CN108271692B - Method for directly differentiating cotton explant into embryogenic callus and culture medium - Google Patents
Method for directly differentiating cotton explant into embryogenic callus and culture medium Download PDFInfo
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- CN108271692B CN108271692B CN201810141299.0A CN201810141299A CN108271692B CN 108271692 B CN108271692 B CN 108271692B CN 201810141299 A CN201810141299 A CN 201810141299A CN 108271692 B CN108271692 B CN 108271692B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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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
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Abstract
The invention discloses a method for directly differentiating cotton explants into embryogenic callus and a culture medium. The method for directly differentiating the cotton explant into the embryogenic callus provided by the invention comprises the following steps: placing the cotton explant in a callus induction culture medium for culture; the callus induction culture medium contains uniconazole with the concentration of 2.5 mg/L. The uniconazole is added into the callus induction culture medium. The method can enable the explant to obtain the embryogenic callus in about 45 days, so that the long process from the explant to the callus and then to the embryogenic callus is skipped, the time for obtaining the cotton embryonic stem cells is greatly shortened, the differentiation efficiency is improved, and the synchronous uniform differentiation of the cotton tissue is realized. The invention will play an important role in the tissue culture, gene function verification and variety improvement of cotton.
Description
Technical Field
The invention relates to the field of plant tissue culture, in particular to a method for directly differentiating cotton explants into embryogenic callus and a culture medium.
Background
The completion of cotton whole genome sequencing makes it faster and simpler to confirm and separate a large number of genes, and the verification of gene functions requires a reliable and effective genetic transformation system to transform the genes into cotton to verify the practicability of candidate genes. The genetic transformation process of cotton mainly comprises the steps of infecting an explant by agrobacterium containing a candidate gene nucleic acid sequence on a plant expression vector, forming undifferentiated callus, inducing embryogenic callus generation, inducing embryo and regenerating seedling generation. The process from callus to embryogenic callus belongs to a bottleneck period in the regeneration process of all cotton varieties, and most of the cotton which can not be regenerated can not pass through the bottleneck period.
The cotton transformation and regeneration process usually takes about 10-11 months, with 1/3(4-5 months) time for embryogenic callus acquisition.
Disclosure of Invention
The invention aims to provide a method and a culture medium for directly differentiating cotton explants into embryogenic callus, wherein the time for obtaining the embryogenic callus can be shortened to 2 months, the embryogenic callus can be synchronously differentiated in a whole block, the tissue has uniform texture, uniform growth state and consistent differentiation time.
First, the present invention claims a method for direct differentiation of cotton explants into embryogenic callus.
The method for directly differentiating the cotton explant into the embryogenic callus provided by the invention specifically comprises the following steps: placing the cotton explant in a callus induction culture medium for culture; the callus induction medium contains uniconazole with the concentration of 1.0-2.5mg/L (such as 2.0-2.5mg/L, more specifically such as 2.5 mg/L).
Second, the present invention claims a method for increasing the efficiency of efficient regeneration of cotton plants.
The method for improving the efficient regeneration efficiency of the cotton plants, provided by the invention, specifically comprises the following steps:
(a1) culturing cotton explants on a callus induction medium to produce callus and embryogenic callus; the callus induction culture medium contains uniconazole with the concentration of 1.0-2.5mg/L (such as 2.0-2.5mg/L, more specifically such as 2.5 mg/L);
(a2) culturing the embryogenic callus on an embryo maturation medium to produce mature embryos;
(a3) culturing the mature embryos on a shoot forming medium to produce regenerated cotton seedlings.
In both methods, the callus induction medium may be specifically a medium obtained by adding uniconazole, KT, 2,4-D, glucose and gel (Gelrite gellan gum) to MSB minimal medium; the final concentration of uniconazole in the callus induction medium is 1.0-2.5mg/L (such as 2.0-2.5mg/L, more specifically such as 2.5mg/L), the final concentration of KT is 0.005-0.15mg/L (such as 0.1mg/L), the final concentration of 2,4-D is 0.001-0.15mg/L (such as 0.1mg/L), the final concentration of glucose is 20-35g/L (such as 30g/L), and the final concentration of gel (Gelrite gellan gum) is 2.0-2.5g/L (such as 2.0 g/L); the callus induction medium has a pH of 5.8-6.5 (e.g., 5.8).
In the second method, the embryo maturation medium may specifically be a medium obtained by adding IAA, KT, glucose and gel (Gelrite gellan gum) to MSB minimal medium; the final concentration of IAA in the embryo maturation medium is 0.001-0.01mg/L (such as 0.005mg/L), the final concentration of KT is 0.005-0.15mg/L (such as 0.005mg/L), the final concentration of glucose is 20-35g/L (such as 30g/L), and the final concentration of Gel is 2.0-2.5g/L (such as 2.0 g/L); the pH of the embryo maturation medium is 6.0-6.8 (e.g., 6.8).
In the second method, the seedling medium may be specifically a medium obtained by adding KT, 6-BA, sucrose and gel (Gelrite gellan gum) to MSB minimal medium; the final concentration of KT in the seedling culture medium is 0.001-0.01mg/L (such as 0.01mg/L), the final concentration of 6-BA is 0.005-0.2mg/L (such as 0.01mg/L), the final concentration of sucrose is 20-35g/L (such as 30g/L), and the final concentration of Gel is 2.0-2.5g/L (such as 2.5 g/L); the pH of the shoot medium is 6.0-6.8 (e.g., 6.8).
In the invention, the solvent of the MSB minimal medium is water, and the solutes and the concentrations are as follows: KNO3 1.90g·L-1；NH4NO3 1.65g·L-1；MgSO4·7H2O 0.37g·L-1；KH2PO4 0.17g·L-1；CaCl2·2H2O0.44g·L-1；MnSO4·H2O 16.900mg·L-1；ZnSO4·7H2O 8.600mg·L-1；H3BO3 6.200mg·L-1；KI 0.830mg·L-1；NaMnO4·2H2O 0.250mg·L-1；CuSO4·5H2O 0.025mg·L-1；CoCl·6H2O0.025mg·L-1；FeSO4·7H2O 37.3mg·L-1；Na2EDTA 27.8mg·L-1；VB1 10.0mg·L-1；VB6 1.0mg·L-1(ii) a Nicotinic acid 1.0 mg.L-1(ii) a Inositol 100.0 mg.L-1。
In the first method, the culture conditions for culturing the cotton explant in the callus induction medium may be: the culture medium is cultured at the temperature of 28 +/-2 ℃ and the illumination intensity of 1500-2000 Lx for 14 hours in illumination and 10 hours in dark every day.
In the second method, in step (a1), the culture conditions for culturing the cotton explant on the callus induction medium may be: the culture medium is cultured at the temperature of 28 +/-2 ℃ and the illumination intensity of 1500-2000 Lx for 14 hours in illumination and 10 hours in dark every day. In step (a2), the culture conditions for culturing the embryogenic callus on the embryo maturation medium may specifically be: the culture medium is cultured at the temperature of 28 +/-2 ℃ and the illumination intensity of 1500-2000 Lx for 14 hours in illumination and 10 hours in dark every day. In the step (a3), the culture conditions for culturing the mature embryo on the seedling medium may specifically be: the culture medium is cultured at the temperature of 28 +/-2 ℃ and the illumination intensity of 1500-2000 Lx for 14 hours in illumination and 10 hours in dark every day.
Thirdly, the following culture medium and a complete set of culture medium are claimed.
The culture medium is the callus induction culture medium described in any one of the preceding paragraphs.
The complete set of medium consists of the callus induction medium as described in any one of the preceding, the embryo maturation medium as described in any one of the preceding and the shoot formation medium as described in any one of the preceding.
Fourthly, the invention claims the use of said medium or said set of media in any of the following:
(b1) cotton tissue culture;
(b2) directly differentiating cotton explants into embryogenic callus;
(b3) improving the regeneration efficiency of cotton plants.
In one embodiment of the invention, the cotton explant is specifically the hypocotyl of cotton. More specifically, the cotton explant is a hypocotyl of a cotton aseptic seedling cultured for 7 days; further, the hypocotyl can be cut into 2-3cm sections for use.
The two methods provided by the invention, the culture medium and the complete set of culture medium provided by the invention are suitable for all cotton varieties, such as medium cotton series, collotype cotton series or sponge series, and the like, and are particularly suitable for the medium cotton station 24.
The uniconazole is added into the callus induction culture medium. The method can lead the explant to be differentiated into the embryogenic callus in about 45 days, and the whole explant is differentiated into the embryogenic callus in 60 days, thereby skipping the lengthy process from the explant to the callus and then to the embryogenic callus, combining the callus induction culture medium and the callus differentiation culture medium, greatly shortening the time for obtaining the cotton embryonic stem cells, improving the differentiation efficiency and realizing the synchronous uniform differentiation of the cotton tissue. Can greatly improve the transformation efficiency of the agrobacterium-mediated method and shorten the transformation period. The invention will play an important role in the tissue culture, gene function verification and variety improvement of cotton.
Drawings
FIG. 1 shows the results of cotton explants cultured on callus induction medium to generate callus and embryogenic callus. A is a treatment group and a control group which are cultured for 45 days; b is the treated group and the control group at 60 days of culture.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The MSB minimal medium used in the following examples was prepared from macroelement mother liquor (MSI), microelement mother liquor (MSII), ferric salt mother liquor, and B5 vitamin mother liquor. The method comprises the following specific steps:
(1) preparation of macroelement mother liquor (MS I)
As shown in table 1. Weighing the following medicines, removing CaCl2·2H2And (3) dissolving O separately, mixing the dissolved O into the mother liquor, dissolving the rest medicines together, finally metering the volume to 1L, and storing at normal temperature for later use, wherein 50ml of the mother liquor is required to be taken if 1L of the MSB minimal medium is prepared.
TABLE 1 composition of macronutrient mother liquor MSI
(2) Preparation of microelement mother liquor (MS II)
As shown in table 2. Weighing the following medicines, dissolving the medicines together, and adding a small amount of 1 mol. L if a small amount of precipitate exists-1Dissolving HCl, diluting to 1L, and storing at 4 deg.C, if preparing 1L MSB minimal medium, taking 5ml of the mother liquid.
TABLE 2 composition of microelement mother liquor MS II
(3) Preparation of mother liquor of iron salt
As shown in table 3. Weighing the following medicines, dissolving one by one, mixing, heating, placing in dark, cooling to room temperature, diluting to 0.5L, and storing at 4 deg.C for use, if preparing 1L MSB minimal medium, absorbing 5ml of the mother liquor.
TABLE 3 composition of mother liquor of iron salts
(4) B5 vitamin mother liquor preparation:
as shown in table 4. Weighing the following medicines, and dissolving one by one, wherein a small amount of 1 mol.L of nicotinic acid is used-1Dissolving HCl, mixing with other medicines, diluting to 0.5L, and refrigerating to obtain 10ml of the mother solution if 1L of MSB minimal medium is prepared.
TABLE 4B5Components of vitamin mother liquor
Example 1 hypocotyl tissue culture in Mitsuga institute 24
One, preferred method of preparation of the medium:
callus induction medium: on the basis of MSB basic culture medium, 2.5mg/L uniconazole, 0.1mg/L KT, 0.1 mg/L2, 4-D, 30g/L glucose, 2g/L Gel and pH 5.8 are added. Each concentration was the final concentration of the corresponding substance in the callus induction medium.
Embryo maturation medium: on the basis of MSB basic culture medium, IAA 0.005mg/L, KT0.005mg/L, glucose 30g/L, Gel 2g/L and pH 6.8 are added. Each concentration is the final concentration of the corresponding substance in the embryo maturation medium.
Seedling culture medium: 0.01mg/L of KT, 0.01mg/L of 6-BA, 30g/L of sucrose, 2.5g/L of Gel and 6.8 of pH are added on the basis of the MSB basic culture medium. Each concentration is the final concentration of the corresponding substance in the seedling medium.
Conventional embryogenic callus induction medium: IAA0.05mg/L, KT 0.15mg/L, glucose 25g/L, Gel 2.5g/L and pH 6.5 are added on the basis of MSB basic culture medium. Each concentration is the final concentration of the corresponding substance in the seedling medium.
The hormone and other components of the culture medium are added into the culture medium before sterilization, and the pH of the culture medium is adjusted with 1MKOH before sterilization, and then the culture medium is heated at 121 deg.C and 1.1kg/cm2High temperature and high pressure sterilization for 14min under the pressure of (1).
Tissue culture of hypocotyl 24 in Zhongmian institute
1. Culturing cotton explants on callus induction medium to produce callus and embryogenic callus
Treatment group: and taking the hypocotyl of the cotton aseptic seedling cultured for 7 days as an explant, cutting the hypocotyl into 2-3cm sections, then placing the sections on the callus induction culture medium prepared in the step one, and culturing the sections under the conditions of the artificial illumination intensity of 1500-2000 Lx at 28 +/-2 ℃ for 14 hours of illumination culture and 10 hours of dark culture every day.
Control group: and (4) removing uniconazole in the callus induction culture medium prepared in the step one, and performing the same operation as the treatment group.
The results show that: after 45 days, embryogenic callus appeared in the treated groups, while differentiated embryogenic callus did not appear in the control group (A in FIG. 1); the treated group cultured for 60 days was differentiated into embryogenic callus in its entire block, whereas the control group required subculture to the conventional embryogenic callus induction medium again for 2 months before differentiation (B in FIG. 1).
2. Culturing the embryogenic callus on an embryo maturation medium to produce a mature embryo
And (3) inoculating the embryogenic callus obtained from the treatment group in the step (1) to the embryo maturation culture medium prepared in the step (I), and culturing at the temperature of 28 +/-2 ℃, the artificial illumination intensity of 1500-2000 Lx under the conditions of 14-hour illumination culture and 10-hour dark culture every day. Mature embryos were obtained after 60 days.
3. Culturing the mature embryos on a seedling medium to produce regenerated cotton seedlings
And (3) inoculating the mature embryo obtained in the step (2) to the seedling culture medium prepared in the step (I), and culturing at the temperature of 28 +/-2 ℃, the artificial illumination intensity of 1500-2000 Lx under the conditions of 14-hour illumination culture and 10-hour dark culture every day. And 3-4 months later, obtaining regenerated cotton seedlings.
Claims (7)
1. A method for directly differentiating cotton explants into embryogenic callus comprises the following steps: placing the cotton explant in a callus induction culture medium for culture;
the callus induction culture medium is obtained by adding uniconazole, KT, 2,4-D, glucose and Gel into an MSB basic culture medium; the final concentration of uniconazole in the callus induction culture medium is 1.0-2.5mg/L, KT and is 0.005-0.15mg/L, the final concentration of 2,4-D is 0.001-0.15mg/L, and the final concentration of glucose is 20-35g/L, Gel and is 2.0-2.5 g/L;
the cotton explant is the hypocotyl of cotton.
2. The method of claim 1, wherein: the culture conditions when the cotton explant is placed in the callus induction culture medium for culture are as follows: the culture medium is cultured at the temperature of 28 +/-2 ℃ and the illumination intensity of 1500-2000 Lx for 14 hours in illumination and 10 hours in dark every day.
3. A method of increasing the regeneration efficiency of a cotton plant comprising the steps of:
(a1) culturing cotton explants on a callus induction medium to produce callus and embryogenic callus;
the callus induction culture medium is obtained by adding uniconazole, KT, 2,4-D, glucose and Gel into an MSB basic culture medium; the final concentration of uniconazole in the callus induction culture medium is 1.0-2.5mg/L, KT and is 0.005-0.15mg/L, the final concentration of 2,4-D is 0.001-0.15mg/L, and the final concentration of glucose is 20-35g/L, Gel and is 2.0-2.5 g/L;
the cotton explant is the hypocotyl of cotton;
(a2) culturing the embryogenic callus on an embryo maturation medium to produce mature embryos;
the embryo maturation culture medium is obtained by adding IAA, KT, glucose and Gel into the MSB minimal medium; the final concentration of IAA in the embryo maturation medium is 0.001-0.01mg/L, KT, 0.005-0.15mg/L and the final concentration of glucose is 20-35g/L, Gel, 2.0-2.5 g/L;
(a3) culturing the mature embryos on a seedling medium to produce regenerated cotton seedlings;
the seedling culture medium is obtained by adding KT, 6-BA, sucrose and Gel into an MSB minimal medium; the final concentration of KT in the seedling culture medium is 0.001-0.01mg/L, the final concentration of 6-BA is 0.005-0.2mg/L, and the final concentration of sucrose is 20-35g/L, Gel is 2.0-2.5 g/L.
4. The method of claim 3, wherein: in step (a1), the culture conditions for culturing the cotton explant on the callus induction medium are: the illumination intensity is 1500-2000 Lx at the temperature of 28 +/-2 ℃, the illumination culture is carried out for 14 hours every day, and the dark culture is carried out for 10 hours;
in step (a2), the culture conditions for culturing said embryogenic callus on said embryo maturation medium are: the illumination intensity is 1500-2000 Lx at the temperature of 28 +/-2 ℃, the illumination culture is carried out for 14 hours every day, and the dark culture is carried out for 10 hours;
in step (a3), the culturing conditions for culturing the mature embryos on the shoot medium are: the culture medium is cultured at the temperature of 28 +/-2 ℃ and the illumination intensity of 1500-2000 Lx for 14 hours in illumination and 10 hours in dark every day.
5. A culture medium characterized by: the culture medium is the callus induction culture medium according to claim 1.
6. The complete set of culture medium is characterized in that: the medium set is composed of the callus induction medium described in claim 3, the embryo maturation medium described in claim 3, and the shoot formation medium described in claim 3.
7. Use of the culture medium of claim 5 or the set of culture media of claim 6 in any one of:
(b1) cotton tissue culture;
(b2) directly differentiating cotton explants into embryogenic callus; the cotton explant is the hypocotyl of cotton;
(b3) improving the regeneration efficiency of cotton plants.
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