CN116548309B - Induction method of gardenia embryogenic callus - Google Patents
Induction method of gardenia embryogenic callus Download PDFInfo
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- CN116548309B CN116548309B CN202310544524.6A CN202310544524A CN116548309B CN 116548309 B CN116548309 B CN 116548309B CN 202310544524 A CN202310544524 A CN 202310544524A CN 116548309 B CN116548309 B CN 116548309B
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- 206010020649 Hyperkeratosis Diseases 0.000 title claims abstract description 97
- 230000000408 embryogenic effect Effects 0.000 title claims abstract description 95
- 230000006698 induction Effects 0.000 title claims abstract description 38
- 238000000034 method Methods 0.000 title claims abstract description 17
- 240000001972 Gardenia jasminoides Species 0.000 title description 6
- 241000157835 Gardenia Species 0.000 claims abstract description 81
- 239000001963 growth medium Substances 0.000 claims abstract description 44
- 230000001939 inductive effect Effects 0.000 claims abstract description 19
- 235000013399 edible fruits Nutrition 0.000 claims abstract description 17
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 238000005286 illumination Methods 0.000 claims description 11
- 238000001914 filtration Methods 0.000 claims description 9
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 8
- 229930006000 Sucrose Natural products 0.000 claims description 8
- 239000005720 sucrose Substances 0.000 claims description 8
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- 239000000706 filtrate Substances 0.000 claims description 4
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- 239000005971 1-naphthylacetic acid Substances 0.000 description 7
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- 241000196324 Embryophyta Species 0.000 description 4
- FAIXYKHYOGVFKA-UHFFFAOYSA-N Kinetin Natural products N=1C=NC=2N=CNC=2C=1N(C)C1=CC=CO1 FAIXYKHYOGVFKA-UHFFFAOYSA-N 0.000 description 4
- QANMHLXAZMSUEX-UHFFFAOYSA-N kinetin Chemical compound N=1C=NC=2N=CNC=2C=1NCC1=CC=CO1 QANMHLXAZMSUEX-UHFFFAOYSA-N 0.000 description 4
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- HXKWSTRRCHTUEC-UHFFFAOYSA-N 2,4-Dichlorophenoxyaceticacid Chemical compound OC(=O)C(Cl)OC1=CC=C(Cl)C=C1 HXKWSTRRCHTUEC-UHFFFAOYSA-N 0.000 description 1
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Classifications
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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
- A01H4/002—Culture media for tissue culture
-
- 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
-
- 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
- A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
- A01H6/76—Rubiaceae, e.g. Pentas
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/14—Measures for saving energy, e.g. in green houses
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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Cell Biology (AREA)
- Health & Medical Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Physiology (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a method for inducing embryogenic callus of gardenia, and relates to the technical field of plant tissue culture. The method comprises the steps of inoculating the gardenia explant into a culture medium for inducing the gardenia explant to generate embryogenic callus, and performing dark culture to obtain the gardenia embryogenic callus. The culture medium comprises 0.1-0.3 mg/L6-BA, 0.1-0.3 mg/L2,4-D and 12mg/L gardenia fruit extract. According to the invention, the influence of different plant growth regulators and different test materials on embryogenic callus induction of gardenia is researched, the proportion and concentration of the plant growth regulators are optimized, and the gardenia fruit extract is applied to embryogenic callus induction culture medium, so that the induction rate of embryogenic callus of gardenia is greatly improved.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a method for inducing gardenia embryogenic callus.
Background
Gardenia jasminoides Ellis, etc., is a plant of Gardenia genus of Rubiaceae family, is a evergreen shrub, and is 0.3-3 m high. The leaves of the plant are opposite or three-leaf wheel-shaped, the leaf shape is various, and the plant is round, needle-shaped and oval; the tender branch is often coated with short hairs, and the branch is cylindrical and gray; big flowers, white, mostly Shan Duoding life, with aromatic flavor. In China, gardenia is widely distributed in southeast and southwest regions such as Jiangsu, zhejiang, jiangxi, guangdong, yunnan, taiwan, hunan and the like, and overseas in Japan, korea, vietnam, laos, cambodia, india, nepal, pakistan, pacific islands, north America and the like. The gardenia has the advantages of strong adaptability, higher survival rate, excellent ornamental property, gorgeous and aromatic plant color, extremely high ornamental value, and extremely high economic value, and is not only a high-quality potted landscape flower, but also widely applied to pharmacology, garden application and the like.
At present, the main propagation method of the gardenia is layering or cutting, the propagation coefficient is low and limited by seasons, high-quality seed resources are difficult to preserve, and the requirement of multi-generation regeneration cannot be met. Previously, zodiac, 2005; wang Yoxuan, 2004; cui Sairan, 2012; peng Ying, 2014; tian Ya, 2020, etc. have been studied on tissue culture of Gardenia jasminoides Ellis in terms of rapid propagation of Gardenia jasminoides Ellis, callus induction, rooting, etc. Lays a foundation for the establishment of a subsequent gardenia regeneration system. Most of researches are focused on single tissue culture, the established rapid propagation system is not efficient enough, and the tissue culture of the gardenia is not researched from the aspects of a regeneration mechanism and the establishment of the efficient regeneration system.
The somatic embryogenesis is the technical means with the best application prospect at present, and the establishment of the fast embryogenesis and propagation system of the gardenia daughter cells not only can propagate high-quality gardenia seedlings in a large quantity, but also provides a technical basis for the breeding and preservation of high-quality gardenia germplasm resources, the regeneration of multiple generations and the verification and research of related gene functions of the gardenia. The induction of gardenia embryogenic callus is an important link for establishing a rapid embryogenic system of gardenia daughter cells, so that the research on the rapid embryogenic system is very necessary.
Disclosure of Invention
The invention aims to provide a method for inducing embryogenic callus of gardenia, which solves the problems in the prior art, and can effectively improve the induction rate of embryogenic callus of gardenia.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a culture medium for inducing gardenia explants to generate embryogenic callus, which comprises 0.1-0.3 mg/L6-BA, 0.1-0.3 mg/L2,4-D and 12mg/L gardenia fruit extract.
Further, the medium comprises 0.1 mg/L6-BA, 0.3mg/L2,4-D and 12mg/L gardenia fruit extract.
The invention also provides application of the culture medium for inducing the gardenia explants to generate embryogenic callus in inducing the gardenia explants to generate embryogenic callus.
The invention also provides an induction method of the gardenia embryogenic callus, which comprises the steps of inoculating the gardenia explant into a culture medium for inducing the gardenia explant to generate the embryogenic callus, and performing dark culture to obtain the gardenia embryogenic callus.
Further, the temperature of the dark culture is 20-25 ℃.
The invention also provides a culture medium for inducing gardenia non-embryogenic callus to generate embryogenic callus, wherein the culture medium comprises 4 mg/L6-BA, 0.2mg/LNAA and 12mg/L gardenia fruit extract.
Further, the culture medium also comprises 0.1mg/L KT.
The invention also provides an application of the culture medium for inducing the gardenia non-embryogenic callus to generate embryogenic callus.
The invention also provides a method for inducing the gardenia non-embryogenic callus to generate embryogenic callus, which comprises the steps of inoculating the gardenia non-embryogenic callus into the culture medium for inducing the gardenia non-embryogenic callus to generate embryogenic callus, and carrying out illumination culture.
Further, the temperature of the illumination culture is 20-25 ℃, the illumination time is 12h/d, and the illumination intensity is 1500-2000 LX.
The invention discloses the following technical effects:
according to the invention, the influence of different plant growth regulators and different test materials on embryogenic callus induction of gardenia is researched, the proportion and concentration of the plant growth regulators are optimized, and the gardenia fruit extract is applied to embryogenic callus induction culture medium, so that the induction rate of embryogenic callus of gardenia is greatly improved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a morphological observation of tender leaf induced embryogenic callus, non-embryogenic callus induced embryogenic callus and non-embryogenic callus; wherein a1, tender leaves induce embryogenic callus morphology; a2, observing the phenotype characteristics of the tender leaf induced embryogenic callus; b1, inducing embryogenic callus morphology by non-embryogenic callus; b2, observing the phenotype characteristics of the non-embryogenic callus induced embryogenic callus; c1, non-embryogenic callus morphology; c2, observing the phenotype characteristics of the non-embryogenic callus; the scales in a2, b2 and c2 are all 1mm;
FIG. 2 shows the results of observing the cell structures of tender leaf-induced embryogenic callus (a), non-embryogenic callus-induced embryogenic callus (b) and non-embryogenic callus (c).
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Example 1
1 test materials
The gardenia callus induced by the stem segments is picked from the gardenia germplasm resource nursery in a teacher and public slope forest farm of the academy of forestry of Hunan province.
2 Experimental methods
2.1 preparation of Gardenia fruit extract
Grinding dried fructus Gardeniae, sieving with 40 mesh sieve, adding 100mL distilled water into 10g powder, boiling, decocting in 80deg.C water bath for 1 hr, filtering, adding 100mL distilled water into the residue, decocting for 1 hr, filtering, mixing the two filtrates, filtering with 0.22 μm filter membrane, and making filtrate to 20mL to obtain fructus Gardeniae extract with concentration of 6mg/mL, and placing into 4 deg.C refrigerator for use.
2.2 preparation of Medium
MS is taken as a basic culture medium, plant growth regulators 2, 4-dichlorophenoxyacetic acid (2, 4-D), 6-benzylaminopurine (6-BA), 1-naphthylacetic acid (NAA) and Kinetin (KT) with different concentrations are added, sucrose is 3%, pH is adjusted to 5.8, agar is 6g/L, the agar is packaged into culture bottles, and the culture bottles are put into a high-pressure steam sterilization pot for sterilization for 20min under the conditions of 121 ℃ and 0.1Mpa, and are cooled and solidified for standby.
2.3 explant selection and pretreatment
The fresh picked tender leaves and stem segments are washed by warm water and detergent, soaked for 10min, and the stem segments are trimmed into small segments of 1.5 cm to 2 cm. The floating dust is brushed off by a small brush, the cleaning is carried out for 1 hour by flowing water, then the cleaning is carried out for three times by sterile water, and the cleaning is put into a shan nong antibacterial solution with the concentration of 3 percent for soaking for 10 hours.
2.4 Induction of callus
Under the aseptic condition, the stem segments after the disinfection treatment are put into a non-embryogenic callus induction culture medium, are subjected to dark culture for 10d, and are transferred into LED illumination culture, and the non-embryogenic callus is obtained through culture. The culture temperature is controlled at 20-25 ℃, the illumination is 12h/d, and the illumination intensity is 1500-2000 LX.
The non-embryogenic callus induction culture medium takes MS as a basic culture medium, 0.2wt% of shannong No. 1 and 3wt% of sucrose are added, 0.2mg/L of 6-BA and 2mg/L of NAA are respectively added, the pH value of the culture medium is 5.8, and the culture medium is placed in an autoclave for sterilization.
2.5 Induction of embryogenic callus
(1) Under the aseptic condition, the sterilized tender leaves and the non-embryogenic callus are respectively inoculated into an embryogenic callus induction culture medium for dark culture, and the culture temperature is 20-25 ℃. And observing and recording the induction condition and the growth change of the embryogenic callus in the culture process.
The embryogenic callus induction medium takes MS as a basic medium, 0.2wt% of shannong No. 1 and 3wt% of sucrose are added, and 0 to 0.3mg/L of 6-BA, 0 to 0.3mg/L of 2,4-D and 0 or 12mg/L of gardenia fruit extract are respectively added (see in particular Table 1). The pH value of the culture medium is 5.8, and the culture medium is placed in a high-pressure steam sterilizing pot for sterilization.
(2) Under the aseptic condition, inoculating the non-embryogenic callus induced by the stem section into an embryogenic callus induction culture medium for LED illumination culture, wherein the culture temperature is controlled to be 20-25 ℃, the illumination time is 12h/d, and the illumination intensity is 1500-2000 LX. The growth change of the calli was observed and recorded during the culture.
The embryogenic callus induction medium takes MS as a basic medium, 3wt% of sucrose is added, and 4mg/L of 6-BA, 0.2mg/L of NAA, 0.1mg/L of KT and 12mg/L of gardenia fruit extract are respectively added. The pH value of the culture medium is 5.8, and the culture medium is placed in a high-pressure steam sterilizing pot for sterilization.
3 results of experiments
Tender leaves of Gardenia jasminoides ellis and non-embryogenic callus are inoculated on embryogenic callus induction medium, and induction condition is observed after dark culture for 30 days. As is clear from Table 1, embryogenic callus could not be obtained without any hormone added to the medium, and the induction effect of 2,4-D on embryogenic callus was significant under dark culture conditions. When only 0.3mg/L of 2,4-D is added into the culture medium, part of embryogenic callus can be induced, and when 0.1mg/L of 6-BA, 0.3mg/L of 2,4-D and 12mg/L of gardenia fruit extract are added into the culture medium, the embryogenic callus induction rate is greatly improved, and the use of the 6-BA and the 2,4-D together can improve the embryogenic callus induction rate of the gardenia on the premise of adding the gardenia fruit extract. Embryogenic callus presents a pale yellow and soft state, and has large cell nucleus and more starch grains; non-embryogenic callus appeared in a white, soft state, water-soaked, no nuclei, and low starch granules (FIGS. 1 and 2).
TABLE 1 Effect of different mass concentrations of 6-BA and 2,4-D on the embryogenic callus induction of Gardenia jasminoides ellis
Note that: the different lower case English letters after the same column of data represent significant differences (P < 0.05); "-" means none.
The gardenia non-embryogenic callus is inoculated on an embryogenic callus induction culture medium, the induction condition is observed after light culture is carried out for 30 days, the result is shown in Table 2, and the induction effect of different hormone ratios on the gardenia embryogenic callus is different. As shown in Table 2, under the condition of adding 12mg/L of gardenia fruit extract, the induction effect of 6-BA and NAA on gardenia embryogenic callus under the condition of light culture is remarkable, and the induction rate can reach 96.67%. Embryogenic callus cannot be obtained without any hormone added to the medium. When 4mg/L of 6-BA, 0.2mg/L of NAA and 12mg/L of gardenia fruit extract are added into the culture medium, the induction rate of gardenia embryogenic callus can be improved. The callus showed pale yellow green compact state, and the nucleus was large, see fig. 1 and 2.
TABLE 2 Effect of different mass concentrations of 6-BA and NAA on embryogenic callus induction under light culture conditions
Note that: the different lower case English letters after the same column of data represent significant differences (P < 0.05); "-" means none.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (8)
1. A culture medium for inducing gardenia explants to generate embryogenic callus is characterized in that the culture medium is prepared by taking MS as a basic culture medium and adding 0.2wt% of shannong No. 1, 3wt% of sucrose, 0.1 mg/L6-BA, 0.3mg/L2,4-D and 12mg/L gardenia fruit extract;
the preparation method of the gardenia fruit extract comprises the following steps: grinding dried fructus Gardeniae, sieving with 40 mesh sieve, adding 100mL distilled water into 10g powder, boiling, decocting in 80deg.C water bath for 1 hr, filtering, adding 100mL distilled water into the residue, decocting for 1 hr, filtering, mixing the two filtrates, and filtering with 0.22 μm filter membrane to obtain fructus Gardeniae extract;
the gardenia explant is gardenia tender leaves.
2. Use of the culture medium according to claim 1 for inducing gardenia explants to produce embryogenic callus, wherein the gardenia explants are gardenia tender leaves.
3. A method for inducing embryogenic callus of gardenia, which is characterized by comprising the steps of inoculating a gardenia explant to the culture medium of claim 1, and performing dark culture to obtain embryogenic callus of gardenia;
the gardenia explant is gardenia tender leaves.
4. The method according to claim 3, wherein the temperature of the dark culture is 20 to 25 ℃.
5. A culture medium for inducing gardenia non-embryogenic callus to generate embryogenic callus is characterized in that MS is taken as a basic culture medium, and 3wt% of sucrose, 4 mg/L6-BA, 0.2mg/L NAA, 12mg/L gardenia fruit extract and 0 mg/L or 0.1mg/L KT are added to prepare the culture medium;
the preparation method of the gardenia fruit extract comprises the following steps: grinding dried fructus Gardeniae, sieving with 40 mesh sieve, adding 100mL distilled water into 10g powder, boiling, decocting in 80deg.C water bath for 1 hr, filtering, adding 100mL distilled water into the residue, decocting for 1 hr, filtering, mixing the two filtrates, and filtering with 0.22 μm filter membrane to obtain fructus Gardeniae extract;
the gardenia non-embryogenic callus is a non-embryogenic callus induced by gardenia stem segments.
6. The use of the culture medium according to claim 5 for inducing gardenia non-embryogenic callus to generate embryogenic callus, wherein the gardenia non-embryogenic callus is obtained by placing stem segments of gardenia into a non-embryogenic callus induction culture medium for culture;
the non-embryogenic callus induction culture medium is prepared by taking MS as a basic culture medium and adding 0.2wt% of shannong No. 1, 3wt% of sucrose, 0.2mg/L of 6-BA and 2mg/L of NAA.
7. A method for inducing gardenia non-embryogenic callus to generate embryogenic callus, which comprises the steps of inoculating the gardenia non-embryogenic callus to the culture medium of claim 5 and carrying out illumination culture;
the gardenia non-embryogenic callus is obtained by placing stem segments of gardenia into a non-embryogenic callus induction culture medium for culture;
the non-embryogenic callus induction culture medium is prepared by taking MS as a basic culture medium and adding 0.2wt% of shannong No. 1, 3wt% of sucrose, 0.2mg/L of 6-BA and 2mg/L of NAA.
8. The method according to claim 7, wherein the temperature of the light culture is 20-25 ℃, the light time is 12h/d, and the light intensity is 1500-2000 LX.
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| CN103430844A (en) * | 2013-08-13 | 2013-12-11 | 广州白云山明兴制药有限公司 | Method for gardenia tissue culture |
| CN104686361A (en) * | 2015-03-20 | 2015-06-10 | 浙江万里学院 | Induction and culture method of embryonic callus of grape |
| CN114747486A (en) * | 2022-04-08 | 2022-07-15 | 湖南省林业科学院 | Somatic embryogenesis and plant regeneration method for gardenia jasminoides ellis |
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