CN101248763A - Method for inducing somatic embryos of jujube leaves in one step - Google Patents
Method for inducing somatic embryos of jujube leaves in one step Download PDFInfo
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- CN101248763A CN101248763A CNA2008100895105A CN200810089510A CN101248763A CN 101248763 A CN101248763 A CN 101248763A CN A2008100895105 A CNA2008100895105 A CN A2008100895105A CN 200810089510 A CN200810089510 A CN 200810089510A CN 101248763 A CN101248763 A CN 101248763A
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- 230000000392 somatic effect Effects 0.000 title claims abstract description 24
- 230000001939 inductive effect Effects 0.000 title claims abstract description 22
- 238000000034 method Methods 0.000 title claims abstract description 16
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- 210000002257 embryonic structure Anatomy 0.000 title description 2
- 241001247821 Ziziphus Species 0.000 claims abstract description 33
- 210000001161 mammalian embryo Anatomy 0.000 claims abstract description 31
- 206010020649 Hyperkeratosis Diseases 0.000 claims abstract description 15
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- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims abstract description 7
- 229920001817 Agar Polymers 0.000 claims abstract description 6
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- 230000006698 induction Effects 0.000 claims abstract description 5
- 238000000338 in vitro Methods 0.000 claims abstract description 4
- 210000003462 vein Anatomy 0.000 claims abstract description 4
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- 230000008929 regeneration Effects 0.000 abstract description 4
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Abstract
The one-step induction method of jujube leaf somatic embryo is to obtain the somatic embryo of winter jujube leaf through tissue culture, including explant selection, shearing and in vitro culture, and the differentiation rate of leaf callus embryoid reaches 84.9%. Selecting strong-growing winter jujube tissue culture seedling leaves with the seedling age of 15-20 days, and cutting the leaves with sterilized scissors perpendicularly to veins. Then inoculating the mixture to an induction culture medium (MS + maltose 15-25 g/L + agar 3.0-5.0 g/L + TDZ 5.0-10.0 mg/L + AgNO)30 to 2.0mg/L) in the dark. Inducing the leaf blade to generate a large amount of yellowish blocky callus at 15-20 days, and gradually protruding from the surface of the blocky callus to form a tumor shape at 20-30 days without changing culture medium to differentiate embryoid. The pH value of the culture medium before sterilization is 5.0-6.0, and the culture temperature is controlled at 25-30 ℃. By using the method, a large amount of leaf embryoid can be obtained after inducing for 20-30 days, and the method is used for in vitro regeneration of the jujube leaves.
Description
Technical field
The present invention relates to a kind of method of an one-step inducing jujube blade somatic embryo.
Background technology
The plant soma embryo is meant somatic cell or reproductive cell under given conditions, merges and by with zygotic embryo the form generating process that similar approach develops into new individuality taking place without sexual cell.Because it is to differentiate from somatic cell, so be called somatic embryo or embryoid.The discussion of somatic embryo mechanism is come from the totipotency of plant cell hypothesis of Haberlandt proposition in 1902, to the real understanding of somatic embryo is to utilize the carrot root to carry out single-cell suspension from Steward in 1958 etc. to cultivate, produce the structure similar, and grow up to that whole plant begins to zygotic embryo.Form regeneration plant by somatic embryo generation approach and in higher plant, proved general phenomenon, in the representative plant of gymnosperm, dicotyledon, the important section of monocotyledon report is arranged both at home and abroad, existing 300 various plants obtained somatic embryo more.At present, most of research data derives from herbaceous plant, report is also arranged, as coniferous tree, birch etc. on the part woody plant.Existing 28 sections on fruit tree, 43 belong to, and 93 kinds (hybrids) have obtained somatic embryo.
Report about jujube somatic embryo approach regeneration induction plant is less, and is explant with rataria or cotyledon how.Utilize the cotyledon of wild jujube rataria to obtain embryoid as Chen Wei human relations the earliest etc., it also is to utilize rataria and cotyledon to obtain embryoid for explant that later Li Dengke etc., Cheng Youfa etc., Hao build equality.Up to the present, be that explant is cultivated all and regenerated in the indefinite bud mode with the jujube excised leaf, yet there are no report by the somatic embryo approach.It is that explant carries out somatic embryo and induces that this research is intended to winter jujube excised leaf, for setting up the blade body blast generation systems establish a firm foundation of efficient stable, this all has crucial meaning for the research that utilizes blade to carry out aspects such as gene engineering, genetic transformation, breed improvement.
Summary of the invention
The present invention has set up jujube excised leaf somatic embryo inductive technology system, can guarantee the somatic embryo of high efficiency acquisition winter jujube blade, for the research of aspects such as the gene engineering of carrying out jujube in a deep going way, genetic transformation, breed improvement provides a kind of new technology platform.
The technical solution adopted for the present invention to solve the technical problems is: at first adopt suitable excised leaf cutting method, again explant is carried out inducing of somatic embryo, regulation and control by different medium components, different plant growth regulator, different culture environment, filter out the appropriate media that winter jujube blade somatic embryo is induced, obtain winter jujube excised leaf somatic embryo.
Particular content of the present invention, promptly the method for in-vitro inducing winter jujube blade somatic embryo is as follows:
It is 15-20 days winter jujube tissue cultivating seedling blade that explant is selected to choose seedling age with preliminary treatment, is seeded in MS+ maltose 15～25g/L+ agar 3～5g/L+TDZ 5.0～10.0mg/L+AgNO
31.0 secretly cultivate on the solid culture medium of～2.0mg/L, the medium pH value is controlled at about 5.0～6.0, environmental temperature can induce the blade body cell stage under 25～30 ℃ of conditions.
The condition of culture minimal medium is the MS medium, maltose 1.5～2.5%, and pH is 5.0～6.0 before the agar 0.3%～0.5%, medium sterilization.Dark surrounds, temperature are controlled at 25～30 ℃.
It is 15-20 days winter jujube tissue cultivating seedling blade that the explant inoculation method is chosen robust growth, seedling age, shears the number cutter with the vertical vein of the scissors of sterilizing, and facing up then is inoculated on the inducing culture.
Inducing culture will be sheared good blade inoculation to MS+ maltose 15～25g/L+ agar 3.0～5.0g/L+TDZ 5.0～10.0mg/L+AgNO
31.0 secretly cultivate on the solid culture medium of～2.0mg/L.Produce the callus of a large amount of faint yellow bulks when inducing 15-20 days, begin when inducing 20-30 days from block callus surface gradually that projection becomes warty, differentiate embryoid.
The beneficial effect of patent of the present invention is, by the cutting method suitable to winter jujube blade explant, be inoculated on the inducing culture then, can obtain winter jujube blade somatic embryo without the conversion medium, realized the somatic embryo regeneration of jujube excised leaf first, and in jujube blade somatic embryo generating process, non-embryonic callus is induced, embryo callus subculture is induced, three steps of body embryo differentiation are combined into a step, promptly on a kind of medium, callus and embryoid differentiation have been realized, utilize the method not only to simplify test procedure, do not need repeatedly to change medium, saved manpower and materials, and with minute one-step inducing reduced in comparison induction time.Therefore, the blade body embryo generation system of this research foundation has great importance to the research of jujube regenerating system.
Description of drawings
Fig. 1 is a winter jujube blade initial inoculation state.
Fig. 2 is the dark cultivation in inducing culture of winter jujube blade produces faint yellow bulk in the time of 15～20 days a callus.
Fig. 3 is that the dark cultivation in inducing culture of winter jujube blade differentiated embryoid gradually from block callus surface in the time of 25～35 days.
Fig. 4 is the globular embryo that the winter jujube blade differentiates.
Fig. 5 is the heart-shape embryo that the winter jujube blade differentiates.
Fig. 6 is the cotyledonary embryos that the winter jujube blade differentiates.
Embodiment
Choose 20 days winter jujube tissue cultivating seedling blade of seedling age, shear the number cutter at the sterile working platform with the vertical vein of the scissors of sterilizing, facing up then to be inoculated in is seeded in MS+ maltose 15～25g/L+ agar 3.0～5.0g/L+TDZ 5.0～10.0mg/L+AgNO
31.0 secretly cultivate on the solid inducing culture of～2.0mg/L, the medium pH value is controlled at about 5.0～6.0, environmental temperature is under 25～30 ℃ of conditions, produce the callus of a large amount of faint yellow bulks when inducing 15-20 days, begin when inducing 20-30 days from block callus surface gradually that projection becomes warty, differentiate embryoid.Differentiation rate is 84.9%.
Claims (3)
1. the method for an one-step inducing jujube blade somatic embryo is characterized in that getting final product a step realization callus induction and embryoid differentiation by the winter jujube blade is carried out cultured in vitro without the conversion medium.
2. in accordance with the method for claim 1, it is characterized in that comprising following incubation step:
(1) choosing robust growth, seedling age is 15-20 days winter jujube tissue cultivating seedling blade, shears the number cutter at the sterile working platform with the vertical vein of the scissors of sterilizing.
(2) will be seeded in MS+ maltose 15～20g/L+TDZ5.0～10.0mg/L+AgNO through the winter jujube excised leaf that step (1) obtains
31.0 secretly cultivate on the medium of～2.0mg/L, produce callus transparent in a large number, faint yellow bulk when inducing 15-20 days, begin when inducing 20-30 days from block callus surface gradually that projection becomes warty, differentiate embryoid gradually.
3. in accordance with the method for claim 1, it is characterized in that minimal medium is MS, contains maltose 15～25g/L, agar 3～5g/L, TDZ5.0～10.0mg/L, AgNO
31.0 pH is 5.5～6.0 before the～2.0mg/L, medium sterilization, cultivation temperature is controlled at 25～35 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100895105A CN101248763A (en) | 2008-04-03 | 2008-04-03 | Method for inducing somatic embryos of jujube leaves in one step |
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| CNA2008100895105A CN101248763A (en) | 2008-04-03 | 2008-04-03 | Method for inducing somatic embryos of jujube leaves in one step |
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| CN101248763A true CN101248763A (en) | 2008-08-27 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103190347A (en) * | 2013-04-26 | 2013-07-10 | 北京林业大学 | Teapot dates tissue culturing method |
| CN105165613A (en) * | 2015-09-24 | 2015-12-23 | 滨州学院 | Culture method of Zhanhua winter jujube leaf calluses |
| CN105941154A (en) * | 2016-05-30 | 2016-09-21 | 沾化县冬枣研究所 | Comprehensive breeding method for superior winter jujube seedlings |
| CN111492979A (en) * | 2020-05-25 | 2020-08-07 | 四川天艺生态园林集团股份有限公司 | Primula forbesii somatic embryo induction method |
-
2008
- 2008-04-03 CN CNA2008100895105A patent/CN101248763A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103190347A (en) * | 2013-04-26 | 2013-07-10 | 北京林业大学 | Teapot dates tissue culturing method |
| CN105165613A (en) * | 2015-09-24 | 2015-12-23 | 滨州学院 | Culture method of Zhanhua winter jujube leaf calluses |
| CN105941154A (en) * | 2016-05-30 | 2016-09-21 | 沾化县冬枣研究所 | Comprehensive breeding method for superior winter jujube seedlings |
| CN111492979A (en) * | 2020-05-25 | 2020-08-07 | 四川天艺生态园林集团股份有限公司 | Primula forbesii somatic embryo induction method |
| CN111492979B (en) * | 2020-05-25 | 2022-03-22 | 四川天艺生态园林集团股份有限公司 | Primula forbesii somatic embryo induction method |
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Open date: 20080827 |