CN103004598B - Method for inducing fructus momordicae tuber tissue culture - Google Patents
Method for inducing fructus momordicae tuber tissue culture Download PDFInfo
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- CN103004598B CN103004598B CN201210548769.8A CN201210548769A CN103004598B CN 103004598 B CN103004598 B CN 103004598B CN 201210548769 A CN201210548769 A CN 201210548769A CN 103004598 B CN103004598 B CN 103004598B
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- momordica grosvenori
- stem tuber
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Abstract
The invention discloses a method for inducing fructus momordicae tuber tissue culture. The method comprises the following steps: 1), obtaining a fructus momordicae virus-free tissue culture seedling according to a conventional method; 2), exscinding the upper terminal bud and the lower root-contained section of the virus-free tissue culture seedling, and slitting the middle robust part into single stems with axillary buds; and 3), finally, slightly cutting the single stems with the axillary buds into modified MS solid media and culturing for 90 to 100 days, wherein the formula of the modified MS solid media is 0.1 to 1.0 mg/L<-1> of MS+LFS, 0.3 to 0.8 mg/L<-1> of SA, and 5 to 12 percent of cane sugar, and the culture condition is 25 minus or plug 2 DEG C in the day, 17 minus or plug 2 DEG C at night, 8 to 10 h photoperiod in the day, 16 to 14 h photoperiod at night and 1200 to 1500 lx illumination intensity. The method is simple to operate, and has the advantages that mass production can be realized; fructus momordicae tubers cultured according to the method are germ-free and nontoxic; convenience is brought for transportation and long-term storage; and the market adaptability is very high.
Description
Technical field
The present invention relates to the tissue culture technique of plant, be specifically related to a kind of method of inducing Momordica grosvenori group training stem tuber.
Background technology
Momordica grosvenori (Siraitia grosvenori (Swingle) C.Jeffrey) is the important special product traditional Chinese medicine of China, its fruit is medicine, food dual-purpose fruit, be the second largest sweetener raw material that is only second to sugarcane, be widely used in the making of Chinese patent drug, health food, beverage, culinary art condiments etc.Because Momordica grosvenori is dioecism, flowering asynchronism again, in addition pollen sticky heavy and be born in flower pesticide and be difficult for the place of being touched by insect, so be difficult to pollinate as common anemophilous flower or entomophilous flower, so under natural conditions, the probability of result extremely low (still necessary artificial pollination of artificial cultivation at present).Meanwhile, it is staminiferous plant that Momordica grosvenori seedling from seed has 70% left and right, and after will waiting until 2～3 years, while blooming, could judge, in production substantially without practical value.But the habit of Momordica grosvenori is, to autumn branch tendril can let droop, and extend rapidly, once landing, will expand and become the stem tuber (being commonly called as " potato piece ", " potato is young ") of making finger size, can survive the winter tenaciously, sprout the coming year and become independently new plant.This is the important way of Momordica grosvenori natural propagation.After artificial cultivation, be evolved into manually and kept down the vines of a creeping plant, little " stem tuber " is still the most important provenance of Momordica grosvenori.Yet the easy transmitted virus of these traditional stem tubers and other damage by disease and insect, be difficult to again produce in enormous quantities, cannot meet the demand of modern large-scale planting.
Last century Mo, aseptic nontoxic Luohanguo With Plantlets of Tissue Culture was studied successfully, and replacing soon traditional stem tuber becomes most important provenance.But Luohanguo With Plantlets of Tissue Culture is easily damage in packed and transported, especially runs into when unmarketable, more need to take a large amount of places and artificial nursing, and be difficult to long-time storage.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of method of inducing Momordica grosvenori group training stem tuber.The method is simple to operation, can realize production in enormous quantities, cultivates the Momordica grosvenori group training stem tuber obtaining aseptic nontoxic, and is convenient to transportation and long-time storage, has very strong market adaptability.
The method of induction Momordica grosvenori group training stem tuber of the present invention, comprises the following steps:
1) obtain according to a conventional method Momordica grosvenori tissue cultural seedlings of free;
2) cut tissue cultural seedlings of free top terminal bud and bottom band root joint, healthy and strong part in the middle of getting, cuts into band axillalry bud list stem segment;
3) by cutting the band axillalry bud list stem segment micro cuttage obtaining, in improvement MS solid culture medium, cultivate 90～100 days, obtain described Momordica grosvenori group training stem tuber; Wherein,
Described improvement MS solid culture based formulas is: MS+LFS 0.1～1.0mgL
-1+ SA 0.3～0.8mgL
-1+ sucrose 5～12%;
Described condition of culture is: temperature: daytime/night 25/17 (± 2) ℃; Periodicity of illumination: daytime/night 8～10h/16～14h; Intensity of illumination 1200～1500lx.
In said method:
Step 3), in, described improvement MS solid culture based formulas is preferably: MS+LFS 0.1～1.0mgL
-1+ SA 0.4～0.6mgL
-1+ sucrose 5～10%; More preferably: MS+LFS 0.3～0.8mgL
-1+ SA 0.5mgL
-1+ sucrose 8%; More preferably: MS+LFS 0.5mgL
-1+ SA0.5mgL
-1+ sucrose 8%.The pH value of improvement MS solid culture medium described in the application is identical with the pH value of conventional MS medium, is generally 5.8～6.0.In above-mentioned formula, the concentration 5～12% of sucrose represents the sucrose that contains 50～120g in every liter of medium.LFS in formula is spirit hair element (Lingfasu, code name PGR-08), has the basic element of cell division and the bioactive feature of growth hormone two class plant growth regulator simultaneously; SA is for spreading acid (Salycylic Acid).
Step 3) in, described condition of culture is preferably: temperature: daytime/night 25/17 (± 2) ℃; Periodicity of illumination: daytime/night 8h/16h; Intensity of illumination 1200～1500lx; Intensity of illumination 1200～1300lx more preferably wherein.Preferred condition of culture is: temperature: daytime/night 25/17 (± 2) ℃; Periodicity of illumination: daytime/night 8h/16h; Intensity of illumination 1200lx.
The Momordica grosvenori group training stem tuber being formed by the method for the invention induction is directly expanding of axillalry bud, and real is cripetura branch, circle after paracone, and likeness in form corncob, just volume ratio tradition stem tuber is little a lot, as soya bean or shelled peanut.Wild stem tuber or the stem tuber of manually keeping down the vines of a creeping plant are the expanding of contact to earth part or buried part of the branch tendril that extended, and two ends morphological differences is not remarkable, and volume is larger, as size as finger.
Compared with prior art, the method for the invention joins LFS and SA in conventional MS solid culture medium, obtains the MS solid culture medium of improvement by the content that spreads acid, sucrose etc. of Optimal Medium; Again by cut with axillalry bud list stem segment minitype cuttage, in improvement MS solid culture medium and under specific condition of culture, cultivate the formation with induction Momordica grosvenori group training stem tuber.Whole method is simple to operation, and can realize production in enormous quantities; By the method, cultivate the Momordica grosvenori group training stem tuber obtaining aseptic nontoxic, and be convenient to transportation (after induction completes, the Momordica grosvenori group training stem tuber obtaining is taken out, clean, directly be placed in the temporary transportation of carton or wooden case) and long-time storage (adopting indoor and outdoor sand to bury storage on clean Momordica grosvenori group training stem tuber), both can be used for planting then, also can store to Second Year plantation, had very strong market adaptability.
Embodiment
With specific embodiment, the invention will be further described below, but the present invention is not limited to these embodiment.
Embodiment 1
1) obtain according to a conventional method Momordica grosvenori tissue cultural seedlings of free;
2) on clean work station, cut tissue cultural seedlings of free top terminal bud and bottom band root joint, healthy and strong part in the middle of getting, cuts into band axillalry bud list stem segment;
3) by cutting the band axillalry bud list stem segment micro cuttage that obtains, in 20, bottledly there are (5 band axillalry bud list stem segments of every bottle of micro cuttage) in improvement MS solid culture medium, cultivate after 95 days, the joint position of each stem middle and lower part of regeneration group training seedling induces the cripetura side stem expanding, circle after paracone, likeness in form corncob, length, within the scope of 0.5～1.5cm, is Momordica grosvenori group training stem tuber of the present invention; Wherein,
Described improvement MS solid culture based formulas is: MS+LFS 0.1mgL
-1+ SA 0.5mgL
-1+ sucrose 8%;
Described condition of culture is: temperature: daytime/night 25 (± 2) ℃/17 (± 2) ℃; Periodicity of illumination: daytime/night 8h/16h; Intensity of illumination 1200lx.
According to total number * 100% of results stem tuber total number/inoculation stem section, calculate stem tuber yield (lower same), stem tuber yield is 75%.
Embodiment 2
Repeat the method for embodiment 1, just will improve MS solid culture medium chemical formulation change and be: MS+LFS0.3mgL
-1+ SA 0.5mgL
-1+ sucrose 8%.
The Momordica grosvenori group training stem tuber form that induction obtains is with embodiment 1, and length is within the scope of 0.5～1.0cm.
As calculated, stem tuber yield is 110%.
Embodiment 3
Repeat the method for embodiment 1, just will improve MS solid culture medium chemical formulation change and be: MS+LFS0.5mgL
-1+ SA 0.5mgL
-1+ sucrose 8%.
The Momordica grosvenori group training stem tuber form that induction obtains is with embodiment 1, and length is within the scope of 0.5～1.0cm.
As calculated, stem tuber yield is 160%.
Embodiment 4
Repeat the method for embodiment 1, just will improve MS solid culture medium chemical formulation change and be: MS+LFS0.8mgL
-1+ SA 0.5mgL
-1+ sucrose 8%.
The Momordica grosvenori group training stem tuber form that induction obtains is with embodiment 1, and length is within the scope of 0.5～1.5cm.
As calculated, stem tuber yield is 148%.
Embodiment 5
Repeat the method for embodiment 1, just will improve MS solid culture medium chemical formulation change and be: MS+LFS1.0mgL
-1+ SA 0.5mgL
-1+ sucrose 8%.
The Momordica grosvenori group training stem tuber form that induction obtains is with embodiment 1, and length is within the scope of 0.5～1.5cm.
As calculated, stem tuber yield is 150%.
Embodiment 6
Repeat the method for embodiment 1, just will improve MS solid culture medium chemical formulation change and be: MS+LFS1.0mgL
-1+ SA 0.3mgL
-1+ sucrose 10%; Condition of culture changes into: temperature: daytime/night 25 (± 2) ℃/17 (± 2) ℃; Periodicity of illumination: daytime/night 10h/14h; Intensity of illumination 1300lx; Incubation time changes 90 days into.
The Momordica grosvenori group training stem tuber form that induction obtains is with embodiment 1, and length is within the scope of 0.5～1.5cm.As calculated, stem tuber yield is 40%.
Embodiment 7
Repeat the method for embodiment 1, just will improve MS solid culture medium chemical formulation change and be: MS+LFS0.3mgL
-1+ SA 0.8mgL
-1+ sucrose 12%; Condition of culture changes into: temperature: daytime/night 25 (± 2) ℃/17 (± 2) ℃; Periodicity of illumination: daytime/night 9h/15h; Intensity of illumination 1500lx; Incubation time changes 100 days into.
The Momordica grosvenori group training stem tuber form that induction obtains is with embodiment 1, and length is within the scope of 0.5～1.0cm.
As calculated, stem tuber yield is 72%.
Field experiment:
2011, applicant induces by the method for the embodiment of the present invention 1, after the Momordica grosvenori group training stem tuber obtaining is taken out and cleans, dries from bottle, directly be seeded in experiment Tanaka, way to manage manages routinely afterwards, germination survival rate >=90% (germination survives number/sowing number * 100%).
Claims (8)
1. a method of inducing Momordica grosvenori group training stem tuber, is characterized in that comprising the following steps:
1) obtain according to a conventional method Momordica grosvenori tissue cultural seedlings of free;
2) cut tissue cultural seedlings of free top terminal bud and bottom band root joint, healthy and strong part in the middle of getting, cuts into band axillalry bud list stem segment;
3) by cutting the band axillalry bud list stem segment micro cuttage obtaining, in improvement MS solid culture medium, cultivate 90～100 days, obtain described Momordica grosvenori group training stem tuber; Wherein,
Described improvement MS solid culture based formulas is: MS+LFS0.1～1.0mgL
-1+ SA0.3～0.8mgL
-1+ sucrose 5～12%;
Described condition of culture is: temperature: 25 ± 2/17 ± 2 ℃ of daytime/nights; Periodicity of illumination: daytime/night 8～10h/16～14h; Intensity of illumination 1200～1500lx.
2. the method for induction Momordica grosvenori group training stem tuber according to claim 1, is characterized in that: step 3) in, described improvement MS solid culture based formulas is: MS+LFS0.1～1.0mgL
-1+ SA0.4～0.6mgL
-1+ sucrose 5～10%.
3. the method for induction Momordica grosvenori group training stem tuber according to claim 2, is characterized in that: described improvement MS solid culture based formulas is: MS+LFS0.3～0.8mgL
-1+ SA0.5mgL
-1+ sucrose 8%.
4. the method for induction Momordica grosvenori group training stem tuber according to claim 3, is characterized in that: described improvement MS solid culture based formulas is: MS+LFS0.5mgL
-1+ SA0.5mgL
-1+ sucrose 8%.
5. according to the method for the induction Momordica grosvenori group training stem tuber described in any one in claim 1～4, it is characterized in that: the pH value of described improvement MS solid culture medium is 5.8～6.0.
6. according to the method for the induction Momordica grosvenori group training stem tuber described in any one in claim 1～4, it is characterized in that: step 3) in, described condition of culture is: temperature: 25 ± 2/17 ± 2 ℃ of daytime/nights; Periodicity of illumination: daytime/night 8h/16h; Intensity of illumination 1200～1500lx.
7. the method for induction Momordica grosvenori group training stem tuber according to claim 6, is characterized in that: described intensity of illumination 1200～1300lx.
8. the method for induction Momordica grosvenori group training stem tuber according to claim 6, is characterized in that: described condition of culture is: temperature: 25 ± 2/17 ± 2 ℃ of daytime/nights; Periodicity of illumination: daytime/night 8h/16h; Intensity of illumination 1200lx.
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| CN104770305A (en) * | 2015-05-08 | 2015-07-15 | 桂林伯林生物技术有限公司 | Method for in vitro conservation of momordica grosvenori genetic resources and method for growth recovery after storage |
| CN106035081A (en) * | 2016-05-27 | 2016-10-26 | 贵州德江易盛农业科技发展有限公司 | Method for cultivating fructus momordicae |
| CN105993953B (en) * | 2016-05-27 | 2018-12-18 | 贵州德江易盛农业科技发展有限公司 | A kind of Siraitia grosvenorii Explant surface sterilizing method |
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