CN1230066C - Bacteria free saffron seedling growth method - Google Patents
Bacteria free saffron seedling growth method Download PDFInfo
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- CN1230066C CN1230066C CN 03142559 CN03142559A CN1230066C CN 1230066 C CN1230066 C CN 1230066C CN 03142559 CN03142559 CN 03142559 CN 03142559 A CN03142559 A CN 03142559A CN 1230066 C CN1230066 C CN 1230066C
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Abstract
The present invention relates to a method for growing saffron aseptic seedlings. In the method, firstly, the influence of rare earth elements on the growth and the differentiation of saffron cells is utilized, and the calluses of the saffron are inoculated on a plant cell culture medium containing the rare earth elements, cytokinin and cell auxin so as to be cultured; secondly, the obtained calluses are inoculated on the plant cell culture medium containing the rare earth elements, the cytokinin and the cell auxin once again to be cultured so as to obtain the somatic embryos or the embryogenic calluses of the saffron; finally, the somatic embryos or the embryogenic calluses are inoculated on the plant cell culture medium containing the rare earth elements once again so as to obtain the saffron aseptic seedlings. In the method, the saffron cells are promoted to form the embryogenic calluses and the somatic embryos in a short time, and the embryogenic calluses and the somatic embryos are differentiated into a great amount of aseptic seedlings so as to solve the problems that in the process of planting the saffron, the seed sources of the saffron are few, and are easily infected by pathogenic bacteria; thereby, the problem of deficient resources of the saffron is solved.
Description
Technical field
The invention belongs to biological technical field, particularly a kind of APPLICATION OF RARE-EARTH ELEMENTS promotes the method for the plantlet that safflower callus differential growth is aseptic.
Technical background
Safflower is Iridaceae (Iridaceae) crocus herbaceos perennial, has another name called Crocus sativus, west safflower.Originate in ground such as Spain, various countries, Greece southern Europe and Iran, import China Tibet into through India, all there are cultivation (Chengdu Traditional Chinese Medical College, Chinese pharmacology evaluation, 1977,271-273 in ground such as present Zhejiang in China, Jiangsu, Shandong, Beijing; The Jiangsu college of traditional Chinese medicine compiles, Chinese medicine dictionary (volume two) 1977:2671).On the one hand, safflower (saffron, Crocussativus L.) on tradition and modern medicine, has very high medical value, studies show that safflower can be used for calmness, eliminates the phlegm, stimulation, spasmolysis, also can be used for stomachache, aphrodisiac, menstruation regulating, and be used for the treatment of dysentery, measles, heating jaundice, hepatosplenomegaly, urethral infection, diabetes, and do to take a risk to do miscarriage medicine etc., also be extensive use of safflower as gynaecology's good medicine in Europe and Asia.On the other hand, safflower has had long history (Warburg E F, the Endeavour of centuries in the application in Europe as a kind of precious flavor enhancement, spices and dyestuff, 1957,16,209), safflower can be used for development functionality food, natural colouring matter, health makeup etc. in addition; Can also cultivate safflower as ornamental plants.In recent years more meaningfully, scientist finds some chemical substances in the safflower column cap, it is synthetic to suppress the startup of proto-oncogene and cancer cell DNA and RNA from molecular level as crocin, thereby safflower has become the research focus (Escribano of new type anticancer medicine, Cancer Letter, 1996,100,1913-1918; Wang C J etc., Molecular Carcinogenesis, 1996,17,235-240).
Although China has abundant resources of medicinal plant, the demand of autonomic drug is grown with each passing day with society along with human, resources of medicinal plant is faced with immense pressure.The atrophy year by year of some Chinese material medicine resources becomes rare endangered plants, thereby has influence on the production of tcm clinical practice medication and pharmacy corporation.Medicinal croceous resource is just extremely limited, China always is the import hyoscine, and the safflower of producing 1 kilogram needs 150000～200000 flowers, also will be through the hand labor more than 400 hours, cause croceous price extremely expensive, per kilogram reaches (PlessnerD, E.BI etc., 1989 about 2000 US dollars, Israel J Botany, 38,1-7), be described as " plant gold " always.Because factors such as regional limited, the condition harshnesses of few, the suitable cultivation of provenance, carrying out introducing and planting only can provide a small amount of commodity; In addition, traditional safflower cultivation and production is wilted easily, and the pathogenic infection microorganism is rotted then, causes the decline of output and quality; China's safflower is sexual infertile simultaneously, can not be solid under the cultivation condition, by propagation by corm, bulb is planted littler and littler in the course of cultivation, and bottom set is bloomed less and spent little, even do not bloom, thereby lose medical value, therefore if single with traditional propagation by corm method, reproduction speed is slow, therefore can not satisfy the demand in market, the situation that supply falls short of demand occur.
Giving birth to metabolite by plant tissue and cell culture parity in next life is address this problem one method preferably, currently reported, approach by plant cell engineering makes Chinese herbal medicines in imminent danger such as Asian puccoon, ginseng, sweet wormwood, digitalis reach pilot scale and industrially scalable (Curtin M E, Biotechnology, 1983,1,649-657).
Equally, domestic and international research person also attempts to solve by the various approach of plant cell engineering the problem of safflower shortage of resources, be inoculated on the MS plant culture that adds plant hormone 6-BA and NAA as the callus of inducing, can obtain embryo callus with croceous bud; Embryo callus is differentiation adult embryo on the medium that adds plant hormone GA; The body embryo transferred to adds plant hormone 6-BA, on the plant culture of NAA and active carbon, can generate aseptic seedling (Ahuja A etc., Indian journal of experiment biology, 1994,32,135-140).But the callus of the method produces embryo callus, and the generation time that forms aseptic seedling is long, needed for 11～15 week, and quantity is few, so still can not meet the need of market.
Rare earth element generally is meant the lanthanide series in the periodic table of elements and the general name of elements such as the scandium similar to the lanthanide series chemical property, yttrium.China is the abundantest country of rare earth resources in the world, account for 80% of world's gross reserves, in order to make full use of this resource, the scientific worker of China is since the research of rare earth application in 1972, and the affirmation rare earth element is the beneficial element of plant growing and growth.Rare earth element has been applied to tens kinds of crops at present, and is applied in the plantation of some Chinese herbal medicines.The practice of using shows that rare earth element can have volume increase, effect such as disease-resistant to Chinese herbal medicines such as genseng, matrimony vine, the barks of eucommia, and remains of pesticide (Chen Hao etc., Guangdong trace element science, 2001 that can improve the content of Chinese herbal medicine effective ingredients and reduce Chinese herbal medicine, 8 (3), 1).Rare earth element is comparatively extensive for the research of culture in vitro zooblast, and rare earth element also increases year by year for the research report of the cultivation of plant cell.These research reports show that rare earth element has certain facilitation to the growth of plant cell and the accumulation of secondary metabolite, as the Eu of low concentration
3+Promote rheum officinale callus Growth (Lu Kekuan etc., journal of Beijing Medical University, 1997,29,289), La has certain facilitation (first Ying Jin etc. to the growth of yew cell and taxol synthetic, China's rare-earth journal, 1998,16,56), rare earth element can partly or entirely replace exogenous hormone to growth of saussurea involucrata cell and synthetic facilitation (Xiaofan Yuan et al, Biotechnology Letters, 2002 of flavonoids, 24,1889).
Summary of the invention
The generation time of formation safflower aseptic seedling that the objective of the invention is to overcome prior art is long, and the few defective of quantity, thereby provides a kind of APPLICATION OF RARE-EARTH ELEMENTS to promote the method for safflower callus differential growth aseptic seedling.The plant hormone of this method by utilizing rare earth element, eliminate free radical, promote effects such as cell differentiation, be differentiated to form aseptic plantlet with promotion safflower callus, thereby fundamentally solve the problem of shortage of safflower provenance and easy infection pathogen.
The objective of the invention is to realize by the following technical solutions:
The invention provides a kind of method that forms the safflower aseptic seedling, comprise the steps:
1) croceous callus is inoculated on the conventional plant cell culture medium of the rare earth element that contains 0.005～0.07mM, and adds the basic element of cell division of 0.5～10.0mg/g and the archusia of 0.5～8mg/g, cultivated 15～20 days down in 15～30 ℃;
2) callus that step 1) is cultivated is inoculated into the conventional plant cell culture medium that contains 0.005～0.07mM rare earth element, and add the basic element of cell division of 1～10.0mg/g and the archusia of 0.05～3mg/g, cultivated 25～30 days down in 15～30 ℃, obtain croceous body embryo or embryo callus;
3) with step 2) body embryo or the embryo callus cultivated be inoculated into the 1/2 conventional plant cell culture medium (promptly the content of all medium components all is 1/2) that contains 0.005～0.02mM rare earth element, cultivated 35～50 days down in 15～30 ℃, obtain the safflower aseptic seedling.
The croceous callus of described step 1) is that croceous bulb, bud, leaf or flower are handled the croceous callus that forms with conventional method.
The rare earth element of described step 1) is Nd, La or Ce, or La
2O
3, CeO
2, Pr
6O
11, Sm
2O
3The mixed rare-earth elements of forming, the mol ratio of each component is La
2O
3: CeO
2: Pr
6O
11: Sm
2O
3=100～355: 10～275: 1～30: 1～40.
Described step 2) rare earth element is Ce, or La
2O
3, CeO
2, Pr
6O
11And Sm
2O
3The mixed rare-earth elements of forming, the mol ratio of each component is La
2O
3: CeO
2: Pr
6O
11: Sm
2O
3=100～355: 10～275: 1～30: 1～40.
The rare earth element of described step 3) is La, or La
2O
3, CeO
2, Pr
6O
11And Sm
2O
3The mixed rare-earth elements of forming, the mol ratio of each component is La
2O
3: CeO
2: Pr
6O
11: Sm
2O
3=100～355: 10～275: 1～30: 1～40.
Described step 1) and step 2) the basic element of cell division be 6-benzyl aminopurine (hereinafter to be referred as 6-BA) or kinetin (hereinafter to be referred as Ki).
Described step 1) and step 2) archusia be that 2,4 dichlorophenoxyacetic acid is (hereinafter to be referred as 2,4-D) or methyl (hereinafter to be referred as NAA).
Described step 1), step 2) and the conventional plant cell culture medium of step 3) be MS medium, B5 medium or N6 medium.Wherein, the MS medium is the bigger medium of salt content, contains high concentration nitrate, potassium ion and ammonium ion, and the kind of trace element is more complete, and its component can be referring to Murashige T, Skoog, and FA, 1962, Physiol.Plant 15:473479 is described.B5 medium is to contain the higher medium of potassium nitrate, and salt content is also higher, and its component can be referring to Gamborg OL, Miller RA, and Ojima K 1968, Exp.Cell Res.50:151-158 is described.The component of N6 medium can be referring to Nitsch JP, Nitsch C, and 1969,, Science 163:85-87 is described.
Use the advantage of method provided by the invention to be: the plant hormone of this method by utilizing rare earth element, to eliminate free radical, promote effects such as cell differentiation, promote the aseptic plantlet of safflower callus differential growth, can obtain relatively large safflower within a short period of time, thereby fundamentally solve the problem of shortage of safflower provenance and easy infection pathogen.
Specific embodiments
Embodiment 1,
Clean the earth on form corm of saffron surface with running water, remove epithelium, if there is scab to remove scab with scalpel, use distilled water flushing again 2 times, blot through filter paper, with 70wt% alcohol-pickled 30 seconds, the liquor natrii hypochloritis that use contains the 2wt% active chlorine sterilized 15 minutes, again with sterile water washing 5 times, blot the moisture on bulb surface at last with aseptic filter paper, to wash good bulb with scalpel and be cut into about 1 square centimeter fritter, be inoculated in respectively and contain 0.25mg/g 2, on the MS plant culture of 4-D and 2.0mg/g 6-BA plant hormone, be positioned over the cultivation indoor cultivation 14 days of 21 ± 0.3 ℃ of climatic cabinates or 27 ± 1 ℃, can obtain croceous bulb callus.
The bulb callus that obtains is inoculated into contains 0.007mM La, 0.005mM on the B5 plant cell culture medium of Ce, and add plant hormone: 2mg/g 2,4-D (promptly 2, the 4-dichlorphenoxyacetic acid) and 0.05mg/g 6-BA (being 6-benzyl aminopurine), cultivated 15 days in 21 ℃;
Be transferred to and contain 0.05mM La, on the B5 plant cell culture medium of 0.05mM Ce 0.05mg/g NAA (being methyl) and 2mg/g 6-BA, cultivated 35 days in 21 ℃;
Be transferred at last on the 1/2MS plant cell culture medium that contains 0.008mM La, in 21 ℃ of cultivations 40 days, obtain the safflower aseptic seedling, its quantity is every blake bottle 29 strains.
And under the similarity condition, just do not add rare earth element to organize in contrast, the quantity that obtains the safflower aseptic seedling is every blake bottle 11 strains.As can be seen, the quantity of using the inventive method to cultivate the safflower aseptic seedling improves 164%.
Embodiment 2,
The callus that the bulb of inducing among the embodiment 1 is formed is inoculated into and contains 0.006mM La, 0.005mM Ce, and 2mg/g 2, on the MS medium of 4-D and 0.5mg/g 6-BA, cultivate 15 days in 15 ℃;
Be transferred to and contain 0.005mM La, 0.01mM Ce on the B5 medium of 0.5mg/g NAA and 3mg/g 6-BA, cultivated 35 days in 15 ℃;
Be transferred at last on the 1/2MS medium that contains 0.005mM La, cultivated 40 days in 15 ℃, the quantity of the safflower aseptic seedling that obtains than under the similarity condition, the control group that do not add rare earth element improves 200-250%.
Embodiment 3,
Induce croceous bud to form callus by the method for embodiment 1.
The callus that the safranine bud is formed is inoculated into and contains 0.007mM La, and 3mg/g 2, on the MS medium of 4-D and 0.5mg/g 6-BA, cultivate 12 days in 30 ℃;
Be transferred to contain the 0.01mM mixed rare-earth elements (its form with mol ratio for being La
2O
3: CeO
2: Pr
6O
11: Sm
2O
3=255: 175: 3: 1), on the MS medium of 0.05mg/g NAA and 2.5mg/g 6-BA, cultivated 35 days in 30 ℃;
Be transferred at last on the 1/2MS medium that contains 0.005mM La, cultivated 40 days in 30 ℃, the quantity of the safflower aseptic seedling that obtains than under the similarity condition, the control group that do not add rare earth element improves 350-450%.
Embodiment 4,
Induce croceous flower to form callus by the method for embodiment 1.
The callus that croceous flower is formed is inoculated into and contains 0.07mM La, and 2.5mg/g 2, on the MS medium of 4-D and 1mg/g 6-BA, cultivate 15 days in 22 ℃;
Be transferred to and contain 0.07mM La, 0.07mM Ce on the B5 medium of 0.5mg/g NAA and 2mg/g 6-BA, cultivated 35 days in 22 ℃;
Be transferred at last on the 1/2MS medium that contains 0.07mM La, cultivated 40 days in 15～30 ℃, the quantity of the safflower aseptic seedling that obtains than under the similarity condition, the control group that do not add rare earth element improves 100-150%.
Embodiment 5,
Induce croceous leaf to form callus by the method for embodiment 1.
The callus that the safflower leaf is formed is inoculated into and contains 0.005mM La, 0.005mM Ce, and 2mg/g 2, on the medium of 4-D and 0.05mg/g 6-BA, cultivate 15 days in 25 ℃;
Be transferred to contain the 0.005mM mixed rare-earth elements (its form with mol ratio for being La
2O
3: CeO
2: Pr
6O
11: Sm
2O
3=255: 175: 3: 1), on the MS medium of 0.5mg/g NAA and 3.5mg/g KT (being kinetin), cultivated 45 days in 25 ℃;
Be transferred at last contain the 0.005mM mixed rare-earth elements (its form with mol ratio for being La
2O
3: CeO
2: Pr
6O
11: Sm
2O
3=255: 175: 3: 1) on the 1/2MS medium, cultivated 40 days in 15～30 ℃, the quantity of the safflower aseptic seedling that obtains than under the similarity condition, the control group that do not add rare earth element improves 400-450%.
Embodiment 6,
Induce croceous leaf to form callus by the method for embodiment 1.
The callus that the safflower leaf is formed is inoculated into and contains 0.05mM La, and 1mg/g 2, on the medium of 4-D and 0.5mg/g 6-BA, cultivate 15 days in 26 ℃;
Be transferred to contain the 0.06mM mixed rare-earth elements (its form with mol ratio for being La
2O
3: CeO
2: Pr
6O
11: Sm
2O
3=255: 175: 3: 10), on the MS medium of 1.0mg/g NAA and 4mg/g 6-BA, cultivated 25 days in 28 ℃;
Be transferred at last on the 1/2MS medium that contains 0.005mmol La, cultivated 35 days in 25 ℃, the quantity of the safflower aseptic seedling that obtains than under the similarity condition, the control group that do not add rare earth element improves 280-300%.
Embodiment 7,
Induce croceous leaf to form callus by the method for embodiment 1.
The callus that the safflower leaf is formed is inoculated into and contains 0.07mM La, and 3mg/g 2, on the medium of 4-D and 0.5mg/g 6-BA, cultivate 25 days in 21 ℃;
Be transferred to contain the 0.07mM mixed rare-earth elements (its form with mol ratio for being La
2O
3: CeO
2: Pr
6O
11: Sm
2O
3=255: 275: 3: 10), on the MS medium of 1.0mg/g NAA and 4mg/g 6-BA, cultivated 35 days in 21 ℃;
Be transferred at last on the 1/2MS medium that contains 0.005mmol La, cultivated 50 days in 21 ℃, the quantity of the safflower aseptic seedling that obtains than under the similarity condition, the control group that do not add rare earth element improves 35-40%.
Embodiment 8,
Induce croceous leaf to form callus by the method for embodiment 1.
The callus that the safflower leaf is formed is inoculated into and contains 0.07mM La, and 0.5mg/g 2, on the medium of 4-D and 0.5mg/g6-BA, cultivate 25 days in 21 ℃;
Be transferred to contain the 0.07mM mixed rare-earth elements (its form with mol ratio for being La
2O
3: CeO
2: Pr
6O
11: Sm
2O
3=255: 275: 3: 10), on the MS medium of 0.05mg/g NAA and 1mg/g 6-BA, cultivated 35 days in 21 ℃;
Be transferred at last on the 1/2MS medium that contains 0.005mmol La, cultivated 50 days in 21 ℃, the quantity of the safflower aseptic seedling that obtains than under the similarity condition, the control group that do not add rare earth element improves 38-40%.
Embodiment 9,
Induce croceous bulb to form callus by the method for embodiment 1.
The callus that the safflower leaf is formed is inoculated into and contains 0.06mM La, and 8mg/g 2, on the medium of 4-D and 10mg/g 6-BA, cultivate 21 days in 21 ℃;
Be transferred to contain the 0.04mM mixed rare-earth elements (its form with mol ratio for being La
2O
3: CeO
2: Pr
6O
11: Sm
2O
3=255: 275: 3: 10), on the MS medium of 3mg/g NAA and 10mg/g 6-BA, cultivated 35 days in 21 ℃;
Be transferred at last on the 1/2MS medium that contains 0.005mmol La, cultivated 50 days in 21 ℃.The quantity of the safflower aseptic seedling that obtains than under the similarity condition, the control group that do not add rare earth element improves 35-40%.
Embodiment 10,
Induce croceous leaf to form callus by the method for embodiment 1.
The callus that the safflower leaf is formed is inoculated into and contains 0.07mM La, and 3mg/g 2, on the medium of 4-D and 0.5mg/g 6-BA, cultivate 25 days in 21 ℃;
Be transferred to contain the 0.07mM mixed rare-earth elements (its form with mol ratio for being La
2O
3: CeO
2: Pr
6O
11: Sm
2O
3=355: 275: 30: 40), on the MS medium of 1.0mg/g NAA and 4mg/g 6-BA, cultivated 35 days in 21 ℃;
Be transferred at last on the 1/2MS medium that contains 0.005mmol La, cultivated 50 days in 21 ℃, the quantity of the safflower aseptic seedling that obtains than under the similarity condition, the control group that do not add rare earth element improves 60-80%.
Embodiment 11,
Induce croceous flower to form callus by the method for embodiment 1.
The callus that the safflower leaf is formed is inoculated into and contains 0.04mM La, and 3mg/g 2, on the medium of 4-D and 0.5mg/g 6-BA, cultivate 25 days in 21 ℃;
Be transferred to contain the 0.05mM mixed rare-earth elements (its form with mol ratio for being La
2O
3: CeO
2: Pr
6O
11: Sm
2O
3=100: 10: 1: 1), on the MS medium of 2.0mg/g NAA and 4mg/g 6-BA, cultivated 35 days in 21 ℃;
Be transferred at last on the 1/2MS medium that contains 0.006mmol La, cultivated 50 days in 21 ℃, the quantity of the safflower aseptic seedling that obtains than under the similarity condition, the control group that do not add rare earth element improves 80-100%.
Claims (4)
1, a kind of method of the safflower aseptic seedling of growing comprises the steps:
1) croceous callus is inoculated on the conventional plant cell culture medium of the rare earth element that contains 0.005～0.07mM, and adds the basic element of cell division of 0.5～10.0mg/g and the archusia of 0.5～8mg/g, cultivated 15～20 days down in 15～30 ℃; The croceous callus of described step 1) is that croceous bulb, bud, leaf or flower are handled the croceous callus that forms with conventional method; Described rare earth element is La or Ce, or La
2O
3, CeO
2, Pr
6O
11And Sm
2O
3The mixed rare-earth elements of forming, the mol ratio of each component is La
2O
3: CeO
2: Pr
6O
11: Sm
2O
3=100～355: 10～275: 1～30: 1～40;
2) callus that step 1) is cultivated is inoculated into the conventional plant cell culture medium that contains 0.005～0.07mM rare earth element, and add the basic element of cell division of 1～10.0mg/g and the archusia of 0.05～3mg/g, cultivated 25～30 days down in 15～30 ℃, obtain croceous body embryo or embryo callus; Described rare earth element is Ce, or La
2O
3, CeO
2, Pr
6O
11And Sm
2O
3The mixed rare-earth elements of forming, the mol ratio of each component is La
2O
3: CeO
2: Pr
6O
11: Sm
2O
3=100～355: 10～275: 1～30: 1～40;
3) with step 2) body embryo or the embryo callus cultivated be inoculated into the 1/2 conventional plant cell culture medium that contains 0.005～0.02mM rare earth element, cultivated 35～50 days down in 15～30 ℃, obtains the safflower aseptic seedling; Described rare earth element is La, or La
2O
3, CeO
2, Pr
6O
11And Sm
2O
3The mixed rare-earth elements of forming, the mol ratio of each component is La
2O
3: CeO
2: Pr
6O
11: Sm
2O
3=100～355: 10～275: 1～30: 1～40.
2, the method for growth safflower aseptic seedling as claimed in claim 1 is characterized in that, described step 1) and step 2) the basic element of cell division be 6-benzyl aminopurine or kinetin.
3, the method for growth safflower aseptic seedling as claimed in claim 1 is characterized in that, described step 1) and step 2) archusia be 2,4 dichlorophenoxyacetic acid or methyl.
4, the method for growth safflower aseptic seedling as claimed in claim 1 is characterized in that, described step 1), step 2) and the conventional plant cell culture medium of step 3) be MS medium, B5 medium or N6 medium.
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| CN102165917A (en) * | 2011-01-11 | 2011-08-31 | 福建农林大学 | Method for raising deguelin content with rare earth element |
| CN105532463A (en) * | 2016-01-09 | 2016-05-04 | 佛山市金蓝领教育科技有限公司 | Proliferation medium for increasing saffron crocus bud proliferation rate |
| CN110999791B (en) * | 2019-12-27 | 2021-11-26 | 青岛大学附属医院 | Saffron crocus culture medium for increasing crocin content |
| CN111837955A (en) * | 2020-07-27 | 2020-10-30 | 湖北省农业科学院中药材研究所 | Culture medium and method for promoting germination of rhubarb seeds for Enshi medicine |
| CN115486362A (en) * | 2022-09-27 | 2022-12-20 | 上海市农业科学院 | Efficient crop rotation planting method for crocus sativus and hippeastrum |
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