CN101554137A - Tissue culture method of arrowhead - Google Patents
Tissue culture method of arrowhead Download PDFInfo
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- CN101554137A CN101554137A CNA2009100943480A CN200910094348A CN101554137A CN 101554137 A CN101554137 A CN 101554137A CN A2009100943480 A CNA2009100943480 A CN A2009100943480A CN 200910094348 A CN200910094348 A CN 200910094348A CN 101554137 A CN101554137 A CN 101554137A
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Abstract
The invention relates to a tissue culture method of arrowhead. The method comprises the following steps of: preparing germless inducing bud culture medium, enrichment medium and budding and rooting culture medium, selecting and placing arrowhead stem eye into the inducing bud culture medium after disinfecting and sterilizing to ensure sprouted growth, and then transferring the stem eye of arrowhead into the enrichment medium and budding and rooting culture medium successively to be cultured into test-tube plantlets; after seedling exercising for about one weak, taking the test-tube plantlets out to be pre-cultured in bacteria free water to obtain breeder seed seeding; transplanting the breeder seed seeding into large field, conducing conventional water and fertilizer management to obtain stock seed seeding, and conducting scale breeding and culturing on the arrowhead stem eye of the fruit of the stock seed seeding to obtain production seeding. By the detoxication and axenic culture to seeding tissue, the original growth potential and the insect pest resistance capability of the breed are rescored, the generation output of the production seeding is averagely improved by 20 to 30 percent, the insect pest resistance capability is obviously enhanced, the applied farm chemical is averagely reduced by 80 percent, and the method has strong operability, stable seeding quality, low culture cost and easy popularization.
Description
Technical field
The present invention relates to a kind of tissue culture method of arrowhead.
Background technology
Arrowhead (Sagittaria sagittrifolia L.) has another name called arrowhead, is Alismataceae aquatic herbaceous plant on the Plant Taxonomy, is the aquatic vegetable kind that people like food.The arrowhead artificial cultivation is existing century-old history, and traditional cultivation method is to protect the bud method of reserving seed for planting, and chooses the bud stem of growth shape reality exactly and breeds as follow-on seedling, belongs to vegetative propagation.Arrowhead is easily infected virus in planting process, forms the spot bubble of yellow-white during morbidity on blade and petiole, and to be full of white emulsus pulp-water in the posterior tuberosity bubble, last emulsus pulp-water becomes black powder, is covered with whole blade.Traditional guarantor's bud method of reserving seed for planting makes virus by the generation accumulation, increases the weight of year by year, causes that arrowhead kind sexual involution, output reduce, the product qualitative change is bad.The plantation family takes the arrowhead tradition freely to reserve seed for planting at present, and the virus infections area accounts for more than 80% of the gross area greatly, makes the relatively low per mu yield of the arrowhead level of production reduce year by year.The spraying pesticide cost is constantly soaring, and causes residue of pesticide to increase the weight of, and influences edible safety.
As hybrid rice, utilize the method for tissue cultivating, select seedling with the obvious growth vigor of a generation, the method that increases output in the present age is applied on some plant varieties.But how to cultivate on a certain concrete kind but is not have the constant way of a dirt to walk, and must be studied at the biological property of certain species, develops suitable process, could produce the effect as the hybrid rice.The group of arrowhead is cultivated the up to the present still unmanned report of studying of seedling, and the production and selling that does not more have tissue cultivating seedling occurs.This problem waits people and researchs and solves.
Summary of the invention
The objective of the invention is to propose a kind of tissue culture method of arrowhead, the seedling that cultivates according to this method has long-living prosperous, output is high, resistance against diseases is strong characteristic, and method itself is workable, and the seedling stay in grade remedies the deficiencies in the prior art with this.
This tissue culture method of arrowhead that the present invention proposes is characterized in that having the following steps:
(1) the following aseptic culture medium of preparation is stand-by:
Induced bud medium MS+6-BA 2-6mg/L+NAA 0.5-1.0mg/L+ sucrose 30g/L, proliferated culture medium MS+6-BA 2-4mg/L+NAA 0.1-0.5mg/L+ sucrose 30g/L, strengthening seedling and rooting medium 1/2MS+NAA 0.1-0.5mg/L+ sucrose 15g/L;
(2) choose healthy and strong arrowhead stem eye and after sterilization is handled, insert earlier in the induced bud medium, allow its growth of sprouting, afterwards stem eye and axillalry bud are transferred to enrichment culture on the proliferated culture medium;
(3) will go up the high seedling of growing thickly of step propagation back plant 2-3cm and receive continuation cultivation on the strengthening seedling and rooting medium, obtain strong plantlets and rootage;
(4) will on go on foot to such an extent that test-tube plantlet is placed on the natural daylight lower refining seedling about 1 week, take out flush away root medium, cultivate in advance in the sterile water, make its stalwartness grow the breeder's stock seedling;
(5) will go up the well-grown breeder's stock seedling replanting of step gained land for growing field crops, and carry out conventional water and fertilizer management, get former seedling.
Choose the arrowhead bud stem of the former seedling fruit of robust growth, implant big Tanaka's scale breeding and cultivate, carry out conventional field management, seedling survive suitable plant after the size produce seedling.
The condition of culture of arrowhead stem eye in the medium in (2)-(3) step is: pH5.8-6.0, cultivation temperature 25-27 ℃, intensity of illumination 1500-2000LX, light application time 10-12 hour/day.
The sterilization way of arrowhead stem eye is in (2) step: stem eye is placed in the beaker rinses well with suds, again with running water flushing 1 hour, immigration is after the husky bed of the river sand of sterilization is trained 15 days in advance, take out and rinse well with suds and running water, move in the aseptic inoculation case, be immersed among 20% the liquor natrii hypochloritis 5-10 minute, take out the back with aseptic water washing 3-5 time, be immersed in concentration expressed in percentage by weight again and be in 0.1% the mercuric chloride solution 8-10 minute, take out the back with aseptic water washing 3-5 time.
The incubation time 20 days (can be 15-25 days) in (2) step, the incubation time in (3) step is 25 days (can be 20-30 days), the incubation time in (3) step is 10-15 days.
The breeder's stock seedling plant height that (5) step was transplanted the land for growing field crops requires at 8-15cm.
Composition and consumption (mg/L) that MS is explained in cultivation in (1) step are:
KNO
31900, NH
4NO
31650, KH
2PO
4170, MgSO
4.7H
2O370, CaCl2.H
2O440, M
nSO
4.4H
2O22.3, Z
nSO
4.7H
2O8.6H
3BO
36.2, KI0.83, Na
2M
0O
4.2H
2O0.25, C
uSO
4.5H
2O0.025, C
0Cl.6H
2O0.025, Na
2-EDTA37.3, F
eSO
4.4H
2O27.8, Cobastab
60.5, Cobastab
10.1, glycine 2, nicotinic acid 0.5, inositol 100.
After (2), growth coefficient can reach 3-4 doubly.It is prominent that white root appears in the seedling base portion after 10-15 days of (3) step, elongation gradually subsequently, and rooting rate reaches more than 90%.
Essence of the present invention with by viral pollution, the arrowhead seedling of original growth characteristics of by long term inhibition carries out detoxification treatment, cut off the transmission of virus between the arrowhead generation-inter-, original growth potential of kind and diseases and insect pests resistance are fully discharged, concentrate on this generation of production seedling and embody.The present invention is by combining the theory of modern plants group training with the actual of habit of growth of this concrete kind of arrowhead, the detoxication and tissue culture arrowhead that has proposed to be fit to produces the method and the technological measure of seedling, solved that people thirst for solving for a long time and the problem that do not have fine solution.
The present invention obtains good effect and is embodied in:
1, the growing way of production seedling obviously is better than traditional seedling, and arrowhead output on average improves 20-30%.
2, the plant diseases and insect pests resistance of production seedling is strong, and the more traditional seedling of the common disease incidence of disease is low more than 50%, and insect pest is few more than 30%, and applying pesticides on average lacks 80%.
3, arrowhead is best in quality, various nutrient composition contents and traditional roughly the same or slightly excellent.
4, method strong operability of the present invention, the seedling stay in grade, it is low to cultivate cost, promotes easily.
Description of drawings
Fig. 1 is a technological process principle block diagram of the present invention.
Fig. 2 is that batch production production seedling increases the process flow diagram of educating.
Among Fig. 2, the arrowhead seedling calculates by 2500 strains/mu; Mu is received the arrowhead bulb by 30000/mu calculating.
Embodiment
The first step is selected good asexual explant (arrowhead bud) stem eye, disinfect according to said method, the husky bed of the river sand that immigration is sterilized is gone up to train in advance to take out after 15 days and is rinsed well with suds and running water, moving into and being soaked in concentration expressed in percentage by weight in the sterile board is among 20% the liquor natrii hypochloritis 5-10 minute, taking out the aseptic clear water in back, to clean with concentration expressed in percentage by weight be 0.1% mercuric chloride solution immersion 8-10 minute again, clean with aseptic water washing again, in induced bud medium, proliferated culture medium and strong plantlets and rootage medium, cultivate successively test-tube plantlet.Test-tube plantlet hardening more promptly got the breeder's stock seedling in 7 days.Conventional water and fertilizer management is carried out in breeder's stock seedling replanting land for growing field crops, the gained fruit, and arrowhead bud stem is former seedling.Former seedling has been the breeding seedling after the detoxification, has obtained inborn good growth characteristics.Former seedling can be directly used in the production seedling of production exactly after scale is cultivated into plantlet.With hybrid rice seed similar essence is arranged.
The above-mentioned specific practice that is seeded in the induction culturing base of this example is: the material shoot tip meristem 3-5mm that intercepting has been sterilized is inoculated on the medium, and 6-8 days stem apexs change green base portion tissue and expand, and bear budlet and stolon.Have axillalry bud to bear about 20 days, at this moment move on in the proliferated culture medium and cultivated about 25 days, growth coefficient reaches 3-4 doubly.Plant strain growth is obviously accelerated.Next the high seedling of growing thickly of 2-3cm being moved on in the strengthening seedling and rooting medium to cultivate has that root is prominent to grow after 10-15 days, and rooting rate reaches about 90%, and after this, root growth is accelerated.Again hardening afterwards, be transplanted to former seedling breed step as above, repeat no more herein.These steps are all carried out at aseptic condition.
Three kinds of medium are: induced bud medium MS+6-BA 2-6mg/L+NAA 0.5-1.0mg/L+ sucrose 30g/L, proliferated culture medium MS+6-BA 2-4mg/L+NAA 0.1-0.5mg/L+ sucrose 30g/L, strengthening seedling and rooting medium 1/2MS+NAA 0.1-0.5mg/L+ sucrose 15g/L.
Wherein cultivating composition and the consumption (mg/L) of explaining MS is:
KNO
31900, NH
4NO
31650, KH
2PO
4170, MgSO
4.7H
2O370, CaCl2.H
2O440, M
nSO
4.4H
2O22.3, Z
nSO
4.7H
2O8.6H
3BO
36.2, KI0.83, Na
2M
0O
4.2H
2O0.25, C
uSO
4.5H
2O0.025, C
0Cl.6H
2O0.025, Na
2-EDTA37.3, F
eSO
4.4H
2O27.8, Cobastab
60.5, Cobastab
10.1, glycine 2, nicotinic acid 0.5, inositol 100.
6-BA is six Bian Ji aminoadenines, and as basic element of cell division usefulness, NAA is a methyl, uses as archusia.
Culture condition is and is pH5.8-6.0 in above-mentioned three kinds of medium, and cultivation temperature 25-27 ℃, intensity of illumination 1500-2000LX, light application time 10-12 hour/day.All the other conditions are the same.
With order shown in Figure 2 and quantity, grow body (common arrowhead bud) outward with 9000, cultivate through this routine method, obtain former seedling 17000 strains, all be transplanted to 6.8 mu of lands for growing field crops, 20.4 ten thousand in results arrowhead kind ball, all plant in 81.6 mu of lands for growing field crops, gather in the crops 2,450,000 arrowhead kind balls again.
Through comparative trial among a small circle, when liquid manure, weather and soil condition were all identical, obvious than tradition arrowhead growing way of the same race with the arrowhead of tissue cultivating seedling plantation of the present invention, output on average improved 20-30%; The plant diseases and insect pests resistance is strong, and the more traditional seedling of the common disease incidence of disease is low more than 50%, and insect pest is few more than 30%, and applying pesticides on average lacks 80-90%; The arrowhead stature is big, and tight is best in quality, various nutrient composition contents and traditional roughly the same or slightly excellent.
Claims (7)
1, a kind of tissue culture method of arrowhead is characterized in that having the following steps:
(1) the following aseptic culture medium of preparation is stand-by:
Induced bud medium MS+6-BA 2-6mg/L+NAA 0.5-1.0mg/L+ sucrose 30g/L, proliferated culture medium MS+6-BA 2-4mg/L+NAA 0.1-0.5mg/L+ sucrose 30g/L, strengthening seedling and rooting medium 1/2MS+NAA 0.1-0.5mg/L+ sucrose 15g/L;
(2) choose healthy and strong arrowhead stem eye and after sterilization is handled, insert earlier in the induced bud medium, allow its growth of sprouting, afterwards stem eye and axillalry bud are transferred to enrichment culture on the proliferated culture medium;
(3) will go up the high seedling of growing thickly of step propagation back plant 2-3cm and receive continuation cultivation on the strengthening seedling and rooting medium, obtain strong plantlets and rootage;
(4) will on go on foot to such an extent that test-tube plantlet is placed on the natural daylight lower refining seedling about 1 week, take out flush away root medium, cultivate in advance in the sterile water, make its stalwartness grow the breeder's stock seedling;
(5) will go up the well-grown breeder's stock seedling replanting of step gained land for growing field crops, and carry out conventional water and fertilizer management, get former seedling.
2, tissue culture method of arrowhead according to claim 1 is characterized in that choosing the arrowhead bud stem of the former seedling fruit of robust growth, implants big Tanaka's scale breeding and cultivates, and carries out conventional field management, seedling survive suitable plant after the size produce seedling.
3, tissue culture method of arrowhead according to claim 1, it is characterized in that the condition of culture of arrowhead stem eye in the medium in (2)-(3) step is pH5.8-6.0, cultivation temperature 25-27 ℃, intensity of illumination 1500-2000LX, light application time 10-12 hour/day.
4, tissue culture method of arrowhead according to claim 1, the sterilization way that it is characterized in that arrowhead stem eye in (2) step is: stem eye is placed in the beaker rinses well with suds, again with running water flushing 1 hour, immigration is after the husky bed of the river sand of sterilization is trained 15 days in advance, take out and rinse well with suds and running water, move in the aseptic inoculation case, be immersed among 20% the liquor natrii hypochloritis 5-10 minute, take out back aseptic water washing 3-5 time, be immersed in concentration expressed in percentage by weight again and be in 0.1% the mercuric chloride solution 8-10 minute, take out the back with aseptic water washing 3-5 time.
5, tissue culture method of arrowhead according to claim 1 is characterized in that (2) incubation time 20 days (can be 15-25 days) that goes on foot, and the incubation time in (3) step is 25 days (can be 20-30 days), and the incubation time in (3) step is 10-15 days.
6, tissue culture method of arrowhead according to claim 1 is characterized in that (5) the breeder's stock seedling plant height that goes on foot the transplanting land for growing field crops requires at 8-15cm.
7, tissue culture method of arrowhead according to claim 1 is characterized in that composition and the consumption (mg/L) of the cultivation note MS in (1) step is:
KNO
31900, NH
4NO
31650, KH
2PO
4170, MgSO
4.7H
2O370, CaCl2.H
2O440, M
nSO
4.4H
2O22.3, Z
nSO
4.7H
2O8.6H
3BO
36.2, KI0.83, Na
2M
0O
4.2H
2O0.25, C
uSO
4.5H
2O0.025, C
0Cl.6H
2O0.025, Na
2-EDTA37.3, F
eSO
4.4H
2O27.8, Cobastab
60.5, Cobastab
10.1, glycine 2, nicotinic acid 0.5, inositol 100.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2009100943480A CN101554137A (en) | 2009-04-09 | 2009-04-09 | Tissue culture method of arrowhead |
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| CNA2009100943480A CN101554137A (en) | 2009-04-09 | 2009-04-09 | Tissue culture method of arrowhead |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103070073A (en) * | 2013-01-22 | 2013-05-01 | 武汉市蔬菜科学研究所 | Method for fast propagation of test tube arrowhead by tissue culture |
| CN104542277A (en) * | 2014-12-19 | 2015-04-29 | 广西壮族自治区农业科学院生物技术研究所 | Rapid propagation method for sagittaria sagittifolia tissue culture seedling and culture medium |
| CN108703068A (en) * | 2018-04-04 | 2018-10-26 | 广西壮族自治区农业科学院生物技术研究所 | Remove method, cultural method and the application of endophyte in arrowhead incubation |
-
2009
- 2009-04-09 CN CNA2009100943480A patent/CN101554137A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103070073A (en) * | 2013-01-22 | 2013-05-01 | 武汉市蔬菜科学研究所 | Method for fast propagation of test tube arrowhead by tissue culture |
| CN104542277A (en) * | 2014-12-19 | 2015-04-29 | 广西壮族自治区农业科学院生物技术研究所 | Rapid propagation method for sagittaria sagittifolia tissue culture seedling and culture medium |
| CN108703068A (en) * | 2018-04-04 | 2018-10-26 | 广西壮族自治区农业科学院生物技术研究所 | Remove method, cultural method and the application of endophyte in arrowhead incubation |
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Application publication date: 20091014 |