CN108112474A - A kind of method that cultured in vitro improves STEVIA REBAUDIANA RA contents - Google Patents
A kind of method that cultured in vitro improves STEVIA REBAUDIANA RA contents Download PDFInfo
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- CN108112474A CN108112474A CN201611106089.5A CN201611106089A CN108112474A CN 108112474 A CN108112474 A CN 108112474A CN 201611106089 A CN201611106089 A CN 201611106089A CN 108112474 A CN108112474 A CN 108112474A
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- stevia rebaudiana
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a kind of cultured in vitro improve STEVIA REBAUDIANA RA contents method, including：STEVIA REBAUDIANA blade is inoculated on the culture medium of evoked callus, is transferred to after generating callus in differential medium, young shoot is transferred to after being formed in root media, is grown after root system by carrying out conventional cultivation management in rooting culture to greenhouse.The invention belongs to cell biologies and biological technical field.The present invention has drawn the method for being quickly obtained the high STEVIA REBAUDIANA plant of RA contents, improves the content of STEVIA REBAUDIANA blade RA, mouthfeel is more preferably.
Description
Technical field
The present invention relates to STEVIA REBAUDIANA (Stevia rebaudiana Bertoni) cultured in vitro to improve RA in STEVIA REBAUDIANA blade
The method of content, belongs to cell biology and biological field.
Background technology
STEVIA REBAUDIANA (Stevia rebaudiana Bertoni) is composite family Stevia herbaceos perennial.STEVIA REBAUDIANA is former
To be produced from South America Paraguay area, steviol glycoside (stevioside, ST) is contained in blade, sugariness is 200-300 times of sucrose,
It is natural high sugariness low energy type sweet substance.Proved through modern science, STEVIA REBAUDIANA to hypertension, diabetes, artery sclerosis,
Patients with coronary heart disease has preferable control effect, drinks stevia rebaudiana tea, can help to release mental fatigue, boost metabolism, be beneficial to people
Body health, is modern safety health care sugar material.At present, some researches show that stevioside glycoside substance has rebaudioside A
A variety ofization such as (Rebaudioside A, RA), RB, RC, RD, RE, RF, RM and stevioside (Stevio Glycoside, SS)
Close object.The chemical composition of steviol glycoside is made of diterpene aglycon (non-saccharide body portion) and glycosyl (mainly glucose).Stevioside
The sweet taste and bitter taste of glycosides be by the saccharide acceptor on human body tongue gustatory and bitterness receptors to the non-saccharide body in steviol glycoside and
Glucityl is identified and felt.Wherein, sweet taste and bitter taste are mainly by the sugar on non-saccharide body portion and the R1 and R2 of C13, C19
Radix amount determines.Steviol glycoside RA more than glycosyl than other steviol glycoside taste more sweet teas closer to the sugariness of sucrose, and bitter taste
It is less.Therefore, high RA STEVIA REBAUDIANAs new varieties are cultivated to be of great significance as sweetener market for further opening up stevioside.
By crossbreeding cultivate high RA contents STEVIA REBAUDIANA new varieties and by cultivate improve STEVIA REBAUDIANA RA contents be all can
Capable scheme.Some researches show that different cultivars STEVIA REBAUDIANA blade RA contents have very big difference, such as SR-2 genotype STEVIA REBAUDIANAs ST
Content reaches 12.87%, and RA contents are only 0.02%；And the RA contents of SR-3 genotype STEVIA REBAUDIANAs have then reached 9.35%,
ST contents only have 1.23%；SR-3 types STEVIA REBAUDIANA has higher quality, while the UGT76G1 genes in SR-3 STEVIA REBAUDIANA blades
Expression quantity also be significantly larger than SR-2.The expression quantity for illustrating to improve 1 genes of UGT76G etc. can improve RA contents.Also there is research
Show that plant modifying agent coordinates NpK complex fertilizer (N: P: K=15%: 15%: 20%) to apply fertilizer, stevioside leaf can be increased
Piece RA contents 11.6%, organic fertilizer mixes application with chemical fertilizer, can improve STEVIA REBAUDIANA blade RA contents more than 10%.Explanation passes through
Hybridization between high RA fine individual plants or the cultivation applied fertilizer by N P and K different ratio can improve STEVIA REBAUDIANA to some extent
Blade RA contents.But cross breeding method needs the time longer, and cannot be stablized by the RA contents improved of applying fertilizer in the next generation
Heredity etc. has certain limitation.
From STEVIA REBAUDIANA biosynthesis pathway angle analysis, stevioside is the kaurene by being synthesized in chloroplaset, is transported to
Endoplasmic reticulum and then steviol is synthesized, steviol synthesizes each component substance of stevioside in vacuole, and steviol is various
Generate stevia rebaudianum monoglycosides, steviolbioside, steviol glycoside and sweet tea under the action of uridine 5'-diphosphate glycosyl transferase respectively by step
Synanthrin A glycosides (RA), the finally largely accumulation storage in vacuole.
On the other hand, it is contemplated that STEVIA REBAUDIANA from blade dedifferentiation form a large amount of callus during intracellular object
Matter needs are recombined and distributed.From above-mentioned RA biosynthesis pathways angle it may be speculated that synthesis of the RA in cell needs very
More intermediate products are synthesized as substrate, if into the cell without enough RA biosynthesis precursors, then external source provides RA
It may also be synthesized for intracellular RA and material base is provided.But grinding for STEVIA REBAUDIANA blade RA contents is improved by callus
Study carefully and have not been reported.Therefore, we are attempted by adding external source RA during tissue culture from the culture medium for starting evoked callus,
Attempt to obtain the strain that RA contents are higher in STEVIA REBAUDIANA plant leaf.
The content of the invention
It is an object of the present invention to the high STEVIA REBAUDIANA plant of RA contents is quickly obtained, to solve the above problems.
Following scheme may be employed to solve in problem solved by the invention：
It is a kind of to be quickly obtained the high STEVIA REBAUDIANA plant method of RA contents, which is characterized in that comprise the following steps：
200mg/g RA is used to be added to evoked callus culture medium MS+30g/L sugarcane of the explant for STEVIA REBAUDIANA blade
In sugar+7g/L agar+1.0mg/L 6-BA+0.5mg/L NAA.
25 DEG C of tissue culture room environmental temperature, relative humidity 75%, 12h/ daytime, 12h night.
Through 30d or so generate green calli after, be transferred to bud inducement cultivation base MS+30g/L sucrose+7g/L agar+
In 0.5mg/L 6-BA+0.5mg/L NAA+200mg/g RA.
Again after 30d generates young shoot, root media 1/2MS+20g/L sucrose+5g/L agar+1.0mg/L IBA+ are transferred to
In 200mg/g RA, after induction generates a large amount of root systems, by carrying out conventional cultivation management in rooting culture to greenhouse.
Blade is gathered when plant buddings, RA contents are detected using high performance liquid chromatograph.
Advantageous effect：Due to using the above method, the invention enables RA contents in STEVIA REBAUDIANA blade averagely to reach 12.8%.
The method that the present invention has drawn the high STEVIA REBAUDIANA plant of fast culture RA contents, this improves RA contents for STEVIA REBAUDIANA, and mouthfeel is more
Sweet tea is closer to the STEVIA REBAUDIANA plant of sucrose sweet taste.
Description of the drawings
Fig. 1 is STEVIA REBAUDIANA blade inoculation.Blade as explant evoked callus culture medium MS+30g/L sucrose+
7g/L agar+1.0mg/L 6-BA+0.5mg/L NAA+200mg/g RA；
Fig. 2 STEVIA REBAUDIANA callus.Callus inducing medium MS+30g/L sucrose+7g/L agar+1.0mg/L 6-
BA+0.5mg/L NAA+200mg/g RA；
Fig. 3 STEVIA REBAUDIANA Bud Differentiations.Bud inducement cultivation base MS+30g/L sucrose+7g/L agar+0.5mg/L 6-BA+0.5mg/
L NAA+200mg/g RA；
Fig. 4 STEVIA REBAUDIANA rooted seedlings.Root induction culture medium 1/2MS+20g/L sucrose+5g/L agar+1.0mg/L IBA+
200mg/g RA；
Fig. 5 tissue-cultured seedling is transplanted to flowerpot.STEVIA REBAUDIANA bottle seedling carries out conventional cultivation into the indoor flowerpot of temperature by rooting culture
Training management；
Fig. 6 STEVIA REBAUDIANA plant blossoms.STEVIA REBAUDIANA plant is colonized the cultivation management into soil in greenhouse.When plant buddings
Blade is gathered, RA contents are detected using high performance liquid chromatograph.
Specific embodiment
In order to which means, creation characteristic, reached purpose and effect for realizing the present invention are easy to understand, further below
Illustrate the present invention.
Domestic stevia rebaudiana kind is collected, after liquid chromatographic detection RA contents, determines the product using high RA contents
The kind STEVIA REBAUDIANA kind of Mingguang City 1, takes blade, routinely tissue culture disinfectant program carries out disinfection.
200mg/g RA solution is added in callus tissue culture base.
Then it is inoculated with.
Keep 25 DEG C of tissue culture room environmental temperature, relative humidity 75%, 12h/ daytime, 12h night.
After 30d or so generates green calli, bud inducement cultivation base culture is transferred to.
Root media culture is transferred to again after 30d or so generates young shoot.
Then after cultivating 30d or so into warm indoor flowerpot by rooting culture, it is colonized into greenhouse in soil and carries out often
Advise cultivation management.
Blade is gathered when STEVIA REBAUDIANA plant buddings, RA contents are detected using high performance liquid chromatograph.
The present invention improves the RA contents of STEVIA REBAUDIANA blade, and mouthfeel is more preferably.
Claims (5)
1. a kind of method that cultured in vitro improves STEVIA REBAUDIANA RA contents, including：STEVIA REBAUDIANA blade is inoculated into evoked callus
Culture medium on, generate callus after be transferred in differential medium, young shoot is transferred to after being formed in root media, is grown
By carrying out conventional cultivation management in rooting culture to greenhouse after root system.
2. cultural method as described in claim 1, which is characterized in that the callus tissue culture base is：MS+30g/L sugarcanes
Sugar+7g/L agar+1.0mg/L 6-BA+0.5mg/L NAA.
3. cultural method as described in claim 1, which is characterized in that the differential medium is：MS+30g/L sucrose+
7g/L agar+0.5mg/L 6-BA+0.5mg/L NAA.
4. cultural method as described in claim 1, which is characterized in that the root media is：1/2MS+20g/L sucrose
+ 5g/L agar+1.0mg/L IBA.
5. the cultural method as described in claim 2,3,4, which is characterized in that add 200mg/g in the culture medium
RA。
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| CN201611106089.5A CN108112474A (en) | 2016-11-28 | 2016-11-28 | A kind of method that cultured in vitro improves STEVIA REBAUDIANA RA contents |
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| CN201611106089.5A CN108112474A (en) | 2016-11-28 | 2016-11-28 | A kind of method that cultured in vitro improves STEVIA REBAUDIANA RA contents |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108142288A (en) * | 2017-11-21 | 2018-06-12 | 山东农业大学 | A kind of method that cultured in vitro improves chrysanthemum for tea use flower sugariness |
| CN108812317A (en) * | 2018-06-27 | 2018-11-16 | 东台润洋甜叶菊高科有限公司 | A method of STEVIA REBAUDIANA RM content is improved using callus |
| CN108918731A (en) * | 2018-06-28 | 2018-11-30 | 安徽蚌埠惠农甜叶菊高科技发展有限公司 | A kind of method of a large amount of acquisition STEVIA REBAUDIANA blade product steviol glycoside RM |
| CN108935103A (en) * | 2018-08-15 | 2018-12-07 | 安徽蚌埠惠农甜叶菊高科技发展有限公司 | A kind of method that in vitro culture improves STEVIA REBAUDIANA steviolbioside SBIO content |
| CN113229136A (en) * | 2021-05-25 | 2021-08-10 | 南京农业大学 | Stevia rebaudiana leaf quality breeding parent matching method |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108142288A (en) * | 2017-11-21 | 2018-06-12 | 山东农业大学 | A kind of method that cultured in vitro improves chrysanthemum for tea use flower sugariness |
| CN108812317A (en) * | 2018-06-27 | 2018-11-16 | 东台润洋甜叶菊高科有限公司 | A method of STEVIA REBAUDIANA RM content is improved using callus |
| CN108918731A (en) * | 2018-06-28 | 2018-11-30 | 安徽蚌埠惠农甜叶菊高科技发展有限公司 | A kind of method of a large amount of acquisition STEVIA REBAUDIANA blade product steviol glycoside RM |
| CN108935103A (en) * | 2018-08-15 | 2018-12-07 | 安徽蚌埠惠农甜叶菊高科技发展有限公司 | A kind of method that in vitro culture improves STEVIA REBAUDIANA steviolbioside SBIO content |
| CN113229136A (en) * | 2021-05-25 | 2021-08-10 | 南京农业大学 | Stevia rebaudiana leaf quality breeding parent matching method |
| CN113229136B (en) * | 2021-05-25 | 2022-02-11 | 南京农业大学 | Stevia rebaudiana leaf quality breeding parent matching method |
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Application publication date: 20180605 |