CN105746344A - Virus-free tissue culture and rapid propagation method of castor oil plants - Google Patents
Virus-free tissue culture and rapid propagation method of castor oil plants Download PDFInfo
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- CN105746344A CN105746344A CN201410800385.XA CN201410800385A CN105746344A CN 105746344 A CN105746344 A CN 105746344A CN 201410800385 A CN201410800385 A CN 201410800385A CN 105746344 A CN105746344 A CN 105746344A
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Abstract
The invention discloses a virus-free tissue culture and rapid propagation method of castor oil plants. The method comprises the following steps: adopting current year's ripe seeds, mixing castor oil plant seeds with coarse sands according to a ratio of 1:5-8, carrying out medium speed grinding and shelling, screening to remove the coarse sands, cleaning the obtained seeds with warm water 2-3 times, immersing the washed seeds in 75% alcohol for 0.5-1.0min, disinfecting the immersed seeds with an aqueous solution of 2% sodium hypochlorite for 20-30min, and flushing the disinfected seeds with sterile water 3-5 times to obtain the flushed seeds used as explants; and carrying out induction culture, propagation culture, rooting culture, transplanting and virus detection to obtain mass propagated virus-free tissue culture seedlings, virus-free plant buds and virus-free small root tubers. Compared with present castor oil plant seedling growing methods, the method disclosed in the invention has the advantages of low inoculation and pollution rate, high transplanting survival rate and high quality.
Description
Technical field
The present invention relates to plant toxic group culturation rapid propagating technology, particularly relate to the side of a kind of Semen Ricini detoxication and tissue culture rapid propagation
Background technology
Semen Ricini is that Euphorbiaceae Ricinus is annual or herbaceos perennial, and current growing of castor oil plant is used for extracting medicine and iundustrial oil, or is used as landscape tree to view and admire its leaf.Wherein, medical value is mainly reflected on ricin, has anticancer, the logical stagnant effect of detumescence that relieves internal heat；Oleum Ricini is used for fine chemistry industry, can control surface activating agent, fatty glyceride, aliphatic glycol, drying oil, decanedioic acid, the stabilizer of polymerization and plasticizer, foam plastics and gum elastic etc..Oleum Ricini viscosity is big, proportion high (0.958～0.968), does not solidify, never degenerates, do not burn under the high temperature of 500～600 DEG C, be the important source material of senior lubricant under-18 DEG C of low temperature.
Semen Ricini is one of big oil crop in the world ten, whole world cultivated area about 1,500,000 hm2, average per unit area yield 1000kg/hm2, total product seed 1,500,000 tons.China's cultivating castor-oil plant area is about 300,000 hm2, the gross output value 25～300,000 tons, occupy the 1/5 of world's total amount.In recent years, along with the resource minimizing of oil savings amount, academy of agricultural sciences of China is renewable as one using Oleum Ricini, and the biomass energy plant of fossil energy can be replaced to study.Oleum Ricini is as energy substance, and it has the advantage that 1) strong adaptability, drought-enduring degeneration-resistant, China's overwhelming majority area all can be planted；2) grow surging, well established and vigorously developing, can improve the ecological environment, it is prevented that soil erosion, increase green coverage；3) Semen Ricini is of many uses, can utilize as energy crop, it is also possible in industry, medicine, sericulture and military use.As can be seen here, cultivating castor-oil plant has wide market prospect and good economic benefit.
At present, cultivating castor-oil plant mainly adopts sowing in the field, and emergence rate is low, and viral infection is serious, strongly limit medicinal and oil mass output, affects the application quality of Semen Ricini.Therefore, it is necessary to the method that a kind of viral infection rate is low, product quality is high detoxication and tissue culture rapid propagation is provided.
Summary of the invention
The present invention seeks to overcome above Shortcomings, it is proposed to a kind of without cutting microphyte stem apex, can the method for fast numerous Semen Ricini tissue cultural seedlings of free in a large number.
The object of the invention is achieved through the following technical solutions:
A kind of method of Semen Ricini detoxication and tissue culture rapid propagation, carries out as follows:
1) preparation of culture medium, trains each stage culture medium including minimal medium and group, and the component of each culture medium is:
1. minimal medium: selection MS culture medium, sucrose or white sugar 20～30g/L, carrageenan 5～7g/L, pH6.0～7.2；
2. inducing culture: add 6-BA0.5～1.5mg/L, NAA0.15～0.5mg/L and chlorella powder 0.5～1.2mg/L in MS；
3. Calli Differentiation culture medium: add 6-BA2.5～3.0mg/L, NAA0.2～0.3mg/L and chlorella powder 0.5～1.2mg/L in MS；
4. proliferated culture medium: add 6-BAl.0mg/L and NAAO.5mg/L in MS；
5. root media: add NAAO.1mg/L and ABT biological bacteria root-inducing powder O.1mg/L in 1/2MS.
2) the choosing and sterilizing of outer implant: taking the seed of Semen Ricini, after sand grinds off shell, sterilized process, as the outer implant of detoxication and tissue culture；
3) inducing culture: by step 2) seed after sterilization treatment is seeded on inducing culture, directly form unrooted tissue cultured seedling or be initially formed callus, callus is migrated to division culture medium again and induces clump bud, form unrooted tissue cultured seedling, bulbil and little tuber；
4) enrichment culture: by step 3) the unrooted tissue cultured seedling that directly formed or be initially formed callus, it is seeded on proliferated culture medium then through the unrooted tissue cultured seedling of division culture medium induced synthesis, bulbil and little tuber, induce clump bud, form unrooted tissue cultured seedling, bulbil and little tuber；
5) root culture: by step 4) the unrooted tissue cultured seedling that formed of propagation, bulbil and little tuber be seeded in root media and carry out root culture；
6) transplant: when step 5) tissue cultured seedling of taking root grow to 8～more than 10cm, bulbil 0.5～more than 0.7cm of taking root, little tuber diameter 0.5～more than 1.0cm of taking root, transplanting medium is cultured to be a seedling；
7) Viral diagnosis: utilize RT-PCR amplification technique, builds its genome sequence clone, and prokaryotic expression prepares the antiserum of virus-specific, sets up ELISA detection technique and carries out the detection of Semen Ricini soybean mosaic virus, Dasheen mosaic virus, Zantedeschia aethiopica Spreng. mosaic virus.
Further, described castor seeds is the mature seed given birth to then, adopts castor seeds: coarse sand is that after the proportioning mixing of 1:5～8, middling speed is ground and shelled, and sieve removes coarse sand, and warm water cleans 2～3 times.
Further, described sterilization treatment be through 75% alcohol-pickled 0.5～l.0min, then the aqueous sodium hypochlorite solution sterilizing 20～30min with 2%, finally with aseptic water washing 3～5 times.
Further, described transplanting medium is field soil: perlite: Vermiculitum: 3:1:1:0.5 by volume is formulated for scale stone.
The invention has the beneficial effects as follows:
1) present invention utilizes castor seeds as the outer implant of detoxication and tissue culture, owing to volume is relatively big, inoculates processing ease；Inoculation pollution rate only has about 3% or lower, and Shoot Tip Culture inoculates in most cases pollution rate about 40～80%；Success ratio of inoculation is high up to 100%, and Shoot Tip Culture inoculation survival rate generally only has 20～40%；Comparable stem apex virus-free culture is done sth. in advance to obtain tissue cultural seedlings of free in 1～2 month, reduces cost, practical easily popularization；
2) detoxification of the present invention is easy, quick, solving the halfway problem of stem apex detoxification, by tissue cultural seedlings of free, detoxification bulbil and the little tuber of detoxification after Viral diagnosis, band poison rate is 0, virus can be solved and cause Semen Ricini kind matter degenerate problem, recover its yield and quality；
3) castor seeds is reserved seed for planting easily, can quickly obtain a large amount of tissue cultural seedlings of free, detoxification bulbil and the little tuber of detoxification, the moon breeding coefficient reach 5～10 times, be suitable for industrial seedling rearing, can merchandized handling.
Detailed description of the invention
By following example, the present invention is described in further detail, but present disclosure is not limited thereto.
Embodiment 1
1) the choosing and sterilizing of outer implant: select mature seed raw then, adopt castor seeds: coarse sand is after the proportioning mixing of 1:5～8, middling speed is ground and is shelled, sieve removes coarse sand, warm water cleans 2～3 times, through 75% alcohol-pickled 0.5～l.0min, then the aqueous sodium hypochlorite solution sterilizing 20～30min with 2%, finally use aseptic water washing 3～5 times, as the outer implant of detoxication and tissue culture；
2) minimal medium: select MS minimal medium, sucrose 25g/L, carrageenan 6.5g/L, pH6.5；
3) inducing culture: aseptically, seed is seeded on the inducing culture of MS+6-BA1.2mg/L+NAA0.35mg/L+ chlorella powder 0.8mg/L, it is initially formed callus, again callus is migrated to and the division culture medium of MS+6-BA2.5mg/L+NAA0.2mg/L+ chlorella powder 1.2mg/L induces clump bud, form unrooted tissue cultured seedling and bulbil；
4) enrichment culture: aseptically unrooted tissue cultured seedling and bulbil are seeded on the proliferated culture medium of MS+6-BAl.0mg/L+NAAO.5mg/L, induced bundle bud, form unrooted tissue cultured seedling and bulbil；
5) root culture: unrooted tissue cultured seedling, bulbil and little tuber are inoculated in 1/2MS+NAAO.1mg/L+ABT biological bacteria root-inducing powder root media O.1mg/L and carry out root culture:
6) transplant: when tissue cultured seedling of taking root grow to 8～more than 10cm, bulbil 0.5～more than 0.7cm of taking root, little tuber diameter 0.5～more than 1.0cm of taking root, transplanting medium is cultured to be a seedling；
7) Viral diagnosis: utilize RT-PCR amplification technique, builds its genome sequence clone, and the anti-ware that prokaryotic expression prepares virus-specific is clear, sets up ELISA detection technique and carries out the detection of Radix Bupleuri soybean mosaic virus, Dasheen mosaic virus, Zantedeschia aethiopica Spreng. mosaic virus.
Testing result: matched group (conventional Seedling) is positive；The present embodiment 1 tissue cultural seedlings of free is negative.
Claims (4)
1. the method for a Semen Ricini detoxication and tissue culture rapid propagation, it is characterised in that: carry out as follows:
1) preparation of culture medium, trains each stage culture medium including minimal medium and group, and the component of each culture medium is:
1. minimal medium: selection MS culture medium, sucrose or white sugar 20～30g/L, carrageenan 5～7g/L, pH6.0～7.2；
2. inducing culture: add 6-BA0.5～1.5mg/L, NAA0.15～0.5mg/L and chlorella powder 0.5～1.2mg/L in MS；
3. Calli Differentiation culture medium: add 6-BA2.5～3.0mg/L, NAA0.2～0.3mg/L and chlorella powder 0.5～1.2mg/L in MS；
4. proliferated culture medium: add 6-BAl.0mg/L and NAAO.5mg/L in MS；
5. root media: add NAAO.1mg/L and ABT biological bacteria root-inducing powder O.1mg/L in 1/2MS.
2) the choosing and sterilizing of outer implant: taking the seed of Semen Ricini, after sand grinds off shell, sterilized process, as the outer implant of detoxication and tissue culture；
3) inducing culture: by step 2) seed after sterilization treatment is seeded on inducing culture, directly form unrooted tissue cultured seedling or be initially formed callus, callus is migrated to division culture medium again and induces clump bud, form unrooted tissue cultured seedling, bulbil and little tuber；
4) enrichment culture: by step 3) the unrooted tissue cultured seedling that directly formed or be initially formed callus, it is seeded on proliferated culture medium then through the unrooted tissue cultured seedling of division culture medium induced synthesis, bulbil and little tuber, induce clump bud, form unrooted tissue cultured seedling, bulbil and little tuber；
5) root culture: by step 4) the unrooted tissue cultured seedling that formed of propagation, bulbil and little tuber be seeded in root media and carry out root culture；
6) transplant: when step 5) tissue cultured seedling of taking root grow to 8～more than 10cm, bulbil 0.5～more than 0.7cm of taking root, little tuber diameter 0.5～more than 1.0cm of taking root, transplanting medium is cultured to be a seedling；
7) Viral diagnosis: utilize RT-PCR amplification technique, builds its genome sequence clone, and prokaryotic expression prepares the antiserum of virus-specific, sets up ELISA detection technique and carries out the detection of Semen Ricini soybean mosaic virus, Dasheen mosaic virus, Zantedeschia aethiopica Spreng. mosaic virus.
2. the method for a kind of Semen Ricini detoxication and tissue culture rapid propagation as claimed in claim 1, it is characterized in that: described castor seeds is the mature seed given birth to then, adopt castor seeds: coarse sand is that after the proportioning mixing of 1:5～8, middling speed is ground and shelled, sieve removes coarse sand, and warm water cleans 2～3 times.
3. the method for a kind of Semen Ricini detoxication and tissue culture rapid propagation as claimed in claim 1, it is characterised in that: described sterilization treatment be through 75% alcohol-pickled 0.5～l.0min, then the aqueous sodium hypochlorite solution sterilizing 20～30min with 2%, finally with aseptic water washing 3～5 times.
4. the method for a kind of Semen Ricini detoxication and tissue culture rapid propagation as claimed in claim 1, it is characterised in that: described transplanting medium is field soil: perlite: Vermiculitum: 3:1:1:0.5 by volume is formulated for scale stone.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106576749A (en) * | 2016-11-22 | 2017-04-26 | 淄博市农业科学研究院 | Castor-oil plant tissue culture plants concentrated propagation method |
| CN106717274A (en) * | 2016-12-15 | 2017-05-31 | 中国热带农业科学院热带作物品种资源研究所 | Francolin tea seed germination method and the culture medium sprouted for francolin tea seed |
| CN111165196A (en) * | 2020-03-06 | 2020-05-19 | 广东海洋大学 | Castor micro-grafting method |
-
2014
- 2014-12-19 CN CN201410800385.XA patent/CN105746344A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106576749A (en) * | 2016-11-22 | 2017-04-26 | 淄博市农业科学研究院 | Castor-oil plant tissue culture plants concentrated propagation method |
| CN106576749B (en) * | 2016-11-22 | 2017-12-19 | 淄博市农业科学研究院 | Castor-oil plant tissue-cultured seedling concentrates expanding propagation method |
| CN106717274A (en) * | 2016-12-15 | 2017-05-31 | 中国热带农业科学院热带作物品种资源研究所 | Francolin tea seed germination method and the culture medium sprouted for francolin tea seed |
| CN111165196A (en) * | 2020-03-06 | 2020-05-19 | 广东海洋大学 | Castor micro-grafting method |
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