CN101595844B - Method for quickly propagating transgenic tuber pinellia by adopting micro adventitious bud technology - Google Patents
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Abstract
The invention discloses a method for quickly propagating transgenic tuber pinellia by adopting micro adventitious bud technology in the field of biotechnical. The method comprises the steps of: placing a disinfected transgenic tuber pinellia explant on a germination medium, and performing lighting culture on the explant to generate leaves and leafstalks; cutting the leaves and the leafstalks down when an apical bud grows to be between 4 and 6 cm; placing the leaves and the leafstalks on a micro adventitious bud induction culture medium; performing lighting culture to generate a large quantity of micro adventitious buds around the explant; transferring the explant onto an adventitious bud rooting medium when the explant grows to be between 3 and 4 cm; allowing the explant to generate adventitious buds; and transplanting a robust-growth intact plant into a flower pot receiving transplanting medium, and obtaining a normal-growth transgenic tuber pinellia plant through the acclimatization. The method screens the culture media which are suitable for the induction and rooting of the micro adventitious buds of the transgenic tuber pinellia, the induction rate of the micro adventitious buds of the transgenic tuber pinellia explant reaches over 90 percent, the rooting rate of the adventitious buds is 90 percent, the transplanting survival rate of a regenerated plant is 90 percent, and the propagation coefficient reaches over 80 percent.
Description
Technical field
What the present invention relates to is a kind of method of biological technical field, specifically is a kind of method that adopts the micro adventitious bud technology quickly propagating transgenic tuber pinellia.
Background technology
The tuber of pinellia (Pinellia ternate (Thunb) Berit (Araceae)) is an Araeceae Pinellia Tenore perennial plant, mainly is distributed in Chinese East China, North China and the Yangtze river basin.Stem tuber contains amino acid, cupreol, choline, cupreol-β-D-glycoside, 3 such as volatile oil, little fat (its fatty acid about 34% is liquid acid for solid acid, 66%), starch, nicotine, mucilaginous substance, asparatate, glutamic acid, arginine, beta-aminobutyric acid; The 4-4-dihydroxy benzaldehyde contains the material of the pharmacology effect alkaloid similar with coniine and nicotine, similar protoanemonin chafe again.Tender shoots contains alcapton and glucoside thereof.The tuber of pinellia is used as medicine with stem tuber, has scorching reducing phlegm, stopping nausea and vomiting by lowering the adverse flow of QI, and the effect that the ruffian that disappears is loose and saved is Chinese conventional medicinal material.It is antitumor to find again that in recent years it has, and anti-early pregnancy, protects the effect of liver, reducing blood lipid.Tuber of pinellia frequency of occurrences in 196 prescriptions of traditional Chinese medicine occupy the 22nd, uses very extensively, has important medical value, and the social demand amount is also very big.
By the tuber of pinellia happiness growth stream, under the dark and damp environment in field, shrub etc., piecemeal stem breeding, bulbil breeding, seminal propagation, but mainly lean on bulbil, the capable vegetative propagation of stem tuber, and reproduction coefficient is lower, and the breeding cycle is longer.
Owing to the continuous growth to tuber of pinellia demand, people excavate in a large number in recent years, and its wild resource sharply reduces; Owing to the scale of its plantations of reason restriction such as provenance shortage, germplasm degeneration and damage by disease and insect, cause the serious lack of tuber of pinellia resource on the other hand, become with the tuber of pinellia bottleneck of the medical product development that is raw material.Along with development of biology, plant tissue culture technique is ripe day by day, on multiple gardens, gardening and medicinal plant, obtains application at present.Because the higher market value of the tuber of pinellia itself, the scientific research personnel breeds fast with the aspect of setting up of cultivating system to it and has launched big quantity research, and obtains a series of achievements.
At present existing various plants is bred into the report of complete regenerated plant fast in the existing document through micro adventitious bud technology.If can pass through the micro adventitious bud technology quickly propagating transgenic tuber pinellia, will promote, ensure that tuber of pinellia seedling, raising output, the shortening breeding cycle of sufficient high-quality is significant to the large tracts of land of transgenic tuber pinellia to keeping the genetic stability of transgenic tuber pinellia.But have in the micro adventitious bud quick propagating technology of plant of report; The medium of sprouting that they are selected, micro adventitious bud inducing culture, adventitious bud rooting medium and transplanting medium all are not suitable for the quick breeding of transgenic tuber pinellia, also find at present the relevant report with the mentioned employing micro adventitious bud technology quickly propagating transgenic tuber pinellia of theme of the present invention as yet.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, a kind of method that adopts the micro adventitious bud technology quickly propagating transgenic tuber pinellia is provided.Relate to sprout medium, micro adventitious bud inducing culture, adventitious bud rooting medium and transplanting medium of the tuber of pinellia among the present invention and be used for the method for micro adventitious bud tissue culture of the present invention; Set up method with the micro adventitious bud technology quickly propagating transgenic tuber pinellia; Can breed a large amount of transgenic tuber pinellia seedlings at short notice, promote, ensure that tuber of pinellia seedling, raising tuber of pinellia output, the shortening breeding cycle of sufficient high-quality established solid foundation for the large tracts of land of transgenic tuber pinellia.
Invention realizes through following technical scheme: the present invention through after sterilizing, places the transgenic tuber pinellia explant on the medium of sprouting, and illumination cultivation generates blade and petiole; When treating terminal bud length to 4cm-6cm, downcut blade and petiole, place on the micro adventitious bud inducing culture, illumination cultivation grows a large amount of micro adventitious buds around explant; When treating explant length, explant is transferred on the adventitious bud rooting medium, made the generation adventive root to 3cm-4cm; The whole plant of robust growth is transplanted in the flowerpot that fills transplanting medium, obtains the normal transgenic tuber pinellia plant of growth through domestication;
The sterilization of said transgenic tuber pinellia explant is meant: strip explant from the transgenic tuber pinellia plant, and with 75% alcohol disinfecting 10 minutes, sterile water wash 3 times, 0.1%HgCl
2Sterilized sterile water wash 8 times 20 minutes.
Said explant is the stem tuber that contains terminal bud, cuts the epidermis part, and being cut into volume is 3cm
3-6cm
3Fritter.
The said medium of sprouting is in the MS medium, to add 1.0mg/L 6-benzyladenine, 1.0mg/L methyl, 30g/L sucrose and 8.5g/L agar powder.Wherein the MS medium is that Murashige and Skoog announces in 1962.
Said through illumination cultivation, generate the blade petiole, be meant: cultivation temperature is 25 ℃ ± 1 ℃, illumination every day 16 hours, explant grows the terminal bud of 4cm-6cm after 10 days.
The said terminal bud of treating is long during to 4cm-6cm, downcuts blade and is meant with petiole and downcuts young tender blade and petiole that the long 1cm-2cm of petiole, blade area are 2cm
2-4cm
2
Said micro adventitious bud inducing culture is in the MS medium, to add 2.0mg/L 6-benzyladenine, 0.1 (blade)/0.25 (petiole) mg/L methyl, 30g/L sucrose and 8.5g/L agar powder;
Said through illumination cultivation, grow a large amount of micro adventitious buds around the explant, be meant: cultivation temperature is 25 ℃ ± 1 ℃, illumination every day 16 hours, per 14 days-20 days successive transfer culture once, through around explant, growing a large amount of micro adventitious buds behind 1 successive transfer culture.
Said adventitious bud rooting medium is in the 1/2MS medium, to add kinetin 1.0mg/L, methyl 2.0mg/L, 30g/L sucrose and 8.5g/L agar powder;
Said through illumination cultivation, make the generation adventive root, be meant: cultivation temperature is 25 ℃ ± 1 ℃, illumination every day 16 hours through 20 angel's adventitious bud rootings, thereby forms complete tuber of pinellia plant.
Said whole plant with robust growth is transplanted in the flowerpot that fills transplanting medium, is meant: choose the plant of robust growth, be transplanted in the cave dish that transplanting medium is housed.
Said transplanting medium is a vermiculite: earth, the volume ratio of vermiculite and earth are 1: 2.
The present invention adopts the method for micro adventitious bud tissue culture; Filter out and be fit to the medium that the transgenic tuber pinellia micro adventitious bud is induced and taken root; Transgenic tuber pinellia explant micro adventitious bud inductivity reaches more than 90%, and the adventitious bud rooting rate is 90%, and it is 90% that regeneration plant is transplanted survival rate; Reproduction coefficient reaches more than 80%; The foundation of transgenic tuber pinellia micro adventitious bud quick propagating technology for breeding a large amount of transgenic tuber pinellia seedlings fast, promotes, ensures that tuber of pinellia seedling, raising tuber of pinellia output, the shortening breeding cycle of sufficient high-quality is significant to the large tracts of land of transgenic tuber pinellia.
Embodiment
Elaborate in the face of embodiments of the invention down: present embodiment provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment being to implement under the prerequisite with technical scheme of the present invention.
Embodiment 1:
Inducing of the sterilization of transgenic tuber pinellia explant and micro adventitious bud:
From the transgenic tuber pinellia plant strip contain terminal bud stem tuber as explant.Strip explant from the transgenic tuber pinellia plant, with 75% alcohol disinfecting 10 minutes, sterile water wash 3 times, 0.1%HgCl
2Sterilized sterile water wash 8 times 20 minutes.The stem tuber that has terminal bud with disinfecting cuts the epidermis part, and being cut into volume is 3cm
3-6cm
3Fritter.Transfer on the medium of sprouting, the said medium of sprouting is in the MS medium, to add 1.0mg/L 6-benzyladenine (6-BA), 1.0mg/L methyl (NAA), 30g/L sucrose and 8.5g/L agar powder and obtain.Illumination cultivation, cultivation temperature are 25 ℃ ± 1 ℃, illumination every day 16 hours, and explant grows the terminal bud of 4cm-6cm after 10 days.When treating terminal bud length to 4cm-6cm, downcut young tender leaf handle and blade, the long 1cm of petiole, blade area are 2cm
2Transfer to the micro adventitious bud inducing culture; Said micro adventitious bud inducing culture is in the MS medium, to add 2.0mg/L 6-benzyladenine (6-BA), 0.1/0.25mg/L methyl (NAA) (if induce micro adventitious bud will add the 0.1mg/L methyl from blade, if induce micro adventitious bud will add the 0.25mg/L methyl from petiole), 30g/L sucrose and 8.5g/L agar powder and obtain; Illumination cultivation, cultivation temperature are 25 ℃ ± 1 ℃, illumination every day 16 hours, per 20 days successive transfer culture once, through around explant, growing a large amount of micro adventitious buds behind 1 successive transfer culture.The micro adventitious bud inductivity reaches 93%.
Taking root of transgenic tuber pinellia indefinite bud:
When treating indefinite bud length to 3cm-4cm; A large amount of indefinite buds are transferred on the adventitious bud rooting medium, and said adventitious bud rooting medium is in the 1/2MS medium, to add kinetin 1.0mg/L (KT), methyl 2.0mg/L (NAA), 30g/L sucrose and 8.5g/L agar powder, through illumination cultivation; Cultivation temperature is 25 ℃ ± 1 ℃; Illumination every day 16 hours through 20 angel's adventitious bud rootings, thereby forms complete tuber of pinellia plant.
The transplanting of transgenic tuber pinellia regeneration plant:
Choose the plant of robust growth, the medium of flush away root is transplanted in the cave dish that transplanting medium is housed, and said transplanting medium is a vermiculite: earth, the volume ratio of vermiculite and earth are 1: 2.Through taming for 1 week, can obtain the normal transgenic tuber pinellia plant of raised growth, transplanting survival rate is 90%, reproduction coefficient reaches 84%.
Embodiment 2:
Inducing of the sterilization of transgenic tuber pinellia explant and micro adventitious bud
From the transgenic tuber pinellia plant strip contain terminal bud stem tuber as explant.Strip explant from the transgenic tuber pinellia plant, with 75% alcohol disinfecting 10 minutes, sterile water wash 3 times, 0.1%HgCl
2Sterilized sterile water wash 8 times 20 minutes.The stem tuber that has terminal bud with disinfecting cuts the epidermis part, and being cut into volume is 3cm
3-6cm
3Fritter.Transfer on the medium of sprouting, the said medium of sprouting is in the MS medium, to add 1.0mg/L 6-benzyladenine (6-BA), 1.0mg/L methyl (NAA), 30g/L sucrose and 8.5g/L agar powder and get.Illumination cultivation, cultivation temperature are 25 ℃ ± 1 ℃, illumination every day 16 hours, and explant grows the terminal bud of 4cm-6cm after 10 days.When treating terminal bud length to 4cm-6cm, downcut young tender leaf handle and blade, the long 1.5cm of petiole, blade area are 3cm
2Transfer to the micro adventitious bud inducing culture; Said micro adventitious bud inducing culture is in the MS medium, to add 2.0mg/L 6-benzyladenine (6-BA), 0.1/0.25mg/L methyl (NAA) (if induce micro adventitious bud will add the 0.1mg/L methyl from blade, if induce micro adventitious bud will add the 0.25mg/L methyl from petiole), 30g/L sucrose and 8.5g/L agar powder and get; Illumination cultivation, cultivation temperature are 25 ℃ ± 1 ℃, illumination every day 16 hours, per 20 days successive transfer culture once, through around explant, growing a large amount of micro adventitious buds behind 1 successive transfer culture.The micro adventitious bud inductivity reaches 96%.
Taking root of transgenic tuber pinellia indefinite bud
When treating indefinite bud length to 3cm-4cm; A large amount of indefinite buds are transferred on the adventitious bud rooting medium, and said adventitious bud rooting medium is in the 1/2MS medium, to add kinetin 1.0mg/L (KT), methyl (NAA) 2.0mg/L, 30g/L sucrose and 8.5g/L agar powder and get, through illumination cultivation; Cultivation temperature is 25 ℃ ± 1 ℃; Illumination every day 16 hours through 20 angel's adventitious bud rootings, thereby forms complete tuber of pinellia plant.
The transplanting of transgenic tuber pinellia regeneration plant:
Choose the plant of robust growth, the medium of flush away root is transplanted in the cave dish that transplanting medium is housed, and said transplanting medium is a vermiculite: earth, the volume ratio of vermiculite and earth are 1: 2.Through taming for 1 week, can obtain the normal transgenic tuber pinellia plant of raised growth, transplanting survival rate is 90%, reproduction coefficient reaches 86%.
Embodiment 3:
Inducing of the sterilization of transgenic tuber pinellia explant and micro adventitious bud:
From the transgenic tuber pinellia plant strip contain terminal bud stem tuber as explant.Strip explant from the transgenic tuber pinellia plant, with 75% alcohol disinfecting 10 minutes, sterile water wash 3 times, 0.1%HgCl
2Sterilized sterile water wash 8 times 20 minutes.The stem tuber that has terminal bud with disinfecting cuts the epidermis part, and being cut into volume is 3cm
3-6cm
3Fritter.Transfer on the medium of sprouting, the said medium of sprouting is in the MS medium, to add 1.0mg/L 6-benzyladenine (6-BA), 1.0mg/L methyl (NAA), 30g/L sucrose and 8.5g/L agar powder and get.Illumination cultivation, cultivation temperature are 25 ℃ ± 1 ℃, illumination every day 16 hours, and explant grows the terminal bud of 4cm-6cm after 10 days.When treating terminal bud length to 4cm-6cm, downcut young tender leaf handle and blade, the long 2cm of petiole, blade area are 4cm
2Transfer to the micro adventitious bud inducing culture; Said micro adventitious bud inducing culture is in the MS medium, to add 2.0mg/L 6-benzyladenine (6-BA), 0.1/0.25mg/L methyl (NAA) (if induce micro adventitious bud will add the 0.1mg/L methyl from blade, if induce micro adventitious bud will add the 0.25mg/L methyl from petiole), 30g/L sucrose and 8.5g/L agar powder and get; Illumination cultivation, cultivation temperature are 25 ℃ ± 1 ℃, illumination every day 16 hours, per 20 days successive transfer culture once, through around explant, growing a large amount of micro adventitious buds behind 1 successive transfer culture.The micro adventitious bud inductivity reaches 100%.
Taking root of transgenic tuber pinellia indefinite bud
When treating indefinite bud length to 3cm-4cm; A large amount of indefinite buds are transferred on the adventitious bud rooting medium, and said adventitious bud rooting medium is in the 1/2MS medium, to add kinetin 1.0mg/L (6-BA), methyl (NAA) 2.0mg/L, 30g/L sucrose and 8.5g/L agar powder and get, through illumination cultivation; Cultivation temperature is 25 ℃ ± 1 ℃; Illumination every day 16 hours through 20 angel's adventitious bud rootings, thereby forms complete tuber of pinellia plant.
The transplanting of transgenic tuber pinellia regeneration plant;
Choose the plant of robust growth, the medium of flush away root is transplanted in the cave dish that transplanting medium is housed, and said transplanting medium is a vermiculite: earth, the volume ratio of vermiculite and earth are 1: 2.Through taming for 1 week, can obtain the normal transgenic tuber pinellia plant of raised growth, transplanting survival rate is 90%, reproduction coefficient reaches 90%.
The foregoing description can breed a large amount of transgenic tuber pinellia plant fast, promotes, ensures that tuber of pinellia seedling, raising tuber of pinellia output, the shortening growth cycle of sufficient high-quality is significant for the large tracts of land of transgenic tuber pinellia.
Claims (9)
1. a method that adopts the micro adventitious bud technology quickly propagating transgenic tuber pinellia is characterized in that, the transgenic tuber pinellia explant through after sterilizing, is placed on the medium of sprouting, and illumination cultivation generates blade and petiole; When treating terminal bud length to 4cm-6cm, downcut blade and petiole, place on the micro adventitious bud inducing culture, illumination cultivation grows a large amount of micro adventitious buds around said blade and petiole; When treating that the long 3cm-4cm of arriving of said micro adventitious bud is high, said micro adventitious bud is transferred on the adventitious bud rooting medium, made the generation adventive root; The whole plant of robust growth is transplanted in the basin that fills transplanting medium again, obtains the normal transgenic tuber pinellia plant of growth through domestication;
Said explant is the underground stem tuber that contains terminal bud;
The said medium of sprouting is for adding 1.0mg/L 6-benzyladenine, 1.0mg/L methyl, 30g/L sucrose and 8.5g/L agar powder in the MS medium;
Said micro adventitious bud inducing culture is for interpolation 2.0mg/L 6-benzyladenine, 30g/L sucrose and 8.5g/L agar powder in the MS medium, if induce micro adventitious bud will add the 0.1mg/L methyl from blade, if induce micro adventitious bud will add the 0.25mg/L methyl from petiole;
Said adventitious bud rooting medium is in the 1/2MS medium, to add kinetin 1.0mg/L, methyl 2.0mg/L, 30g/L sucrose and 8.5g/L agar powder.
2. according to the method for the said employing micro adventitious bud technology of claim 1 quickly propagating transgenic tuber pinellia, it is characterized in that the sterilization of said transgenic tuber pinellia explant is meant: strip explant from the transgenic tuber pinellia plant, disinfect in alcohol, sterile water wash, HgCl again
2Sterilization, sterile water wash.
3. according to the method for the said employing micro adventitious bud technology of claim 2 quickly propagating transgenic tuber pinellia, it is characterized in that earlier with volumetric concentration 75% alcohol disinfecting 10 minutes, sterile water wash 3 times was used mass concentration 0.1%HgCl again
2Sterilized sterile water wash 8 times 20 minutes.
4. according to the method for claim 1 or 2 said employing micro adventitious bud technology quickly propagating transgenic tuber pinellias, it is characterized in that the said underground stem tuber that contains terminal bud cuts the epidermis part, being cut into volume is 3cm
3-6cm
3Fritter.
5. according to the method for claim 1 or 2 said employing micro adventitious bud technology quickly propagating transgenic tuber pinellias; It is characterized in that; The said terminal bud of treating is grown when arriving 4cm-6cm, downcuts blade and petiole and is meant cutting-out young leaflet tablet and petiole, and the long 1cm-2cm of petiole, blade area are 2cm
2-4cm
2
6. according to the method for claim 1 or 2 said employing micro adventitious bud technology quickly propagating transgenic tuber pinellias, it is characterized in that, said through illumination cultivation; Generate blade and petiole; Be meant: cultivation temperature is 25 ℃ ± 1 ℃, illumination every day 16 hours, and explant grows the terminal bud of 4cm-6cm after 10 days.
7. according to the method for claim 1 or 2 said employing micro adventitious bud technology quickly propagating transgenic tuber pinellias; It is characterized in that, said through illumination cultivation, around blade and petiole, grow a large amount of micro adventitious buds; Be meant: cultivation temperature is 25 ℃ ± 1 ℃; Illumination every day 16 hours, per 14 days-20 days successive transfer culture once, through around blade and petiole, growing a large amount of micro adventitious buds behind 1 successive transfer culture.
8. according to the method for claim 1 or 2 said employing micro adventitious bud technology quickly propagating transgenic tuber pinellias; It is characterized in that the said generation adventive root that makes is meant: cultivation temperature is 25 ℃ ± 1 ℃, illumination every day 16 hours; Through 20 angel's adventitious bud rootings, thereby form complete tuber of pinellia plant.
9. according to the method for claim 1 or 2 said employing micro adventitious bud technology quickly propagating transgenic tuber pinellias, it is characterized in that said transplanting medium is vermiculite and earth, both volume ratios are 1: 2.
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| CN102668829B (en) * | 2011-03-14 | 2013-05-29 | 华中农业大学 | Rapid propagation method for Pinellia ternate buds |
| CN110278871B (en) * | 2019-07-01 | 2021-03-23 | 长江大学 | Tissue culture method for one-step planting of tissue-cultured cluster seedlings by using pinellia ternata |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1475107A (en) * | 2002-08-14 | 2004-02-18 | 北京金长河科技发展有限公司 | Normalization tissue culturing and fast breeding method of pinellia ternata |
| CN1799342A (en) * | 2005-12-30 | 2006-07-12 | 浙江省农业科学院 | Pinellia detoxification, tissue culture and quick propagation method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1475107A (en) * | 2002-08-14 | 2004-02-18 | 北京金长河科技发展有限公司 | Normalization tissue culturing and fast breeding method of pinellia ternata |
| CN1799342A (en) * | 2005-12-30 | 2006-07-12 | 浙江省农业科学院 | Pinellia detoxification, tissue culture and quick propagation method |
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