CN100381044C - Beiwuweizi cell embryo growth and strain regeneration - Google Patents
Beiwuweizi cell embryo growth and strain regeneration Download PDFInfo
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- CN100381044C CN100381044C CNB2006100097820A CN200610009782A CN100381044C CN 100381044 C CN100381044 C CN 100381044C CN B2006100097820 A CNB2006100097820 A CN B2006100097820A CN 200610009782 A CN200610009782 A CN 200610009782A CN 100381044 C CN100381044 C CN 100381044C
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Abstract
The present invention relates to a method for generating the somatic embryos of northern schisandra and regenerating plants, particularly to a method for generating cell embryos and regenerating plants. The present invention solves the problems of the existing asexual propagation method of the northern schisandra, for example, the culturing and the breeding period is long, the survival rate is low, and the genic properties of the regenerated plants can not keep accordant with that of female parent. The present invention comprises the following methods: (1) good individual nutritive organs of the northern schisandra are fetched and pre-treated; (2) the nutritive organs are cultured; (3) somatic embryos are subcultured; (4) embryonic calluses are cultured to obtain germinated somatic embryos; (5) the germinated somatic embryos are cultured to obtain complete regenerated plants; (6) the regenerated plants are transplanted in soil to culture, and then seedlings of the northern schisandra are obtained. The present invention has the advantages that the fetching material range of the somatic embryogenesis is board, the breeding speed is high, the proliferation multiple is high, the survival rate reaches above 80 percent, and the present invention can produce the regenerated plants having the same good properties as the female parent in scale.
Description
Technical field
The present invention relates to a kind of blast takes place and plant regeneration method.
Technical background
Fructus schisandrae (Schisandra Chinensis Baill) belongs to the perennial wild woody climber of Schisandraceae (Schisandraceae), and its fruit is important traditional Chinese medicine, also can be used for making wine and making fruit drink.Find the drug ingedient couplet benzene ring octadiene system lignans in recent years in fructus schisandrae fruit, seed and stem, so fructus schisandrae comes into one's own day by day, development prospect is very wide.Because environmental destruction and long-term predation formula are plucked, wild resource destroys serious, and supply falls short of demand in fructus schisandrae market.China fructus schisandrae clone kind " red pearl " has good proterties, but because of it adopts the breeding of growing directly from seeds, does not have ripe vegetative propagation system, is difficult to obtain promote.There are many defectives in the asexual process that adopts at present, as: the cottage propagation method is transplanted a cutting and must just can be taken root with the branch of life in 2 years, and branch quantity is few, and cultivation period is long; Ground stem breeding method root system too a little less than, leave parent after survival rate low, can not be applied to produce; Induce axillalry bud sproutung method propagation multiple low, have only 3.2 times, the long speed of blastogenesis is slow, so also be not suitable for being applied to producing; Zygotic embryo inductor blast take place and the method for plant regeneration method loaded down with trivial details, and be the somatic embryo inducement material with the zygotic embryo, it is consistent with female parent that the genetic character of regeneration plant can not keep.
Summary of the invention
The objective of the invention is the problem that present fructus schisandrae asexual process cultivation period is long, survival rate is low in order to solve, the propagation multiple is low, the long speed of blastogenesis genetic character slow, regeneration plant can not keep and female parent is consistent, and a kind of fructus schisandrae somatic embryo that provides takes place and plant regeneration method.The fructus schisandrae somatic embryo takes place and plant regeneration method carries out according to the following steps: (one) cuts good fructus schisandrae individual nutrition organ and carries out preliminary treatment, be inoculated into then on the MS medium of the 2,4 dichlorophenoxyacetic acid, 10～60g/L sucrose and the 5.0～8.0g/L agar that add 0.5～10mg/L; (2) be 29 ± 1 ℃ in temperature, the photoperiod is to cultivate for 7～9 weeks in the culturing room of the dark 8h of bright 16h/, obtains somatic embryo; (3) somatic embryo is transferred to the identical solid culture medium of (one) step composition in successive transfer culture 2～3 months, obtain callus; (4) the translucent milky embryo callus that successive transfer culture is obtained changes over to and is added with 2 of 0.5～10mg/L, cultivated 25～35 days on the MS medium of 4-dichlorphenoxyacetic acid, transfer to inorganic salt concentration afterwards again and be MS 1/4～1/2 and added on the MS medium of 10～60g/L sucrose and 5.0～8.0g/L agar, be to cultivate for 4～5 weeks, the somatic embryo that obtains germinateing under the condition of the dark 8h of bright 16h/ at 25 ± 1 ℃, photoperiod; (5) somatic embryo that germinates is transferred to (four) and gone on foot in the identical medium of composition and cultivated for 4～5 weeks, obtain complete regeneration plant; (6) regeneration plant is transplanted into the soil cultivation, promptly obtains the fructus schisandrae nursery stock; Wherein preliminary treatment is that nutrition organs is put into concentration with the running water flushing after one day be that 70% alcohol soaks 30s in the step (), use distilled water flushing again 2 times, the liquor natrii hypochloritis who the puts into concentration 3% then 10min that sterilizes is afterwards with sterile distilled water flushing 4～6 times.The fructus schisandrae somatic embryo takes place and plant regeneration method also can carry out according to the following steps: (one) cuts good fructus schisandrae individual nutrition organ and carries out preliminary treatment, be inoculated into then on the MS medium of the 2,4 dichlorophenoxyacetic acid, 10～60g/L sucrose and the 5.0～8.0g/L agar that add 0.5～10mg/L; (2) be 29 ± 1 ℃ in temperature, the photoperiod is to cultivate for 7～9 weeks in the culturing room of the dark 8h of bright 16h/, obtains somatic embryo; (3) somatic embryo is transferred to the identical solid culture medium of (one) step composition in successive transfer culture 2～3 months, obtain callus; (4) the translucent milky embryo callus that successive transfer culture is obtained changes over to and adds 2 of 0.5～10mg/L, cultivated 4～5 months on the MS medium of 4-dichlorphenoxyacetic acid, choose afterwards little group that eugonic embryo callus is cut into 25～35mg transfer to inorganic salt concentration be MS 1/4～1/2 and added on the MS medium of 10～60g/L sucrose and 5.0～8.0g/L agar, be to cultivate for 4～5 weeks, the somatic embryo that obtains germinateing under the condition of the dark 8h of bright 16h/ at 25 ± 1 ℃, photoperiod; (5) somatic embryo that germinates is transferred to (four) and gone on foot in the identical medium of composition and cultivated for 4～5 weeks, obtain complete regeneration plant; (6) regeneration plant is transplanted into the soil cultivation, promptly obtains the fructus schisandrae nursery stock; Wherein preliminary treatment is that nutrition organs is put into concentration with the running water flushing after one day be that 70% alcohol soaks 30s in the step (), use distilled water flushing again 2 times, the liquor natrii hypochloritis who the puts into concentration 3% then 10min that sterilizes is afterwards with sterile distilled water flushing 4～6 times.
The successive transfer culture cycle of fructus schisandrae somatic embryo was 4 weeks.The present invention can induce the bipolar structure with young root, young shoot, than two kinds of different hormones of needs respectively the organ occurring mode of evoking adventive bud, adventive root be more conducive to realize the automated production of nursery stock.Somatic embryo generation scope of selecting material of the present invention is wide, and little to female parent destruction, reproduction speed is fast, propagation multiple height, the 30mg embryo callus can obtain 1800～2200 individual cells embryos in 2 months, bud fast growth, cultivation growth in 15 days 2.0～3.0cm, and survival rate is up to more than 80%.But the present invention's hybrid vigor fixing, a large amount of production and maternal regeneration plant with identical merit used for many years and be need not the breeding process of complexity such as three series mating.And the present invention also can be used as the modular system of different biological events in the research plant embryos generating process.
Embodiment
Embodiment one: present embodiment fructus schisandrae somatic embryo takes place and plant regeneration method carries out according to the following steps: (one) cuts good fructus schisandrae individual nutrition organ and carries out preliminary treatment, is inoculated into then on the medium that adds 0.5～10mg/L growth hormone, 10～60g/L sucrose and 5.0～8.0g/L agar; (2) be 29 ± 1 ℃ in temperature, the photoperiod is to cultivate for 7～9 weeks in the culturing room of the dark 8h of bright 16h/, obtains somatic embryo; (3) somatic embryo was transferred in the solid culture medium identical successive transfer culture 2～3 months, obtained callus with first step composition; (4) the translucent milky embryo callus of successive transfer culture is changed over to cultivated on the medium that adds 0.5～10mg/L growth hormone 25～35 days, transfer to again afterwards that to have added 10～60g/L sucrose and 5.0～8.0g/L agar, inorganic salt concentration be on 1/4～1/2 the medium, be to cultivate for 4～5 weeks, the somatic embryo that obtains germinateing under the condition of the dark 8h of bright 16h/ at 25 ± 1 ℃, photoperiod; (5) somatic embryo that germinates is transferred in the medium identical and cultivated for 4～5 weeks, obtain complete regeneration plant with the 4th step composition; (6) regeneration plant soil is transplanted and is cultivated, and promptly obtains the fructus schisandrae nursery stock.
The successive transfer culture cycle of fructus schisandrae somatic embryo was 4 weeks.Present embodiment can be passed through the bioreactor culture somatic embryo, has enlarged the cultivation scale, has reduced cost, has realized the batch production production of good fructus schisandrae and the quick popularization of new varieties.
Embodiment two: the difference of present embodiment and embodiment one is: step () selects for use the fructus schisandrae individual nutrition organ of " red pearl " strain to carry out preliminary treatment.Other step is identical with embodiment one.
Present embodiment has been set up the vegetative propagation system of fructus schisandrae clone improved seeds " red pearls ".
Embodiment three: the difference of present embodiment and embodiment one is: the nutrition organs in the step () is that sterile tissue is cultivated root, stem, leaf, the bud of seedling or is the root of perennial plant, stem, leaf, flower, bud or apical meristem.Other step is identical with embodiment one.
Embodiment four: the difference of present embodiment and embodiment one is: preliminary treatment is that nutrition organs is put into concentration with the running water flushing after one day be that 70% alcohol soaks 30s in the step (), use distilled water flushing again 2 times, the liquor natrii hypochloritis who puts into concentration 3% 10min that sterilizes is afterwards with sterile distilled water flushing 4～6 times.Other step is identical with embodiment one.
Embodiment five: the difference of present embodiment and embodiment one is: medium is earlier with adding agar again after the NaOH of 1N or the HCl adjusting pH value to 5.75 in the step ().Other step is identical with embodiment one.
Embodiment six: the difference of present embodiment and embodiment one is: the growth hormone among step () and (four) be 2,4 dichlorophenoxyacetic acid (2,4-D), methyl or indolebutyric acid.Other step is identical with embodiment one.
Embodiment seven: the difference of present embodiment and embodiment one is: the 2,4 dichlorophenoxyacetic acid of every liter of medium adding 2～8mg among step () and (four).Other step is identical with embodiment one.
Embodiment eight: the difference of present embodiment and embodiment one is: the 2,4 dichlorophenoxyacetic acid of every liter of medium adding 4mg among step () and (four).Other step is identical with embodiment one.
Present embodiment is selected the scheme that adds the 4mg 2,4 dichlorophenoxyacetic acid in every liter of medium for use by test.2,4-D concentration is as shown in table 1 to the test result of frequency of embryonic callus induction influence.
Table 1
Embodiment nine: the difference of present embodiment and embodiment one is: use in the step () to (five) with a kind of medium, medium is a kind of in MS (Murashige and Skoog) medium, SH medium, Gamborg B5 medium, N6 medium or the Heller medium.Other step is identical with embodiment one.
Present embodiment is only used a kind of medium, the step that can simplify the operation like this, can save the labour and reduces production costs, and helps realizing the batch production production of nursery stock.
Embodiment ten: the difference of present embodiment and embodiment one is: use the MS medium in the step () to (five).Other step is identical with embodiment one.
Embodiment 11: the difference of present embodiment and embodiment one is: 3 running water flush away medium of the regeneration plant with blade will be taken root and have to step (six), be transplanted in the container that turfy soil is housed, overlay film 2～4 weeks of cultivation, every day water 1 time under greenhouse experiment, remove the plastic film of covering after the young leaves sheet grows.Other step is identical with embodiment one.
Present embodiment regeneration plant survival rate is higher than 80%.
Embodiment 12: the difference of present embodiment and embodiment 11 is: be transplanted to the regeneration plant overlay film under 20 ℃ of conditions that is equipped with in the turfy soil container in the step (six) and cultivated for 2～4 weeks.Other step is identical with embodiment 11.
Present embodiment regeneration plant survival rate can be up to 100%.
Embodiment 13: present embodiment fructus schisandrae somatic embryo takes place and plant regeneration method, the fructus schisandrae somatic embryo takes place and plant regeneration method carries out according to the following steps: (one) cuts good fructus schisandrae individual nutrition organ and carries out preliminary treatment, is inoculated into then on the medium that adds 0.5～10mg/L growth hormone, 10～60g/L sucrose and 5.0～8.0g/L agar; (2) be 29 ± 1 ℃ in temperature, the photoperiod is to cultivate for 7～9 weeks in the culturing room of the dark 8h of bright 16h/, obtains somatic embryo; (3) somatic embryo was transferred in the solid culture medium identical successive transfer culture 2～3 months, obtained callus with first step composition; (4) the translucent milky embryo callus of successive transfer culture is changed over to cultivated on the medium that adds 0.5～10mg/L growth hormone 4～5 months, choose little group that eugonic embryo callus is cut into 25～35mg afterwards and transfer to that to have added 10～60g/L sucrose and 5.0～8.0g/L agar, inorganic salt concentration be on 1/4～1/2 the medium, be to cultivate for 4～5 weeks, the somatic embryo that obtains germinateing under the condition of the dark 8h of bright 16h/ at 25 ± 1 ℃, photoperiod; (5) somatic embryo that germinates is transferred in the medium identical and cultivated for 4～5 weeks, obtain complete regeneration plant with the 4th step composition; (6) regeneration plant soil is transplanted and is cultivated, and promptly obtains the fructus schisandrae nursery stock.
Present embodiment is carried out repeatedly successive transfer culture to embryo callus, and its result is as shown in table 2.Embryo callus is through successive transfer culture repeatedly, and it is vigorous to grow, and inoculum concentration increases, and the somatic embryo number significantly increases.
Table 2
| The successive transfer culture number of times of embryo callus | The upgrowth situation of embryo callus | Embryo callus inoculum concentration (mg fresh weight) | The somatic embryo number (individual) that obtains |
| 1 time | Slowly | 3mg | 20～35 |
| More than 4 times | Vigorous | 30mg | 1800～2200 |
Embodiment 14: present embodiment fructus schisandrae somatic embryo takes place and plant regeneration method carries out according to the following steps: the full hibernaculum that () cuts on the annotinous branches of 4 years living above plant of " red pearl " fructus schisandrae carries out preliminary treatment, be inoculated into then on the MS medium that adds 4mg/L 2,4 dichlorophenoxyacetic acid, 30g/L sucrose and 5.5g/L agar; (2) be 29 ℃ in temperature, the photoperiod is to cultivate for 8 weeks in the culturing room of the dark 8h of bright 16h/, obtains somatic embryo; (3) somatic embryo was transferred in the solid culture medium identical successive transfer culture 2～3 months, obtained callus with first step composition; (4) the translucent milky embryo callus of successive transfer culture is changed over to add 4mg/L 2, cultivated 4～5 months on the MS medium of 4-dichlorphenoxyacetic acid, choose little group that eugonic embryo callus is cut into 28～32mg afterwards and transfer to that to have added 20g/L sucrose and 5.5g/L agar, inorganic salt concentration be on 1/2 the medium, be to cultivate for 4～5 weeks, the somatic embryo that obtains germinateing under the condition of the dark 8h of bright 16h/ at 25 ℃, photoperiod; (5) somatic embryo that germinates is transferred in the medium identical and cultivated for 4～5 weeks, obtain complete regeneration plant with the 4th step composition; (6) will take root and have 3 running water flush away medium of the regeneration plant with blade, be transplanted in the container that turfy soil is housed, overlay film cultivated for 3 weeks under greenhouse experiment, after growing, the young leaves sheet removes the plastic film of covering, transfer to and continue in 20 ℃ of environment to cultivate, water water every day 1 time in soil transplanting incubation, promptly obtains the fructus schisandrae nursery stock.
The incidence of somatic embryo is 1.7～3.5% in the present embodiment, cultivates 20 days through step (three), and the fresh weight of somatic embryo increases more than 10 times.
Claims (6)
1. the fructus schisandrae somatic embryo takes place and plant regeneration method, it is characterized in that the fructus schisandrae somatic embryo takes place and plant regeneration method carries out according to the following steps: (one) cuts good fructus schisandrae individual nutrition organ and carries out preliminary treatment, be inoculated into then on the MS medium of the 2,4 dichlorophenoxyacetic acid, 10～60g/L sucrose and the 5.0～8.0g/L agar that add 0.5～10mg/L; (2) be 29 ± 1 ℃ in temperature, the photoperiod is to cultivate for 7～9 weeks in the culturing room of the dark 8h of bright 16h/, obtains somatic embryo; (3) somatic embryo is transferred to the identical solid culture medium of (one) step composition in successive transfer culture 2～3 months, obtain callus; (4) the translucent milky embryo callus that successive transfer culture is obtained changes over to and is added with 2 of 0.5～10mg/L, cultivated 25～35 days on the MS medium of 4-dichlorphenoxyacetic acid, transfer to inorganic salt concentration afterwards again and be MS 1/4～1/2 and added on the MS medium of 10～60g/L sucrose and 5.0～8.0g/L agar, be to cultivate for 4～5 weeks, the somatic embryo that obtains germinateing under the condition of the dark 8h of bright 16h/ at 25 ± 1 ℃, photoperiod; (5) somatic embryo that germinates is transferred to (four) and gone on foot in the identical medium of composition and cultivated for 4～5 weeks, obtain complete regeneration plant; (6) regeneration plant is transplanted into the soil cultivation, promptly obtains the fructus schisandrae nursery stock; Wherein preliminary treatment is that nutrition organs is put into concentration with the running water flushing after one day be that 70% alcohol soaks 30s in the step (), use distilled water flushing again 2 times, the liquor natrii hypochloritis who the puts into concentration 3% then 10min that sterilizes is afterwards with sterile distilled water flushing 4～6 times.
2. fructus schisandrae somatic embryo according to claim 1 takes place and plant regeneration method, it is characterized in that step () carries out preliminary treatment with the fructus schisandrae individual nutrition organ of red pearl strain.
3. fructus schisandrae somatic embryo according to claim 1 takes place and plant regeneration method, it is characterized in that the nutrition organs in the step () is root, stem, leaf or the bud that sterile tissue is cultivated seedling; Perhaps nutrition organs is root, stem, leaf, flower, bud or the apical meristem of perennial plant.
4. fructus schisandrae somatic embryo according to claim 1 takes place and plant regeneration method, it is characterized in that the 2,4 dichlorophenoxyacetic acid of every liter of medium adding 4mg among step () and (four).
5. fructus schisandrae somatic embryo according to claim 1 takes place and plant regeneration method, it is characterized in that the transplanting in the step (six) is to take root and 3 running water flush away medium of the regeneration plant with blade are arranged, be transplanted in the container that turfy soil is housed, overlay film cultivated for 2～4 weeks under greenhouse experiment, water every day 1 time, after the young leaves sheet grows, remove the plastic film of covering.
6. fructus schisandrae somatic embryo according to claim 5 takes place and plant regeneration method, it is characterized in that, is transplanted to the regeneration plant overlay film under 20 ℃ of conditions that is equipped with in the turfy soil container and cultivates for 2～4 weeks.
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| Application Number | Priority Date | Filing Date | Title |
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| CNB2006100097820A CN100381044C (en) | 2006-03-08 | 2006-03-08 | Beiwuweizi cell embryo growth and strain regeneration |
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| CNB2006100097820A CN100381044C (en) | 2006-03-08 | 2006-03-08 | Beiwuweizi cell embryo growth and strain regeneration |
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| CN1817106A CN1817106A (en) | 2006-08-16 |
| CN100381044C true CN100381044C (en) | 2008-04-16 |
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101803568B (en) * | 2010-04-28 | 2012-05-30 | 吉林大学 | Quickly breeding method of schisandra chinensis hybridized homozygous lines |
| CN101904302B (en) * | 2010-07-13 | 2012-06-27 | 沈阳农业大学 | Method for somatic cell embryogeny and plant regeneration of medicinal plant schisandga chinensis baill |
| CN101878737B (en) * | 2010-07-14 | 2012-05-30 | 长春百瑞蓝莓科技发展有限公司 | Schisandra tissue culture method |
| CN103299904B (en) * | 2013-05-30 | 2014-12-24 | 辽宁万亩五味子科技产业园区有限公司 | Artificial schisandra chinensis seed preparation and seedling culture method |
| CN106171991B (en) * | 2016-07-15 | 2018-12-11 | 中国农业科学院特产研究所 | A kind of expanding propagation method of fructus schisandrae |
| CN111387059B (en) * | 2020-05-12 | 2022-11-22 | 沈阳农业大学 | Method for regenerating plants from callus of schisandra chinensis |
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2006
- 2006-03-08 CN CNB2006100097820A patent/CN100381044C/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| 五味子的组织培养. 周鑫.中国林副特产，第4期. 2001 * |
| 五味子研究进展. 张彩丽，贺学礼.保定师范专科学校学报，第17卷第4期. 2004 * |
| 药用植物北五味子的组织培养. 陈雅君，吴秀菊，关政华，杨永富.植物研究，第19卷第3期. 1999 * |
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