CN108812317A - A method of STEVIA REBAUDIANA RM content is improved using callus - Google Patents
A method of STEVIA REBAUDIANA RM content is improved using callus Download PDFInfo
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- CN108812317A CN108812317A CN201810680621.7A CN201810680621A CN108812317A CN 108812317 A CN108812317 A CN 108812317A CN 201810680621 A CN201810680621 A CN 201810680621A CN 108812317 A CN108812317 A CN 108812317A
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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
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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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Abstract
The invention discloses a kind of methods for improving STEVIA REBAUDIANA RM content using callus, belong to cytology and field of biology, include the following steps：(1) it using STEVIA REBAUDIANA blade as explant, is inoculated into callus tissue culture base and cultivates；(2) after callus tissue culture base culture generates green calli, it is transferred to plus the callus tissue culture base of RA carries out squamous subculture, the step (2) is repeated 1-7 times, completes squamous subculture 1-7 generation, obtains the STEVIA REBAUDIANA callus of high RM content.The present invention is by the way that STEVIA REBAUDIANA blade to be inoculated on the culture medium of evoked callus, it is transferred in the callus tissue culture base containing RA after generating callus, callus is set to absorb the RA in culture medium, pass through the route of synthesis of itself, conversion accumulates more RM, is improved with achieving the purpose that improve RM content in callus.
Description
Technical field
The invention belongs to cell biologies and biological field, and in particular to a kind of to be contained using callus raising STEVIA REBAUDIANA RM
The method of amount.
Background technique
STEVIA REBAUDIANA (Stevia rebaudianaBertoni) is composite family Stevia herbaceos perennial.STEVIA REBAUDIANA originates in
South America Paraguay is regional, contains steviol glycoside (stevioside, ST) in blade, and sugariness is 200-300 times of sucrose, is
Natural high sugariness low energy type sweet substance.It is proved through modern science, STEVIA REBAUDIANA is to hypertension, diabetes, artery sclerosis, hat
Cardiaopath has preferable control efficiency, drinks stevia rebaudiana tea, can help to release mental fatigue, boost metabolism, be beneficial to human body
Health is modern safety health care sugar material.
Currently, existing research show steviol glycoside substance have rebaudioside A (Rebaudioside A, RA), RB, RC,
The multiple compounds such as RD, RE, RF, RM and stevioside (Stevio Glycoside, SS).Wherein stevioside (ST) and auspicious Bao
Enlightening glycosides A (RA) proportion maximum (generally 90% or more), rebaudioside C (RC), Dole gram glycosides A (DA), steviolbioside
(RM) content is less.The chemical component of steviol glycoside is by diterpene aglycon (non-saccharide body portion) and glycosyl (mainly glucose) group
At.The sweet taste and bitter taste of steviol glycoside be by human body tongue gustatory saccharide acceptor and bitterness receptors in steviol glycoside
Non-saccharide body and glucose identify and feel.Wherein, sweet taste and bitter taste mainly by non-saccharide body portion and C13, C19 R1 and
Glycosyl quantity on R2 determines.It is analyzed from the mouthfeel of steviol glycoside, the sweet tea steviol glycoside RA and RM more than glycosyl fewer than glycosyl
Synanthrin glycosides ST taste more sweet tea.Steviolbioside RM in STEVIA REBAUDIANA, sugariness is higher than steviolbioside RA, and bitter taste is than stevia rebaudianum disaccharide
Glycosides RA and ST is weak, thus is expected to having wider array of utilization in the future.But the RM content in STEVIA REBAUDIANA is few, and supply falls short of demand, so, how
The yield for improving RM becomes crucial problem.
Crossbreeding and reasonable cultivation management are all one of the approach of raising RM content, but because not having high RM in nature
STEVIA REBAUDIANA parent, crossbreeding is limited and time used is long.Reasonable planting type can only improve in steviol glycoside on a small quantity
RM content, effect is limited, and cannot stablize heredity.
Summary of the invention
The present invention provides a kind of method for improving STEVIA REBAUDIANA RM content using callus, and purpose is that being quickly obtained RM contains
High STEVIA REBAUDIANA callus is measured, to solve problems of the prior art.
For the present invention from STEVIA REBAUDIANA biosynthesis pathway angle analysis, stevioside is the kaurene by synthesizing in chloroplaset,
It is transported to endoplasmic reticulum and then synthesizes steviol, steviol synthesizes each component substance of stevioside, steviol in vacuole
Stevia rebaudianum monoglycosides, steviolbioside, stevioside are generated respectively by step under the action of various uridine 5'-diphosphate glycosyl transferases
Glycosides and stevioside M glycosides (RM), the finally largely accumulation storage in vacuole.
On the other hand, the present invention in view of STEVIA REBAUDIANA from blade dedifferentiation form a large amount of callus during it is intracellular
Material demand is recombined and is distributed, and the reproductive efficiency of callus is high.It can be pushed away from above-mentioned RM biosynthesis pathway angle
It surveys, synthesis of the RM in cell needs many intermediate products (such as RA) to be used as substrate, if into the cell without enough RM biologies
Precursor is synthesized, then external source, which provides steviol glycoside such as RA, to provide material base for intracellular RM synthesis.But it is logical
Callus raising STEVIA REBAUDIANA blade RM is crossed to have not been reported containing quantifier elimination.Therefore, the present invention is by callus tissue culture base
Middle addition external source RA, and successive propagation to be to obtain the higher callus of RM content, and by extract the RM in callus with
Improve the yield of RM in STEVIA REBAUDIANA.
The present invention provides a kind of method for improving STEVIA REBAUDIANA RM content using callus, includes the following steps：
(1) it using STEVIA REBAUDIANA blade as explant, is inoculated into callus tissue culture base and cultivates；The callus tissue culture
The ingredient of base is MS+30g/L sucrose+5g/L agar+0.6mg/L 6-BA+0.6mg/LNAA, the callus tissue culture base training
15-35 DEG C of the environment temperature (preferably 25 ± 1 DEG C) of tissue culture room, light intensity 1500-3000Lux (preferably 2000Lux), light when supporting
7-17h/ daytime in period, 7-17h night (photoperiod is preferably daytime 12h/ night 12h), callus tissue culture base culture 10-30 days is (preferably
20 days).
(2) it after generating green calli, is transferred to plus the callus tissue culture base of RA carries out squamous subculture；It is described plus RA
The ingredient of callus tissue culture base is MS+30g/L sucrose+5g/L agar+0.6mg/L 6-BA+0.6mg/L NAA+200mg/g
RA, condition of culture are 15-35 DEG C of temperature (preferably 25 ± 1 DEG C)；Light intensity 1500-3000Lux (preferably 2000Lux)；Light week
7-17h/ daytime phase, 7-17h night (photoperiod is preferably daytime 12h/ night 12h), callus tissue culture base carry out squamous subculture number of days and are
10-30 days (being preferably 20 days).
(3) step (2) are repeated to carry out squamous subculture squamous subculture 1-7 times, the ingredient of condition of culture and culture medium
Identical as step (2), the number of days cultivated every time is also 10-30 days, finally obtains the STEVIA REBAUDIANA callus of high RM content.
RM content is detected using high performance liquid chromatograph, RM content increases to 0.78% or so by 0.36% or so, increases
1.17 times, significant effect.
Present invention has the advantage that：
The present invention provides a kind of method for improving STEVIA REBAUDIANA RM content using callus, from RM biosynthesis pathway angle
It sets out, thus it is speculated that synthesis of the RM in cell needs many intermediate products (such as RA) to synthesize as substrate, then external source provides RA
It may also be synthesized for intracellular RM and material base be provided, the RM content in Stevia leaves is increased with this.The present invention attempts
By the way that STEVIA REBAUDIANA blade to be inoculated on the culture medium of evoked callus, the callus group containing RA is transferred to after generating callus
It knits in culture medium, callus is made to absorb the RA in culture medium, by the route of synthesis of itself, conversion accumulates more RM, with
Achieve the purpose that improve RM content in callus to improve.Callus is cured by callus culture medium subculture 1-7 times with expanding
The cell number of injured tissue amount and callus, so that RM total amount and unit callus RM content be made to increase.Utilize this
The method for improving STEVIA REBAUDIANA RM content using callus that invention provides, STEVIA REBAUDIANA RM content increases significant after culture,
Each culture medium is also easy to get in incubation, and incubation is simply easily implemented.
Detailed description of the invention
Shape appearance figure when Fig. 1 is STEVIA REBAUDIANA blade inoculation；
Fig. 2 is the shape appearance figure that STEVIA REBAUDIANA generates callus；
Fig. 3 is using the steviol glycoside content in high performance liquid chromatography survey callus (using the reality of the method for the present invention
Apply example)；
Fig. 4 is that the steviol glycoside content surveyed in callus using high performance liquid chromatography (does not use the method for the present invention
Control group)
Specific embodiment
In order to preferably illustrate present patent application, the present invention is realized now in conjunction with attached drawing and specific embodiment
Means are illustrated：
The present embodiment takes blade as explant using domestic stevia rebaudiana kind.Pass through high performance liquid chromatograph
After detection, the method for improving STEVIA REBAUDIANA RM content using callus is specific as follows：
(1) explant sterilizes：Blade is first clean with distilled water flushing, is then disappeared on superclean bench with 70% alcohol
Malicious 10s, aseptic water washing 1-2 times, then 10min is impregnated with 1% liquor natrii hypochloritis, after aseptic water washing 4-5 times；
(2) callus induction：Explant is inoculated on callus inducing medium, as shown in Figure 1, callus
The ingredient of induced medium is MS+30g/L sucrose+5g/L agar+0.6mg/L 6-BA+0.6mg/L NAA, tissue culture room environmental temperature
25 ± 1 DEG C, light intensity 2000LUX, daytime photoperiod 12h/ night 12h are spent, the cultivated days of callus are 20 days；
(3) after green calli, as shown in Fig. 2, being transferred in the callus subculture medium for adding RA, add the callus of RA
The ingredient for organizing subculture medium is MS+30g/L sucrose+5g/L agar+0.6mg/L 6-BA+0.6mg/L NAA+200mg/g
RA, the number of days that callus tissue culture base carries out squamous subculture is 20 days；
(4) then every 20 days subcultures are primary, are no more than 3 times, obtain enough STEVIA REBAUDIANA callus.
Callus is taken, RM content is detected using high performance liquid chromatograph, the implementation of the method for the present invention is used by comparing
The comparative example (Fig. 4) of example (Fig. 3) and unused the method for the present invention finds that STEVIA REBAUDIANA blade RM content is increased to 0.78%, specifically
Content data is detailed in shown in the following table 1 and table 2.Therefore, the side provided by the invention that STEVIA REBAUDIANA RM content is improved using callus
Method can significantly provide STEVIA REBAUDIANA blade RM content.
Table 1：Detect the detection data of the STEVIA REBAUDIANA blade RM content after callus tissue culture
Table 2：Detect the detection data of STEVIA REBAUDIANA blade RM content
These are only the preferred embodiment of the present invention, is not intended to restrict the invention, for those skilled in the art
For member, the invention may be variously modified and varied.All within the spirits and principles of the present invention, it is made it is any modification,
Equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (8)
1. a kind of method for improving STEVIA REBAUDIANA RM content using callus, which is characterized in that include the following steps：
(1) it using STEVIA REBAUDIANA blade as explant, is inoculated into callus tissue culture base and cultivates；
(2) through callus tissue culture base culture generate green calli after, be transferred to plus RA callus tissue culture base carry out after
It is commissioned to train feeding, obtains the STEVIA REBAUDIANA callus of high RM content.
2. the method according to claim 1 for improving STEVIA REBAUDIANA RM content using callus, which is characterized in that described to be cured
The ingredient of injured tissue culture medium is MS+30g/L sucrose+5g/L agar+0.6mg/L 6-BA+0.6mg/LNAA.
3. the method according to claim 1 or 2 for improving STEVIA REBAUDIANA RM content using callus, which is characterized in that institute
15-35 DEG C of environment temperature of tissue culture room when stating callus tissue culture base culture, light intensity 1500-3000Lux, daytime photoperiod 7-17h/
Night 7-17h；Callus tissue culture base cultivated days are 10-30 days.
4. the method according to claim 3 for improving STEVIA REBAUDIANA RM content using callus, which is characterized in that described to be cured
25 ± 1 DEG C, light intensity 2000Lux, daytime photoperiod 12h/ night 12h of the environment temperature of tissue culture room when injured tissue culture medium culture；Callus
Tissue culture medium (TCM) cultivated days are 20 days.
5. the method according to claim 1 for improving STEVIA REBAUDIANA RM content using callus, which is characterized in that described to add
The ingredient of the callus tissue culture base of RA is MS+30g/L sucrose+5g/L agar+0.6mg/L 6-BA+0.6mg/L NAA+
200mg/g RA。
6. the method for improving STEVIA REBAUDIANA RM content using callus according to claim 1 or 5, which is characterized in that institute
Condition of culture is 15-35 DEG C of temperature when stating the callus tissue culture base progress squamous subculture for adding RA；Light intensity 1500-3000Lux；Light
7-17h/ daytime in period, 7-17h night, the number of days that callus tissue culture base carries out squamous subculture is 10-30 days.
7. the method for improving STEVIA REBAUDIANA RM content using callus according to claim 1 or 5, which is characterized in that institute
Condition of culture is 25 ± 1 DEG C of temperature when stating the callus tissue culture base progress squamous subculture for adding RA；Light intensity 2000Lux；Photoperiod
12h/ daytime, 12h night, the number of days that callus tissue culture base carries out squamous subculture is 20 days.
8. the method according to claim 1 for improving STEVIA REBAUDIANA RM content using callus, which is characterized in that the step
Suddenly (2) are repeated 1-7 times, complete squamous subculture 1-7 generation, obtain the STEVIA REBAUDIANA callus of high RM content.
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| CN201810680621.7A CN108812317A (en) | 2018-06-27 | 2018-06-27 | A method of STEVIA REBAUDIANA RM content is improved using callus |
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| CN201810680621.7A CN108812317A (en) | 2018-06-27 | 2018-06-27 | A method of STEVIA REBAUDIANA RM content is improved using callus |
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Cited By (1)
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| 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 |
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| CN105104207A (en) * | 2015-09-22 | 2015-12-02 | 安徽科技学院 | Method for obtaining regenerated plants of stevia rebaudiana Bertoni |
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