CN102349445B - Method for producing tissue culture seedlings on assembly line with low cost - Google Patents
Method for producing tissue culture seedlings on assembly line with low cost Download PDFInfo
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- CN102349445B CN102349445B CN 201110229381 CN201110229381A CN102349445B CN 102349445 B CN102349445 B CN 102349445B CN 201110229381 CN201110229381 CN 201110229381 CN 201110229381 A CN201110229381 A CN 201110229381A CN 102349445 B CN102349445 B CN 102349445B
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Abstract
The invention discloses a method for producing tissue culture seedlings on an assembly line with low cost, which comprises the following steps of: preparing a culture medium; sub-packaging and sterilizing the culture medium; separating and inoculating an explant by sterile operation; and carrying out tissue culture and the like. In the invention, a polypropylene (PP) film bag replaces the traditional glass container, and an illumination source for tissue culture is improved. The PP film bag culture container adopted in the method has the advantages of good light permeability, good air permeability, low cost, light weight and great effective weight, and is more convenient to carry. By adopting the improved method, the production cost of the tissue culture seedlings is lowered, and the production efficiency is obviously improved.
Description
Technical field
The present invention relates to a kind of method of producing group training seedling, particularly a kind of method of low-coat scale production group training seedling.
Background technology
Plant Tissue Breeding is the plant Sterile Culture Methods Used, to have totipotent theory according to plant cell, organ (such as root, stem, leaf, stem apex, flower, the fruit etc.) tissue (as forming layer, epidermis, cortex, marrow cell, endosperm etc.) or cell (such as megaspore, microspore, somatic cell etc.) and the protoplast that utilize plant corpus to exsomatize, under the artificial conditions such as aseptic and suitable synthetic medium and illumination, temperature, can induce callus, indefinite bud, adventive root, form at last complete plant.
But the employed glass culture vessel of traditional mode of production group training seedling light transmission ventilation is poor, cost is high, weight is large, effective weight is little, difficult, and is fragile.Production cost is higher, and production efficiency is lower, and the further improvement of low-coat scale production group training seedling technology can be arranged.
Summary of the invention
The object of the present invention is to provide a kind of low-cost tissue culture vessel that is easy to promote, set up a kind of method for tissue culture of easy, low-cost, streamlined.
In order to realize the foregoing invention purpose, the technical solution used in the present invention is as follows:
The present invention includes following steps:
(1) preparation of medium:
(1) medicine that takes by weighing following mass fraction places respectively beaker:
KH
2PO
4 3.75
NH
4NO
3 36.43
KNO
3 41.94
CaCl
2·2H
2O 9.71
MgSO
4·7H
2O 8.17
In each beaker, add respectively medicine gross weight 33-34 doubly after the heavy dissolved in purified water, according to listed each medicine sequencing solution slowly is mixed in the larger beaker, constantly stir simultaneously, stand-by;
(2) take by weighing the medicine of following mass fraction, the medicine gross mass be in (1) medicine gross mass 1/22:
MnSO
4·4H
2O 10.8 ZnSO
4·7H
2O 4.17
H
3BO
3 3.00 KI 0.40
Na
2MoO
4·2H
2O 0.12 CoCl
2·6H
2O 0.012
CuSO
4·5H
2O 0.012 FeSO
4·7H
2O 13.47
Na
2-EDTA 18.07 nicotinic acid 0.24
Vitamin B6 0.24 vitamin B1 0.05
All the other are inositol for glycine 0.97
Above medicine is placed beaker, adds the dissolved in purified water of 1000 times of weights of medicine gross weight,
Slowly pour in the solution for preparing in (1), behind the stirring and evenly mixing, stand-by;
(3) compound concentration is 6-benzyl aminoadenine (6-BA) and methyl α-naphthyl acetate (NAA) solution of 0.2mg/ml;
(4) taking by weighing the agar of 30 times of medicine gross weights in (2), 150 times sucrose pours in the solution in (1) step and stirs, behind the ebuillition of heated, the 6-benzyl aminoadenine (6-BA) and methyl α-naphthyl acetate (NAA) solution that add (3) preparation of different volumes according to the test-tube plantlet of different plants, at last by medicine gross weight in (1), every 4.53g constant volume dropwise splashes into the pH value to 5.8 that the 1N potassium hydroxide solution is regulated medium again to 1000mL;
(2) packing and sterilization:
The medium for preparing is distributed in the polypropylene film bag while hot, is fixed on the Simple framework made from iron wire with Small clamp and makes it upright, then put into the high-pressure sterilizing pot sterilization, sterilization time keeps 18～20min to get final product at 121 ℃;
(3) sterile working:
(1) preparation of sterile working experimental apparatus and material: comprise the aseptic process of superclean bench, sterilization both hands and cutter and tweezers;
(2) after sterilization finishes, simple and easy shelf is placed on the superclean bench, the arrangement bag film can be upright, seals sack with plastic bag sealer, and bag film leaves standstill to culture medium solidifying, and is stand-by;
(3) continuous productive process is carried out separation and the inoculation of explant: culture bag, operation utensil, culture dish and the plastic bag sealer that test-tube plantlet is housed put into superclean bench after with bromogeramine or the sterilization of 75% alcohol wipe; No. 1 the tissue culture technology workman takes out test-tube plantlet in superclean bench from culture bag, puts on the aseptic filter paper, and the test-tube plantlet for the treatment of breeding according to the breeding requirement of different test-tube plantlets with cutter cuts; Simultaneously No. 2 skilled workers cut off the bag film sealing part that can medium and medium have solidified, and the test-tube plantlet of the breeding of No. 1 tissue culture technology workman being cut with the tweezers of sterilizing and cooling off sandwiches in the bag film; If stem tuber or the little insertion medium of stem section that No. 3 tissue culture technology workmans put into No. 2 tissue culture technology workmans are the petiole horizontal positioned; No. 4 tissue culture technology workmans, are positioned over No. 3 ready-made bag films of tissue culture technology workman on the culturing rack in the culturing room with its bag film sealing and numbering with plastic bag sealer;
(4) carry out sterile working by (three) described step, the test-tube plantlet of bag film encapsulation is positioned on the culturing rack in the culturing room organizes cultivation, the lighting source that tissue is cultivated adopts electricity-saving lamp, is preferably 11 watts electricity-saving lamp.
Compared with prior art, the invention has the beneficial effects as follows:
1, adopt polypropylene film bag as culture vessel, the light transmission ventilation that its tool is good than glass, culture is than viridescent, the stalwartness of traditional glass container.Polypropylene film conducts heat good, is conducive to culture and does sth. in advance hardening, makes it more reform of nature temperature, and transplanting survival rate is high.
2, the traditional glass container weight is large and effective weight is little, general empty bottle is heavily approximately about 240 grams, and seedling and medium only have about 55 grams, effective weight is about 23% of empty bottle weight, and the carrying of vial has become the manual labor of a burdensome, transplants if group training seedling will be transported into the greenhouse, cost of transportation increases, and the shared volume of vial is larger, puts into culturing room and cultivates, and has increased the expense of depositing.And polypropylene film bag is lightweight, has not only reduced cost of transportation, and has alleviated workman's labour intensity, and is also convenient during transplanting.
3, the traditional glass container cost is high, and the glass container of a 350ml needs about 0.8～1.0 yuan, and easily damaged, need to constantly replenish, and the bag film cost is low only approximately 0.035 yuan/.In addition, use traditional glass cleaning container and sterilization expense high, and polypropylene film bag as container removed from cleaning charge with and sterilize to take and also reduced.
4, the bag film light transmission is good, adds improving the source of light, uses electricity-saving lamp, if be placed in culturing room on 1.0m * 2.0m culturing rack, only needs totally 44 watts electricity-saving lamp, and culture gets final product normal growth.
5, the tissue culture technology set up of the present invention is easy and be easy to grasp.The inoculation step of streamlined has improved operating efficiency, adopts this kind method not only to reduce the production cost of group training seedling, and has improved the production efficiency of group training seedling, compares with conventional method and has played the effect of getting twice the result with half the effort.
Embodiment
Below in conjunction with embodiment foregoing invention content of the present invention is described in further detail.
But this should be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following embodiment.Not breaking away from the above-mentioned technological thought situation of the present invention, according to ordinary skill knowledge and customary means, make various replacements and change, all should comprise within the scope of the invention.
Embodiment 1
The present embodiment is the preparation of medium:
(1) takes by weighing KH
2PO
4170mg, NH
4NO
31650mg, KNO
31900mg, CaCl
22H
2O 440mg, MgSO
47H
2O 370mg places respectively the beaker of 200mL, add pure water 150 mL dissolving after, solution slowly is mixed in the beaker of 2000mL according to listed each medicine sequencing, constantly stir simultaneously, stand-by.
(2) take by weighing MnSO
44H
2O 22.3 mg, ZnSO
47H
2O 8.6 mg, H
3BO
36.2 mg,
KI 0.83 mg, Na
2MoO
42H
2O 0.25 mg, CoCl
26H
2O 0.025 mg, CuSO
45H
2O 0.025 mg, FeSO
47H
2O 27.8 mg, Na
2-EDTA 37.3 mg, nicotinic acid 0.5 mg, vitamin B6 0.5mg, vitamin B1 0.1mg, glycine 2.0 mg, inositol 100 mg in the beaker of 500mL, add pure water 200 mL dissolving after, slowly pour in the beaker of the 2000mL in 1, behind the stirring and evenly mixing, stand-by.
(3) compound concentration is 6-benzyl aminoadenine (6-BA) and methyl α-naphthyl acetate (NAA) solution of 0.2mg/ml;
(4) accurately take by weighing in the beaker that agar 6g, sucrose 30g pour the 2000mL in 1 step into and stir, behind the ebuillition of heated, the 6-benzyl aminoadenine (6-BA) and methyl α-naphthyl acetate (NAA) solution that add different volumes according to the test-tube plantlet of different plants, add at last pure water to beaker scale 1000mL place, dropwise splash into again the pH value to 5.8 that potassium hydroxide solution is regulated medium.
Embodiment 2
The present embodiment is packing and sterilization
The medium for preparing is distributed in the polypropylene film bag while hot.Be fixed on the Simple framework made from iron wire with Small clamp and make it upright, then put into the high-pressure sterilizing pot sterilization, 121 ℃ of maintenance 20min sterilizes.
Embodiment 3
The present embodiment is sterile working
(1) preparation of sterile working experimental apparatus and material: with bromogeramine or 75% alcohol wipe superclean bench, open superclean bench and indoor uviol lamp, sterilization 20～30min.Articles for use on the work top can not be placed too many or overlapping placement, in order to avoid reduce sterilization effect; With bromogeramine or 75% wipes of alcohol table top and sterilize both hands and test tools made, open the inoculation sterilizer, when temperature reaches 290 ℃～300 ℃ operation tool (cutter and tweezers) was put into sterilization approximately 2 minutes, take out, be placed on the tool rack and cool off.
(2) after sterilization finishes, immediately simple and easy shelf is placed on the superclean bench, the arrangement bag film can be upright, seals sack with plastic bag sealer, and bag film leaves standstill to culture medium solidifying, and is stand-by.
(3) continuous productive process is carried out separation and the inoculation of explant: culture bag, operation utensil, culture dish and the plastic bag sealer that test-tube plantlet is housed put into superclean bench after with bromogeramine or the sterilization of 75% alcohol wipe.No. 1 the tissue culture technology workman takes out test-tube plantlet in superclean bench from culture bag, puts on the aseptic filter paper, and the test-tube plantlet for the treatment of breeding according to the breeding requirement of different test-tube plantlets with cutter cuts: if the stem section is cut to 1cm, if the stem tuber rip cutting is 1cm
3If petiole fragments 1～1.2cm is if blade is cut into the square of 1cm * 1cm; Simultaneously No. 2 skilled workers cut off the bag film sealing part that can medium and medium have solidified, and the test-tube plantlet of the breeding of No. 1 tissue culture technology workman being cut with the tweezers of sterilizing and cooling off sandwiches in the bag film; If stem tuber or the little insertion medium of stem section that No. 3 tissue culture technology workmans put into No. 2 tissue culture technology workmans are the petiole horizontal positioned; No. 4 tissue culture technology workmans with its bag film sealing, write the test-tube plantlet numbering of cultivation with plastic bag sealer with No. 3 ready-made bag films of tissue culture technology workman (test-tube plantlet that breeding is arranged in the bag), are positioned on the culturing rack of putting in the culturing room.
Embodiment 4
The present embodiment is cultivated for tissue
Organize cultivation according to the sterile working method that embodiment 3 introduces, the test-tube plantlet of bag film encapsulation is positioned on the culturing rack in the culturing room organizes cultivation, lighting source is improved: lighting source adopts 11 watts electricity-saving lamp to substitute former 40 watts fluorescent tube.
Embodiment 5
The present embodiment is the Estimation To The Economic Returns to the described technology of embodiment 1-4:
(1) 10,000 bottles of (traditional glass container 250ml) medium of preparation need 500 liters, with 3 yuan of medium component costs/rise calculating, need 1500 yuan.
Prepare 10,000 bags of (polypropylene film bag) medium and need 400L, with 3 yuan of medium component costs/rise calculating, need 1200 yuan.
(2) 10,000 bottles of (traditional glass container 350ml) medium of preparation calculate with the 50ml/ bottle, need 500 liters, and a workman can prepare 15 liters in one day, and 40 yuan/day of labour costs need 1340 yuan altogether.
Prepare 10,000 bags of (polypropylene film bag) medium, calculate with the 40ml/ bag, need 400 liters, a workman can prepare 15 liters in one day, and 40 yuan/day of labour costs need 1070 yuan altogether.
(3) 120L vertical pressure steam sterilization pan 150 vials of once can sterilizing, and sack can be sterilized 200 bags.Equally once sterilization needs 12 degree electricity, and kilowatt-hour one yuan amounts to 12 yuan.Can sterilize in one day 15 liters medium of workman, 40 yuan/day of labour costs.10,000 vials of sterilizing, 1340 yuan of labour costs, the electricity charge consume 800 yuan, amount to 2140 yuan; 10,000 bag films of sterilizing, 1070 yuan of labour costs, the electricity charge consume 600 yuan, amount to 1670 yuan.
(4) the vial sealed membrane is 0.10 yuan/, and 10,000 bottles of medium need 1000 yuan.0.035 yuan/of polypropylene film bag, 350 yuan of 10,000 bag films.
(5) inoculation: every bag (bottle) inoculation 18 seedlings, 40 yuan/day of wage for workmen use vial, and bag film is used in 120 bottles of people inoculations in a day, can inoculate 200 bags, and inoculating 10,000 bottles of test-tube plantlets need labour cost 3334 yuan.Connect 10,000 bags of test-tube plantlets and need 2000 yuan of labour costs.
(6) if use vial, then need clean bottle.A workman washed 300 vials in one day, 40 yuan/day of wages, 1340 yuan of 10,000 bottles of need.
(7) the illumination cultivation electricity charge are adjusted: 1.0m * 2.0m culturing rack can be put 325 bag culture mediums, because the bag film light transmission is good, use 4 different electricity-saving lamps of power to amount to 44 watts, group training seedling can normally be cultivated, calculate like this, 10,000 bags of groups were trained the seedling illumination cultivation 30 days, and lighting hours was 10 hours in one day, power consumption 410 degree, 410 yuan of the electricity charge (1 degree electricity is by 1 yuan of calculating); Equally, 1.0m * 2.0m culturing rack is placed 160 bottles of medium, needs three 40 watts of fluorescent tubes, power consumption 2268 degree, 2668 yuan of the electricity charge.
(8) totle drilling cost: adopt bag film to do culture vessel, the cost of producing 180,000 test-tube plantlets is 6700 yuan, and 0.0372 yuan of average every seedling cost adopts vial to do culture vessel, and the cost of producing 180,000 test-tube plantlets is 13322 yuan, 0.0744 yuan of average every seedling cost.Adopt bag film to do culture vessel, save production cost 6622 yuan, the every seedling cost of test-tube plantlet be vial do culture vessel 1/2nd.
(9) traffic expense: the lorry of load-carrying 1.0L, average 2 yuan/kilometer, a car can fill 20000 bags, and if adopt vial, a car only fills 7200 bottles.By one kilometer calculating of use 1.0L Freight Transport, 50,000 bags of test-tube plantlet traffic expense 5 yuans, 50,000 bottles of test-tube plantlets then need 14 yuan, save 9 yuan, and the cost of transportation that adopts bag film to do culture vessel is to adopt vial to make 35.7% of culture vessel cost of transportation.
In sum, employed polypropylene film bag culture vessel light transmission ventilation is good among the present invention, cost is low, lightweight, effective weight large, carrying is simpler.By technology of the present invention, so that group training seedling production cost reduces, production efficiency significantly improves.
Claims (2)
1. the method for a low-cost streamlined production group training seedling may further comprise the steps:
(1) preparation of medium:
(1) medicine that takes by weighing following mass fraction places respectively beaker:
KH
2PO
4 3.75
NH
4NO
3 36.43
KNO
3 41.94
CaCl
2·2H
2O 9.71
MgSO
4·7H
2O 8.17
In each beaker, add respectively medicine gross weight 33-34 doubly after the heavy dissolved in purified water, according to listed each medicine sequencing solution slowly is mixed in the larger beaker, constantly stir simultaneously, stand-by;
(2) take by weighing the medicine of following mass fraction, the medicine gross mass be in (1) medicine gross mass 1/22:
MnSO
4·4H
2O 10.8 ZnSO
4·7H
2O 4.17
H
3BO
3 3.00 KI 0.40
Na
2MoO
4·2H
2O 0.12 CoCl
2·6H
2O 0.012
CuSO
4·5H
2O 0.012 FeSO
4·7H
2O 13.47
Na
2-EDTA 18.07 nicotinic acid 0.24
Vitamin B6 0.24 vitamin B1 0.05
All the other are inositol for glycine 0.97
Above medicine is placed beaker, adds the dissolved in purified water of 1000 times of weights of medicine gross weight,
Slowly pour in the solution for preparing in (1), behind the stirring and evenly mixing, stand-by;
(3) compound concentration is 6-benzyl aminoadenine (6-BA) and methyl α-naphthyl acetate (NAA) solution of 0.2mg/ml;
(4) taking by weighing the agar of 30 times of medicine gross weights in (2), 150 times sucrose pours in the solution in (1) step and stirs, behind the ebuillition of heated, the 6-benzyl aminoadenine (6-BA) and methyl α-naphthyl acetate (NAA) solution that add (3) preparation of different volumes according to the test-tube plantlet of different plants, at last by medicine gross weight in (1), every 4.53g constant volume dropwise splashes into the pH value to 5.8 that the 1N potassium hydroxide solution is regulated medium again to 1000mL;
(2) packing and sterilization:
The medium for preparing is distributed in the polypropylene film bag while hot, is fixed on the Simple framework made from iron wire with Small clamp and makes it upright, then put into the high-pressure sterilizing pot sterilization, sterilization time keeps 18～20min to get final product at 121 ℃;
(3) sterile working:
(1) preparation of sterile working experimental apparatus and material: comprise the aseptic process of superclean bench, sterilization both hands and cutter and tweezers;
(2) after sterilization finishes, simple and easy shelf is placed on the superclean bench, the arrangement bag film can be upright, seals sack with plastic bag sealer, and bag film leaves standstill to culture medium solidifying, and is stand-by;
(3) continuous productive process is carried out separation and the inoculation of explant: culture bag, operation utensil, culture dish and the plastic bag sealer that test-tube plantlet is housed put into superclean bench after with bromogeramine or the sterilization of 75% alcohol wipe; No. 1 the tissue culture technology workman takes out test-tube plantlet in superclean bench from culture bag, puts on the aseptic filter paper, and the test-tube plantlet for the treatment of breeding according to the breeding requirement of different test-tube plantlets with cutter cuts; Simultaneously No. 2 skilled workers cut off the bag film sealing part that can medium and medium have solidified, and the test-tube plantlet of the breeding of No. 1 tissue culture technology workman being cut with the tweezers of sterilizing and cooling off sandwiches in the bag film; If stem tuber or the little insertion medium of stem section that No. 3 tissue culture technology workmans put into No. 2 tissue culture technology workmans are the petiole horizontal positioned; No. 4 tissue culture technology workmans, are positioned over No. 3 ready-made bag films of tissue culture technology workman on the culturing rack in the culturing room with its bag film sealing and numbering with plastic bag sealer;
(4) carry out sterile working by (three) described step, the test-tube plantlet of bag film encapsulation is positioned on the culturing rack in the culturing room organizes cultivation, the lighting source that tissue is cultivated adopts electricity-saving lamp.
2. the method for a kind of low-cost streamlined production group training seedling according to claim 1 is characterized in that: the electricity-saving lamp that the lighting source employing that tissue is cultivated is 11 watts.
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|---|---|---|---|
| CN 201110229381 CN102349445B (en) | 2011-08-11 | 2011-08-11 | Method for producing tissue culture seedlings on assembly line with low cost |
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|---|---|---|---|
| CN 201110229381 CN102349445B (en) | 2011-08-11 | 2011-08-11 | Method for producing tissue culture seedlings on assembly line with low cost |
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| CN102349445B true CN102349445B (en) | 2013-02-06 |
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102936579A (en) * | 2012-09-23 | 2013-02-20 | 四川省农业科学院园艺研究所 | Liquid culture medium for promoting fruit tree tissue culture seedling growth |
| CN105638467B (en) * | 2016-01-06 | 2018-06-05 | 南平市华科生物科技有限公司 | A kind of method that roxburgh anoectochilus terminal bud is cultivated with culture bag |
| CN109076959A (en) * | 2018-08-31 | 2018-12-25 | 湖州德清玖沐农业科技有限公司 | A kind of generic plant tissue culture culture medium and preparation method |
| CN112167064A (en) * | 2020-11-11 | 2021-01-05 | 山东农业大学 | Tissue culture and rapid propagation method for salvia miltiorrhiza |
| CN112772412A (en) * | 2020-12-30 | 2021-05-11 | 吴有光 | Method for performing open type plant tissue culture and rapid propagation by utilizing self-standing self-sealing film bag |
| CN116458426A (en) * | 2023-03-20 | 2023-07-21 | 云浮市南药研究院 | Energy-saving simple plant tissue culture aseptic operation method |
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| CN2262801Y (en) * | 1996-01-10 | 1997-09-24 | 付翔 | Culture bag by tissue culture |
| CN1586169A (en) * | 2004-08-06 | 2005-03-02 | 上海光兆植物速生技术有限公司 | Poplar tissue cultivation quick breeding industrial producing plan controlled digital model |
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|---|---|---|---|---|
| CN2262801Y (en) * | 1996-01-10 | 1997-09-24 | 付翔 | Culture bag by tissue culture |
| CN1586169A (en) * | 2004-08-06 | 2005-03-02 | 上海光兆植物速生技术有限公司 | Poplar tissue cultivation quick breeding industrial producing plan controlled digital model |
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