CN1545862A - Krishum tissue cultivation rapid breed system fabricating technology - Google Patents
Krishum tissue cultivation rapid breed system fabricating technology Download PDFInfo
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- CN1545862A CN1545862A CNA2003101184771A CN200310118477A CN1545862A CN 1545862 A CN1545862 A CN 1545862A CN A2003101184771 A CNA2003101184771 A CN A2003101184771A CN 200310118477 A CN200310118477 A CN 200310118477A CN 1545862 A CN1545862 A CN 1545862A
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Abstract
The invention relates to a krishum tissue culture fast breeding architecture construction technology, which comprises explant preparation, callus evoking, green sprout differentiation, step generation propagation root-producing culture, adding agar powder to the elementary composition of MS culture medium during test tube sprout transplanting, charging cane sugar and special-purpose phytohormone, adjusting pH to be 6.0.
Description
Technical field
The present invention relates to greenly by plant field, especially the biotechnology of quick reproduction technique
Technical background
Chinese small iris [Iris lacteal pall.var chinensis (Fisch.)] is an Iridaceae Jris perennial herb perennial plant; have very strong drought resisting, cold-resistant, salt tolerant alkali; resistant to diseases and insects is used as the excellent material that water conservation slope, afforestation are built with viewing and admiring in recent years gradually.Yet, owing to have germination rate and the low breeding limitation of germination vigor under chinensis seed hard seed rate height and the normal temperature condition of culture, and cutcross makes seminal propagation can not guarantee the shortcoming of variety and genetype unanimity, therefore, very urgently go to seek the cover Chinese small iris technical system of breeding fast, not only can accelerate the Chinese small iris reproduction speed, save cost, be not subject to seasonal restrictions listing in batch, for the industrialization of Chinese small iris seedling provides technical guarantee, and in time provided seedling to lay the foundation in a large number by construction and road, river levee bank protection etc. for urban landscaping with viewing and admiring.This technology is that the insider payes attention to and appeal the urgent important technological problems that solves.
Summary of the invention
The purpose of this invention is to provide a kind of fast asexual propagation; the survival rate height; be not subjected to seasonal effect; significantly reduce the planting cost; the Chinese small iris tissue-culturing rapid propagation system construction technology that is easy to promote in by construction in water conservation slope and city comprises the optimal medium and the operating process of each link in the whole process with viewing and admiring.
The technical scheme of the purpose of realization invention is as follows:
Chinese small iris tissue-culturing rapid propagation system construction technology comprises the explant preparation, callus induction, green seedling differentiation, shoot proliferation, culture of rootage and the constructing technology in six stages of test-tube seedling transplanting.
After when (1) explant prepares chinensis seed being removed hard sclerotesta, soak sterilization, use sterile water wash again through 2.33% liquor natrii hypochloritis,
(2) callus induction is inoculated into explant on the callus inducing medium, and under no optical condition, room temperature 24-26 ℃ is carried out callus of induce,
(3) green seedling differentiation is transferred to callus on the differentiation bud medium, is to carry out 24hr/d illumination under the 1000LX in intensity of illumination, and 24-26 ℃ is broken up the bud cultivation,
(4) shoot proliferation will break up the bud clump and transfer on the proliferated culture medium, be to carry out 24hr/d illumination under the 1000LX in intensity of illumination, carry out shoot proliferation at room temperature 24-26 ℃,
(5) culture of rootage will be transferred on the root media through the differentiation bud that shoot proliferation is cultivated, and be to carry out 24hr/d illumination under the 1000LX in intensity of illumination, and 24-26 ℃ is carried out culture of rootage,
(6) test-tube seedling transplanting, when root density on average reaches 3.33, when the long 6-10cm of root, average plant height 9-10cm, can open-air direct transplanting, tissue cultivating seedling transplanted bury, keep soil gentle, and take fluffy protection, tear open fluffy behind the first quarter moon.
Various medium preparation methods involved in whole reproductive process are as follows:
Callus inducing medium is to add the 6g/l agar powder in the MS medium, and 60g/l sucrose, and plant hormone are regulated PH to 6.0 with INNaOH solution and can be used through the high-pressure steam sterilization sterilization.In the MS medium ammonium nitrate under normal circumstances, hormone prescription be 2,4 dichlorophenoxyacetic acid (2,4-D) 4mg/l+6-benzyladenine (BA) 2-5mg/l, or 2,4-D 2mg/l+6 furfuryl group adenine (KT) 1.5mg/l; When ammonium nitrate content doubled in the MS medium, the plant hormone prescription was: 2, and 4-D 2-4mg/l+BA1-2mg/l, or 2,4-D 4mg/l+KT 0.5-1.5mg/l.
Differentiation bud medium is to add the 6g/l agar powder in the MS medium, and 30g/l sucrose and plant hormone are regulated pH value to 6.0 with INNaOH solution again, again through the disinfection with high pressure steam sterilization.
Growth hormone/mitogen in callus inducing medium (2,4-D:KT) be at 1: 0.75 o'clock, the plant hormone prescription is: BA4mg/l+ naa (NAA) 0.5mg/l, or BA 2mg/l+NAA 0.5mg/l.
The shoot proliferation medium adopts MS cultivation basis, adds the 6g/l agar powder again, and 30g/l sucrose, and plant hormone are regulated PH to 6.0 with INNaOH solution again, sterilizes the back as the shoot proliferation medium through high pressure steam sterilization again.As plant hormone component in the callus differentiation bud medium is BA4mg/l+ (NAA) 0.5mg/l, and then the plant hormone in the shoot proliferation medium adopts BA2-4mg/l+NAA0-0.5mg/l; Or adopt BA2-4mg/l, change BA0.5-1mg/l+NAA 0-0.5mg/l again over to; As plant hormone component in the callus differentiation bud medium is BA2mg/l+NAA0.5mg/l, and then the plant hormone in the shoot proliferation medium adopts BA2mg/l+NAA 0.1-0.2mg/l.
Root media is to add the 6g/l agar powder in the 1/2MS medium, and 30g/l sucrose is regulated PH to 6.0 with INNaOH solution, sterilizes through high-pressure steam sterilization again.
The present invention has following characteristics:
(1) emerge in advance, save time, seed breaks up bud behind callus induction cultivates, and can break up the clump that sprouts through 1-2 month callus, has shortened clump time of sprouting.
(2) survival rate height: with traditional seed propagation method, survival rate is low, general＜50%, and survival rate is brought up to more than 95% behind employing the present invention.
(3) be not subjected to the influence in season, be suitable for annual breeding, can supply seedling in enormous quantities.
(4) when test-tube seedling transplanting, do not need hardening, the method that adopts direct bottle outlet to transplant is buried the tissue cultivating seedling transplanting, has simplified program, has reduced cost.
Embodiment
Now preferred embodiment the present invention is further illustrated by following.
(1) choose the chinensis seed of mature and plump, remove sclerotesta after, bind up with gauze, soak through 2.33% liquor natrii hypochloritis, stir, sterilization 25 minutes, on superclean bench with sterile water wash 7 times, each 2-3 minute.
(2) adopt MS medium basis, add the 6g/l agar powder, 60g/l sucrose, and plant hormone are regulated PH to 6.0 with the IN sodium hydroxide solution, insert the sterilization of culture dish or triangular flask mesohigh steam sterilization after, as callus inducing medium.Explant is inoculated on the medium, and condition of culture is controlled at dark unglazed, under 26 ℃ of the room temperatures, induces about 1 month good callus to occur.
(3) callus is transferred on the differentiation bud medium that hormone-content is respectively BA 4mg/l+NAA (naa) 0.5mg/l or BA 2mg/l+NAA 0.5mg/l, condition of culture is controlled at 24hr/d illumination, under 26 ℃ of the room temperatures, under the intensity of illumination 1000lx, can break up the clump that sprouts through 1-2 month callus.
(4) will in containing the medium that plant hormone is BA 4mg/l+NAA (naa) 0.5mg/l, changing over to of callus differentiation bud cultivation contain BA 2-4mg/l+NAA 0-0.5mg/l; Cultivating 25 days in the shoot proliferation medium of plant hormone, is 25hr/d illumination under the 1000LX in intensity of illumination, and temperature remains on 24-26 ℃, obtains good shoot proliferation.
(5) will transfer on the green seedling rooting medium through the differentiation bud clump of shoot proliferation, be to carry out 24hr/d illumination under the 1000LX condition with the intensity of illumination, and temperature remains on 24 ℃, takes root through one month regrowth.
(6) the root density of taking root when regrowth on average reaches 3.33, and when the long 6-10cm of root, average plant height 9-10cm, leaf growth is dense, healthy, reaches open-air direct transplanting standard.Adopt the method do not need the direct bottle outlet of hardening to transplant, tissue cultivating seedling is transplanted bury, sand soil is good, and transplanting the back, need to build plastics early spring big fluffy or need build sunshade summer with the simulation closed environment, tears open fluffy behind the first quarter moon.Maintenance ground moistening during this time will water.The phase survival rate reaches more than 95%.
Claims (9)
1. Chinese small iris tissue-culturing rapid propagation system construction technology comprises the explant preparation, callus induction, and green seedling differentiation, shoot proliferation, culture of rootage and test tube are transplanted, and it is characterized by:
After when (1) explant prepares chinensis seed being removed hard sclerotesta, soak sterilization, use sterile water wash again through 2.33% liquor natrii hypochloritis,
(2) callus induction is inoculated into explant on the callus inducing medium, and under no optical condition, room temperature 24-26 ℃ is carried out callus of induce,
(3) green seedling differentiation is transferred to callus on the differentiation bud medium, is to carry out 24hr/d illumination under the 1000LX in intensity of illumination, and 24-26 ℃ is broken up the bud cultivation,
(4) shoot proliferation will break up the bud clump and transfer on the enrichment culture stem, be to carry out 24hr/d illumination under the 1000LX in intensity of illumination, carry out shoot proliferation at room temperature 24-26 ℃,
(5) culture of rootage will be transferred on the root media through the differentiation bud that shoot proliferation is cultivated, and be to carry out 24hr/d illumination under the 1000LX in intensity of illumination, and 24-26 ℃ is carried out culture of rootage,
(6) test-tube seedling transplanting, when root density on average reaches 3.33, when the long 6-10cm of root, average plant height 9-10cm, can open-air direct transplanting, tissue cultivating seedling transplanted bury, keep soil gentle, and take fluffy protection, tear open fluffy behind the first quarter moon.
2. Chinese small iris tissue-culturing rapid propagation system construction technology according to claim 1, its characteristics are that callus inducing medium is to add the 6g/l agar powder in the MS medium, 60g/l sucrose, and plant hormone are regulated PH to 6.0 with INNaOH solution and are sterilized through high-pressure steam sterilization.
3. Chinese small iris tissue-culturing rapid propagation system construction technology according to claim 2, it is characterized by that ammonium nitrate is that hormone prescription is as follows under the normal condition in the MS medium: 2,4-dichlorphenoxyacetic acid (2,4-D) 4mg/l+6-benzyladenine (BA), or 2,4-D2mg/l+6 furfuryl group adenine (KT) 1.5mg/l.
4. according to claims 2 described Chinese small iris tissue-culturing rapid propagation system construction technology, it is characterized by that ammonium nitrate content doubles in the MS medium, the plant hormone prescription is as follows: 2,4-D2-4mg/l+BA1-2mg/l, or 2,4-D4mg/l+KT 0.5-1.5mg/l.
5. according to the described Chinese small iris tissue-culturing rapid propagation of claim 1 system construction technology, it is characterized by differentiation bud medium is to add the 6g/l agar powder in the MS medium, 30g/l sucrose and plant hormone are regulated pH value to 6.0 with INNaOH solution again, again through the disinfection with high pressure steam sterilization.
6. Chinese small iris tissue-culturing rapid propagation system construction technology according to claim 5, it is characterized by growth hormone/mitogen (2 in callus inducing medium, when 4-D:KT) being 1:0.75, the plant hormone prescription is as follows: BA4mg/l+ naa (NAA) 0.5mg/l, or BA 2mg/l+NAA 0.5MG/l.
7. Chinese small iris tissue-culturing rapid propagation system construction technology according to claim 1 is characterized by the shoot proliferation medium and adopts MS medium basis, adds the 6g/l agar powder again, 30g/l sucrose, and plant hormone are regulated PH to 6.0 with INNaOH solution again, again through the disinfection with high pressure steam sterilization
8. according to the described Chinese small iris tissue-culturing rapid propagation of claim 7 system construction technology, it is characterized by in the shoot proliferation medium plant hormone prescription and be BA 2-4mg/l+NAA 0-0.5mg/l or, change the medium that contains plant hormone BA0.5-1mg/l+NAA-0.5mg/l again over to earlier through containing the medium of plant hormone BA2-4mg/l.
9. according to the described Chinese small iris tissue-culturing rapid propagation of claim 1 system construction technology, it is characterized by root media is to add the 6g/l agar powder in the 1/2MS medium, and 30g/l sucrose is regulated PH to 6.0 with INNaOH solution, sterilizes through autoclaving again.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200310118477 CN1284443C (en) | 2003-12-18 | 2003-12-18 | Krishum tissue cultivation rapid breed system fabricating technology |
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| CN 200310118477 CN1284443C (en) | 2003-12-18 | 2003-12-18 | Krishum tissue cultivation rapid breed system fabricating technology |
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| CN1545862A true CN1545862A (en) | 2004-11-17 |
| CN1284443C CN1284443C (en) | 2006-11-15 |
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| CN 200310118477 Expired - Fee Related CN1284443C (en) | 2003-12-18 | 2003-12-18 | Krishum tissue cultivation rapid breed system fabricating technology |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101099422B (en) * | 2007-07-25 | 2011-06-15 | 毕慧滨 | Method for improving iris lactea seed germination rate |
| CN104429963A (en) * | 2014-12-08 | 2015-03-25 | 邱林 | Free pollen cultivation method of iris ensata thunb |
| CN104839019A (en) * | 2015-04-29 | 2015-08-19 | 浙江农林大学 | Method for in-vitro rapid propagation of iris laevigata by using immature fruits |
| CN108496800A (en) * | 2018-04-10 | 2018-09-07 | 黑龙江省科学院自然与生态研究所 | Chinese small iris high frequency regenerating system method is established based on callus induction |
| CN108719072A (en) * | 2018-06-21 | 2018-11-02 | 四川千草生物技术股份有限公司 | Rhizoma Et Radix Notopterygii seed tissue culture fast breeding technology |
-
2003
- 2003-12-18 CN CN 200310118477 patent/CN1284443C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101099422B (en) * | 2007-07-25 | 2011-06-15 | 毕慧滨 | Method for improving iris lactea seed germination rate |
| CN104429963A (en) * | 2014-12-08 | 2015-03-25 | 邱林 | Free pollen cultivation method of iris ensata thunb |
| CN104839019A (en) * | 2015-04-29 | 2015-08-19 | 浙江农林大学 | Method for in-vitro rapid propagation of iris laevigata by using immature fruits |
| CN108496800A (en) * | 2018-04-10 | 2018-09-07 | 黑龙江省科学院自然与生态研究所 | Chinese small iris high frequency regenerating system method is established based on callus induction |
| CN108496800B (en) * | 2018-04-10 | 2021-07-16 | 黑龙江省科学院自然与生态研究所 | Method for establishing iris high-frequency regeneration system based on callus induction |
| CN108719072A (en) * | 2018-06-21 | 2018-11-02 | 四川千草生物技术股份有限公司 | Rhizoma Et Radix Notopterygii seed tissue culture fast breeding technology |
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| Publication number | Publication date |
|---|---|
| CN1284443C (en) | 2006-11-15 |
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