CN116584381A - Litsea cubeba embryogenic callus induction medium and induction method - Google Patents
Litsea cubeba embryogenic callus induction medium and induction method Download PDFInfo
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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
-
- 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
- Y02P60/40—Afforestation or reforestation
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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a litsea cubeba embryogenic callus induction culture medium and an induction method, wherein 6-BA, 2,4-D, IAA and pH 5.85 are added in an MS culture medium, fresh and tender litsea cubeba fruits which are provided with embryos and are immature are collected, the surface dirt of the fruits is thoroughly cleaned and removed, the outer skin of the meat is peeled off under the aseptic condition, then 75% ethanol is used for soaking for 3-5 min, and sterile water is used for flushing for a plurality of times after shaking sterilization; sucking the sterilized litsea cubeba with sterile filter paper, peeling off the epicarp, and cutting the seeds along the central axis of the embryo; inoculating the embryogenic callus to an induction culture medium for induction culture to obtain embryogenic callus; fresh embryogenic callus with good growth condition is selected and placed in a proliferation culture medium for proliferation culture. The invention realizes the embryogenic callus induction of litsea cubeba for the first time, the embryogenic callus induction rate reaches more than 17.50 percent, and a new way is opened up for the utilization and industrialization development of litsea cubeba resources.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, relates to an induction culture medium and an induction method based on the culture medium, and in particular relates to a litsea cubeba embryogenic callus induction culture medium and a litsea cubeba embryogenic callus induction method based on the litsea cubeba embryogenic callus induction culture medium.
Background
Litsea cubeba is woody spice tree species with development and utilization values in the south of China, but the fine variety breeding and industrial scale is still a problem to be solved urgently.
At present, the litsea cubeba artificial cultivation scale is smaller, and is mainly limited by an efficient breeding technology of excellent seedlings, wherein the seedling breeding technology mainly comprises 4 modes of seed propagation, cutting propagation, grafting propagation and tissue culture.
Seed propagation is low in germination rate, high in economic cost, serious in offspring property separation, difficult to maintain the excellent properties of parents, and unsuitable for large-scale forestation or industrialized development.
The wound of the litsea cubeba branch is easily oxidized due to the influence of metabolites such as citral, and the propagation process is limited by natural factors, so that the cutting and grafting propagation seedling raising coefficients of the litsea cubeba are low.
Compared with the three propagation technologies, the tissue culture has the advantages of high propagation coefficient, short period, no influence of natural conditions, stable heredity and the like. An existing tissue culture method, for example, a litsea cubeba tissue culture method disclosed in CN111642391A, uses stem segments with buds of litsea cubeba spring as explants for culture.
Placing the explant in a culture medium with a specific hormone ratio, inducing embryogenic callus, differentiating the embryogenic callus to form somatic embryos, and finally forming complete plants. This process is known as an indirect pathway of somatic embryogenesis. Somatic embryogenesis is one of the modes of tissue culture, has the advantages of large quantity, high speed, complete structure, high regeneration rate and the like, and has wide application prospects in the aspects of nursery stock rapid propagation, genetic engineering, germplasm resource preservation and the like. The high-quality embryogenic callus is a precondition for obtaining somatic embryos, and is also an ideal material for researching single-cell differentiation and gene totipotency expression of plants. However, no study on embryogenic callus culture of litsea cubeba is known in the prior art.
Disclosure of Invention
In view of the above, an object of the present invention is to provide a litsea cubeba embryogenic callus induction medium.
The second purpose of the invention is to provide a litsea cubeba embryogenic callus induction culture method.
The inventor continuously reforms and innovates through long-term exploration and trial and repeated experiments and efforts, and the technical scheme provided by the invention is that in order to solve the technical problems, a litsea cubeba embryogenic callus induction culture medium is provided, and 0.5-1 mg.L is added in an MS culture medium -1 6-BA、0.2～1.0mg·L -1 2,4-D、0.2～0.5mg·L -1 IAA，pH 5.85。
According to one embodiment of the litsea cubeba embryogenic callus induction medium, 20-30 g/L sucrose or 20-40 g/L glucose is added into the induction medium.
According to one embodiment of the litsea cubeba embryogenic callus induction medium according to the present invention, 700mg/L acid hydrolyzed casein is also added to the induction medium.
According to one embodiment of the litsea cubeba embryogenic callus induction medium according to the invention, 7g/L agar is also added to the induction medium.
The invention also provides a litsea cubeba embryogenic callus induction culture method, which comprises the following steps:
s1, collecting fresh and tender fruits of immature litsea cubeba with embryos, and immediately processing or preserving at 4-8 ℃ for treatment;
s2, pretreatment of explants
Thoroughly cleaning to remove the mud on the surface of the fruit, peeling off the outer seed coats of the meat under the aseptic condition, soaking for 3-5 min by 75% ethanol, shaking and sterilizing, and washing for a plurality of times by using sterile water;
s3, induction culture
Sucking the sterilized litsea cubeba with sterile filter paper, peeling off the epicarp, and cutting the seeds along the central axis of the embryo; inoculating the strain to the induction culture medium according to any one of claims 1 to 4 for induction culture to obtain embryogenic callus;
s4, embryogenic callus proliferation and differentiation induction culture
Fresh embryogenic callus with good growth condition is selected and placed in a proliferation culture medium for proliferation culture.
According to one embodiment of the litsea cubeba embryogenic callus induction culture method of the invention, the proliferation medium comprises the following components: the MS culture medium is added with 0.5 mg.L -1 6-BA、0.1mg·L -1 2,4-D、0.25mg·L -1 IAA。
According to one embodiment of the method for inducing culture of embryogenic litsea cubeba callus, the proliferation medium component further comprises 30g/L sucrose or 40g/L glucose, 700mg/L acid hydrolyzed casein and 7g/L agar.
According to one embodiment of the method for inducing and culturing litsea cubeba embryogenic callus, the embryo in the immature litsea cubeba fresh and tender fruit is a young embryo.
According to one embodiment of the litsea cubeba embryogenic callus induction culture method of the invention, the young embryo is a spherical embryo, a heart-shaped embryo, a torpedo embryo or a cotyledon-shaped embryo.
According to one embodiment of the litsea cubeba embryogenic callus induction culture method, the culture condition is dark culture, and the temperature is 24+/-2 ℃.
Compared with the prior art, one of the technical schemes has the following advantages:
a) The invention realizes the induction of the litsea cubeba embryogenic callus for the first time, and opens up a new way for the utilization and industrialization development of litsea cubeba resources. The embryogenic callus induction stage is a key stage of recovering totipotency of differentiated somatic cells, is influenced by a plurality of internal and external factors such as explant types, explant development stage, proportion of hormone additives, culture mode, culture conditions and the like, and determines whether the later stage somatic embryogenesis can be successful or not, and is an important link in somatic embryogenesis research.
b) Compared with the organogenesis way, the somatic embryo produced by the method has obvious bipolarity, and has the advantages of larger propagation amount, higher propagation efficiency, more stable inheritance and the like.
c) The embryogenic callus induction scheme provided by the invention is a primary link in an indirect way of somatic embryogenesis and is also a premise for obtaining a large number of somatic embryos.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some examples of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a morphological diagram of litsea cubeba fruits, seeds and embryos.
FIG. 2 is a diagram of a litsea cubeba immature embryo induced callus wherein, a. White compact form; b. white loose type; c. embryogenic callus; d. somatic embryos; NEC, non-embryogenic callus group; EC embryogenic callus; SE, somatic embryo.
FIG. 3 is a process of generating embryogenic callus and somatic embryos from litsea cubeba. Wherein, EC is embryogenic callus; SE, somatic embryo.
Detailed Description
The following description is of one embodiment with reference to the accompanying drawings.
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, based on the embodiments of the invention, which are apparent to those of ordinary skill in the art without inventive faculty, are intended to be within the scope of the invention. Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention.
It should be noted that: like reference numerals and letters denote like items in the following figures, and thus, once an item is defined in one figure, it may not be further defined and explained in the following figures.
Example 1
The litsea cubeba embryogenic callus induction medium described in the example is a theoretical optimal medium formula deduced based on the analysis of the stepwise test data: the MS culture medium is added with 0.5 mg.L -1 6-BA、1.0mg·L -1 2,4-D、0.2mg·L -1 IAA, 40g/L glucose, 700mg/L acid hydrolyzed casein and 7g/L agar, pH 5.85.
The litsea cubeba seed development stage has obvious influence on the litsea cubeba embryogenic callus induction effect, and the immature seed preservation mode and preservation time also have obvious influence on the litsea cubeba embryogenic callus induction effect, so that the induction material limits the verification of the inventor team on the theoretical optimal culture medium formula.
Nevertheless, the inventors have demonstrated through experiments that the hormone combination of 6-BA, 2,4-D and IAA is capable of achieving the induction of embryogenic callus of litsea cubeba, and have achieved induction effects that can be applied in industry.
According to experimental data obtained during the course of the inventors' study: the inventors used MS as a minimal medium, and added different concentrations (mg.L) -1 ) 2,4-D (0.2, 0.5, 1.0, 2.0), 6-BA (0.5, 1.0)1.5, 2.0), IAA (0.2, 0.5), NAA (0.2, 0.5), KT (1.0, 2.0) hormone combination screening experiments, by L 9 (3 4 ) Orthogonal experiment design and single factor completely random experiment design, each treatment set is repeated 4 times, 5 litsea cubeba embryos are inoculated in a culture dish for each repetition to carry out experiments, and the addition of 0.5 mg.L in MS culture medium is finally confirmed by data analysis -1 6-BA、1.0mg·L -1 2,4-D、0.2mg·L -1 The combination of IAAs is the best combination, see table 1.
TABLE 1 litsea cubeba callus induction L 9 (3 4 ) Analysis of the results of orthogonal experiments
Different hormone ratios can produce callus, but the difference of callus induction rate is obvious. For L 9 (3 4 ) The result shows that the primary and secondary orders of the factors are IAA after the result of the callus induction of the orthogonal experiment is subjected to extremely poor analysis>2,4-D>The 6-BA, IAA concentration had a more significant effect on callus induction rate when IAA concentration was 0.2 mg.L -1 When the callus is healed, the average induction rate is highest and reaches 65.99 percent; the concentration of 2,4-D is 1.0mg.L -1 When the callus is healed, the average induction rate is highest and reaches 65.81 percent; 6-BA concentration was 0.5 mg.L -1 When the callus is used, the average induction rate is highest and reaches 64.11 percent.
In the orthogonal test, 1.5 mg.L -1 6-BA、1.0mg·L -1 2,4-D、0.2mg·L -1 The callus induction rate of IAA treatment is 79.37%, and based on data analysis, 0.5 mg.L can be reasonably expected -1 6-BA、1.0mg·L -1 2,4-D、0.2mg·L -1 The IAA hormone combination has better callus induction effect.
Referring to Table 2, different carbon sources can generate callus, and the difference of callus induction rate is remarkable (P < 0.05), wherein from the viewpoint of callus induction rate, glucose > sucrose > maltose, and when the glucose concentration is 40g/L, the callus induction rate is highest and can reach 100.00%.
TABLE 2 Effect of different carbon sources on Litsea cubeba callus and embryogenic callus induction
Example 2
The litsea cubeba embryogenic callus induction culture medium described in the embodiment is a staged result obtained by screening based on single-factor test design in the research and test process of the inventor, and is found by different hormone type selection and concentration setting induction tests that the hormone ratio has very important influence on whether litsea cubeba embryogenic callus is successfully induced or not, and is shown in table 3.
As is clear from Table 3, in the culture medium (treatments 1, 2, 3, 4) in which KT and another hormone (2, 4-D, IAA, NAA) were simultaneously added, the callus induction rates of treatments 1 and 2 were 0.00%, the callus induction rates of treatments 3 and 4 were 30% or less, and the callus was all white or off-white compact, and had no embryony. In the culture medium (treatments 3, 4, 5, 6) with simultaneous addition of 2,4-D and another hormone (IAA, NAA, KT), the callus induction rate was below 30%, treatments 3, 4, 5 were all non-embryogenic callus, and treatment 6 could induce both embryogenic and non-embryogenic callus, with embryogenic callus induction rate of 12.50% in pale yellow loose granules, and not tightly connected to the explant. The non-embryogenic callus is white and loose, and is tightly connected with the explant. In the culture medium (treatments 7, 8, 10) with the simultaneous addition of 6-BA and the other hormone (2, 4-D, IAA, NAA), the induction rate of the callus is obviously improved, wherein the treatments 7, 10 can induce embryogenic callus, and the induction rates are 5.00% and 12.50%, respectively. In treatment 9, three hormones (6-BA, 2,4-D, IAA) were added simultaneously, the callus induction rate was up to 79.63% and the embryogenic callus induction rate was also up to 17.50%.
TABLE 3 influence of different hormone ratios on Litsea cubeba callus and embryogenic callus
Due to limitations of experimental conditions, particularly limitations of development time of immature seeds of litsea cubeba on material collection, the inventors actually used the culture medium in this example in subsequent experiments: 1.0 mg.L of the medium was added to the MS medium -1 6-BA、0.2mg·L -1 2,4-D、0.5mg·L -1 IAA,30g/L sucrose, 700mg/L acid hydrolyzed casein and 7g/L agar, pH 5.85.
Example 3
The method for inducing and culturing litsea cubeba embryogenic callus described in the embodiment comprises the following specific steps.
S1, collecting fresh and tender fruits of immature litsea cubeba.
In the embodiment, in the scientific and technological garden of the agricultural university of Jiangxi, branches of the fine single plant of litsea cubeba with high citral are cut by using high branch scissors, the picked fruits are packaged by using a sealing bag, and then are placed in a medical refrigerated cabinet for storage at 4-8 ℃ and brought back to a laboratory for immediate treatment.
In this example, the actual fruit collection time was 7 months and 10 days. The cotyledonary embryos of the fruits picked during this period have begun to differentiate and even some fruits have tended to mature, see FIG. 1, with different degrees of litsea fruit development in treatments 1-6. The embryogenic callus is not obtained by the induction test using treatments 1-4 as materials, the induction rate of the treated 5 embryogenic callus is 6.67%, and the induction rate of the treated 6 embryogenic callus is 7.04%. According to the parallel researches of the applicant, the litsea cubeba heart-shaped embryo period is found to be 5-6 months, and the test result of the invention can reasonably infer that the litsea cubeba fruits of spherical embryo, heart-shaped embryo or torpedo embryo are picked in 5-6 months, and the induction rate is obviously higher than the test result data shown in the embodiment.
S2, pretreatment of explants
Selecting fruits which are not bitten and blackened, flushing with detergent in running water, slightly stirring with a brush with fluff, and flushing with gauze in running water for one hour to ensure that the surface mud and dirt are sufficiently removed. After washing with running water, the meat is placed on a clean table top, and the outer pericarp of the meat is gently peeled off by wearing sterile gloves. Soaking in 75% ethanol solution for 5min, shaking, sterilizing, and washing with sterile water for 5 times.
Sterilization of explants is an important step in embryogenic callus induction. The proper disinfection mode can effectively avoid the original microorganism source, thereby ensuring the embryogenic callus induction rate. The endocarp was sterilized with 75% alcohol for 5min, with the lowest contamination rate.
TABLE 4 influence of different disinfection modes on litsea cubeba pollution Rate
S3, induction culture
Sucking the sterilized litsea cubeba with sterile filter paper, peeling off the epicarp, and cutting the seeds along the central axis of the embryo; inoculating to the induction culture medium described in the example 2 for induction culture, and obtaining embryogenic callus. Dark culture at 24 ℃.
Referring to fig. 2, NEC is a non-embryogenic callus group; SE is somatic embryo; EC is embryogenic callus. The litsea cubeba embryo is used as an explant to induce 2 types of callus, and the callus is observed under a split microscope to find that the first type of callus is mostly white compact (a picture) or white loose (b picture), is not easy to separate from the explant, is extremely easy to brown and die after subculture, cannot differentiate somatic embryos, and is non-embryogenic callus. The second type of callus is pale yellow granule, loose in texture, not tightly connected with the matrix, and easy to peel (c picture). After being taken out from the induction culture medium and subjected to proliferation culture, the callus can be greatly proliferated and successfully differentiated into transparent or white somatic embryos (d picture), so that the callus is judged to have embryogenesis and is embryogenic callus. Furthermore, under the same induction conditions, embryogenic and non-embryogenic callus may coexist, but embryogenic callus may appear significantly later in time than non-embryogenic callus.
S4, embryogenic callus proliferation and differentiation induction culture
Fresh embryogenic callus with good growth condition is selected and placed in a proliferation culture medium for proliferation subculture. The proliferation medium comprises the following components: the MS culture medium is added with 0.5 mg.L -1 6-BA、0.1mg·L -1 2,4-D、0.25mg·L - 1 IAA,30g/L sucrose, 700mg/L acid hydrolyzed casein and 7g/L agar.
See fig. 3. The embryogenic callus differentiation process is continuously observed under a split microscope, the embryogenic callus can be obviously proliferated, the original faint yellow and loose granular embryogenic callus (a picture) is gradually expanded after 15d, the spherical embryo is differentiated to form yellow spherical embryo (b picture), the spherical embryo is further differentiated into two-flap transparent somatic embryo (c picture), the somatic embryo gradually becomes white cotyledon-shaped (e picture) along with the increase of the culture time, and finally, the germination of adventitious buds can be observed at the basal part of the cotyledon-shaped somatic embryo (f picture).
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that the above-mentioned preferred embodiment should not be construed as limiting the invention, and the scope of the invention should be defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.
Claims (10)
1. A litsea cubeba embryogenic callus induction culture medium is characterized in that 0.5-1 mg.L is added into an MS culture medium -1 6-BA、0.2～1.0mg·L -1 2,4-D、0.2～0.5mg·L -1 IAA, pH 5.85-6.
2. The litsea cubeba embryogenic callus induction medium according to claim 1, wherein 20-30 g/L sucrose or 20-40 g/L glucose is also added into the induction medium.
3. The litsea cubeba embryogenic callus induction medium according to claim 1 or 2, characterized in that 700mg/L acid hydrolyzed casein is also added to the induction medium.
4. The litsea cubeba embryogenic callus induction medium according to claim 3, wherein 7g/L agar is added to the induction medium.
5. The litsea cubeba embryogenic callus induction culture method is characterized by comprising the following steps of:
s1, collecting fresh and tender fruits of immature litsea cubeba with embryos, and immediately processing or preserving at 4-8 ℃ for treatment;
s2, pretreatment of explants
Thoroughly cleaning to remove the mud on the surface of the fruit, peeling off the outer seed coats of the meat under the aseptic condition, soaking for 3-5 min by 75% ethanol, shaking and sterilizing, and washing for a plurality of times by using sterile water;
s3, induction culture
Sucking the sterilized litsea cubeba with sterile filter paper, peeling off the epicarp, and cutting the seeds along the central axis of the embryo; inoculating the strain to the induction culture medium according to any one of claims 1 to 4 for induction culture to obtain embryogenic callus;
s4, embryogenic callus proliferation and differentiation induction culture
Fresh embryogenic callus with good growth condition is selected and placed in a proliferation culture medium for proliferation culture.
6. The litsea cubeba embryogenic callus induction medium according to claim 5, wherein the proliferation medium components are: the MS culture medium is added with 0.5 mg.L -1 6-BA、0.1mg·L -1 2,4-D、0.25mg·L -1 IAA。
7. The litsea cubeba embryogenic callus induction medium according to claim 6, wherein the proliferation medium composition further comprises 30g/L sucrose or 40g/L glucose, 700mg/L acid hydrolyzed casein and 7g/L agar.
8. The litsea cubeba embryogenic callus induction medium according to claim 6, wherein the embryo in the immature litsea cubeba fresh tender fruit is a young embryo.
9. The litsea cubeba embryogenic callus induction medium according to claim 8, wherein the young embryo is a spherical embryo, a heart embryo, a torpedo embryo or a cotyledon embryo.
10. The litsea cubeba embryogenic callus induction medium according to claim 5, wherein the culture conditions are dark culture at a temperature of 24+ -2 ℃.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117178896A (en) * | 2023-10-26 | 2023-12-08 | 江西农业大学 | Method for inducing adventitious bud regeneration plants by eupatorium sinensis leaves |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102227976A (en) * | 2011-04-11 | 2011-11-02 | 广西壮族自治区林业科学研究院 | Litsea cubeba pers asexual quick propagation and seedling breeding method |
| CN107223565A (en) * | 2017-06-13 | 2017-10-03 | 中国林业科学研究院亚热带林业研究所 | Cubeb litsen tree tissue-cultured seedling root media and cultural method |
| CN111642391A (en) * | 2020-04-27 | 2020-09-11 | 中南林业科技大学 | Tissue culture method for litsea cubeba |
| CN112335508A (en) * | 2020-11-06 | 2021-02-09 | 中国科学院成都生物研究所 | Application method of moss sporophyte suspension containing chitosan/glucan in bare land greening |
-
2023
- 2023-05-10 CN CN202310521800.7A patent/CN116584381B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102227976A (en) * | 2011-04-11 | 2011-11-02 | 广西壮族自治区林业科学研究院 | Litsea cubeba pers asexual quick propagation and seedling breeding method |
| CN107223565A (en) * | 2017-06-13 | 2017-10-03 | 中国林业科学研究院亚热带林业研究所 | Cubeb litsen tree tissue-cultured seedling root media and cultural method |
| CN111642391A (en) * | 2020-04-27 | 2020-09-11 | 中南林业科技大学 | Tissue culture method for litsea cubeba |
| CN112335508A (en) * | 2020-11-06 | 2021-02-09 | 中国科学院成都生物研究所 | Application method of moss sporophyte suspension containing chitosan/glucan in bare land greening |
Non-Patent Citations (5)
| Title |
|---|
| A. A. MAO等: "In vitro propagation of Litsea cubeba (Lours.) Pers., a multipurpose tree", 《PLANT CELL REPORTS》, vol. 19, pages 263 - 267 * |
| 徐健: "山苍子种子繁育和组织培养技术初步研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》, no. 08, pages 047 - 83 * |
| 杨文新等: "乌塌菜耐寒变异植株无性系的建立", 《西北师范大学学报(自然科学版)》, vol. 41, no. 04, pages 55 - 58 * |
| 赵振玲等: "滇黄芩体胚诱导与植株再生", 《西南农业学报》, vol. 19, no. 04, pages 714 - 718 * |
| 顾关云等: "山苍子的生药学鉴别及其质量的研究", 《上海第一医学院学报》, no. 03, 29 June 1984 (1984-06-29), pages 205 - 211 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117178896A (en) * | 2023-10-26 | 2023-12-08 | 江西农业大学 | Method for inducing adventitious bud regeneration plants by eupatorium sinensis leaves |
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