elements:
0.025mg/L copper sulfate;
6.2mg/L boric acid;
16.9mg/L manganese sulfate;
0.25mg/L sodium molybdate;
8.6mg/L zinc sulfate;
0.025mg/L cobalt chloride;
0.83mg/L potassium iodide;
iron salt:
27.8mg/LFeSO4·7H2O；
37.3mg/L Na2-EDTA·2H2O；
organic components:
2.0mg/L glycine;
100mg/L inositol;
0.50mg/L niacin;
0.1mg/L thiamine VB 1;
0.5mg/L pyridoxine VB 6;
the balance of water.
The test material was the well-grown explant leaf from example 1 after sterilization, and the leaf was divided into 0.5-1.0cm using sterile scissors2And inoculating the small leaf tissue blocks of the explants to an induction culture medium for culture, wherein the induction culture medium takes the MS liquid culture medium as a basic culture medium (wherein the concentration of sucrose is 30g/L, the concentration of agar powder is 7g/L), the pH is 5.8, the concentrations of 6-BA and NAA are shown in table 3, 9 bottles of each group and 3 explants of each bottle are contained, and 10 groups of 90 bottles are total. The basic culture medium is MS liquid culture medium, and the illumination culture conditions are unified: the temperature is 25 +/-2 ℃, the daily illumination time is 14h, the illumination intensity is 2000-3000 LX, and the culture days are 25 d. The induction rate is shown in Table 4.
TABLE 3 design of culture medium components for Sedum plumbizincicola induction test
Results of different induction medium tests
TABLE 4 results of different induction medium tests
As can be seen from Table 4, the induction rate of the Sedum plumbizincicola explant is 88.89% in the treatment 5, so that the callus induction culture medium of the Sedum plumbizincicola is MS +6-BA2.5mg/L + NAA0.1mg/L + sucrose 30g/L + agar powder 7g/L, and the pH is 5.8.
It will be appreciated by those skilled in the art that the invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The embodiments disclosed above are therefore to be considered in all respects as illustrative and not restrictive. All changes which come within the scope of or equivalence to the invention are intended to be embraced therein.
Claims (10)
1. A sterilization method of an explant of a sedum plant comprises the following steps:
(i) obtaining an exophyte of the sedum;
(ii) soaking the Crassulaceae plant explant with alcohol;
(iii) using 0.08-0.12 w/w% of HgCl2Sterilizing the Crassulaceae plant explant with water solution for 2.8-3.2 min;
(iv) washing the Crassulaceae plant explant by sterile water to obtain a sterilized Crassulaceae plant explant.
2. The sterilization method according to claim 1, wherein in step (i), the sedum explant is washed clean and used after being washed with running water;
preferably, in step (i), the cleaning is carried out by surface disinfection for 4-6min by 0.4-0.6 w/w% NaClO aqueous solution;
preferably, in step (ii), the alcohol is 70-80% alcohol aqueous solution by volume fraction;
preferably, in step (ii), the soaking time is 25-35 s;
preferably, in step (ii), the soaking is carried out under stirring conditions;
preferably, in step (ii), after the soaking, the sedum explants are rinsed with sterile water for 1.8-2.2 min;
preferably, in step (ii), the rinsing with sterile water is carried out under stirring conditions;
preferably, in step (iii), the sterilization is carried out under stirring conditions;
preferably, in step (iv), the Crassulaceae explants are rinsed with sterile water for 2-6 times, each for 0.8-2.2 min.
Preferably, in step (iv), the sedum explants are rinsed with sterile water for 4 times, the first rinse being 1.8-2.2 min; washing for the second time for 0.8-1.2 min; washing for the third time for 1.8-2.2 min; washing for 1.8-2.2min for the fourth time;
preferably, in step (iv), the rinsing with sterile water is carried out under stirring conditions.
3. The sterilization method according to claim 1, wherein the plant of the genus rhodiola is rhodiola crenulata.
4. The sterilization method according to claim 1, wherein the rhodiola explant is a leaf of a rhodiola.
5. An induction medium for callus induction in a plant of the genus sedum, the induction medium comprising: liquid basal culture medium and phytohormones 6-BA and NAA, wherein the concentration of the phytohormones in the induction culture medium is 2.4-2.6mg/L of 6-BA and 0.09-0.11mg/L of NAA.
6. The induction medium of claim 5, wherein the concentration of said hormone in said induction medium is 2.5mg/L6-BA and 0.1mg/L NAA;
preferably, the liquid basal medium is MS liquid medium;
preferably, the composition of the MS liquid medium is:
1900mg/L potassium nitrate, 285.85mg/L calcium chloride, 1650mg/L ammonium nitrate, 180mg/L magnesium sulfate, 170mg/L monopotassium phosphate, 0.025mg/L CoCo-6H2O, 0.025mg/L bluestone 5H2O, 6.2mg/L boric acid, 16.9mg/L manganese sulfate, 0.25mg/L sodium molybdate 2H2O, 8.6mg/L Zinc sulfate 7H2O, 0.83mg/L potassium iodide, 27.8mg/LFeSO4·7H2O、37.3mg/L Na2-EDTA·2H2O, 2.0mg/L glycine, 100mg/L inositol, 0.1mg/L thiamine VB1, 0.50mg/L nicotinic acid, 0.5mg/L pyridoxine VB6, and the balance water;
preferably, the induction medium further comprises: 5-8g/L agar and 25-35g/L sucrose;
preferably, the induction medium further comprises: 7g/L agar, 30g/L sucrose;
preferably, the pH of the MS liquid medium is 5.75-5.95;
preferably, the pH of the MS liquid medium is 5.8.
7. The induction medium of claim 5, wherein the Crassulaceae plant is Sedum plumbizincicola;
preferably, the induction medium is used to induce callus formation from leaves of the sedum plant.
8. A sedum plant callus induction method comprises the following steps: inducing explants of a cultured Crassulaceae plant to form the Crassulaceae plant callus using the induction medium of any one of claims 5-7.
9. The induction method according to claim 8, wherein the induction culture is performed after the explant of the plant of Crassulaceae is sterilized by the sterilization method according to any one of claims 1 to 4.
10. The induction method according to claim 8, wherein the temperature of the induction culture is 25 ± 2 ℃;
preferably, the temperature of the induction culture is 25 ℃;
preferably, the illumination intensity of the induction culture is 1800-3200 LX;
preferably, the illumination intensity of the induction culture is 2000-3000 LX;
preferably, the illumination time of the induction culture is 12-16h per day;
preferably, the illumination time of the induction culture is 14h per day;
preferably, the induction culture is carried out for 23-27 days;
preferably, the induction culture is performed for 25 days.
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Application publication date: 20210622 |