CN1276424A - Process for generating natural rooting agent by tissue culture of cape jasmine cells - Google Patents

Process for generating natural rooting agent by tissue culture of cape jasmine cells Download PDF

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Publication number
CN1276424A
CN1276424A CN 99112969 CN99112969A CN1276424A CN 1276424 A CN1276424 A CN 1276424A CN 99112969 CN99112969 CN 99112969 CN 99112969 A CN99112969 A CN 99112969A CN 1276424 A CN1276424 A CN 1276424A
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China
Prior art keywords
cape jasmine
culture
gardenoside
cell
cell tissue
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CN 99112969
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Chinese (zh)
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CN1120886C (en
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白雪芳
杜昱光
王靖楣
王毓福
朱亮
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Dalian Institute of Chemical Physics of CAS
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Dalian Institute of Chemical Physics of CAS
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Abstract

A process for generating natural rooting agent by tissue culture of cape jasmine cell includes constructing cape jasmine cell tissue, culturing and extracting jasminoidin. It features that used cape jasmine cell tissue is the callus generated by cape jasmine leaf induction, its culture medium is modified MS one, and its culture condition is modified solid MS culture medium, inoculated amount of 1-5 g/100 ml, 20-30 deg.c, 60-85% humidity and light intensity of 1000-3000lx for solid culture, or modified liquid MS culture medium, inoculated amount of 1-5 g/100 ml, 20-30 deg.C, rotation speed of 50-150 rpm and light intensity of 1000-3000 lx for liquid culture.

Description

The method of generating natural rooting agent by tissue culture of cape jasmine cells
The invention provides a kind of promotion plant establishment material--gardenoside (Geniposide that generates by the cape jasmine cell cultures, Jasminoidin) method, utilize the method for culture plant cell, can obtain the vegetable cell of higher yields, and then extract the gardenoside that promotes the plant establishment effect.
Particularly all contain the material iridoid glycoside constituents that can promote plant establishment in the cape jasmine fruit in the cape jasmine body, as gardenoside (Geniposide, Jasminoidin), gardenoside (Gardenoside) and genipin-1-0-gentiobioside (Genipin1-0-1-gentiobioisde) etc.But no matter be wild or artificial culture all exists wild resource few, lack environment protection, cultivation technique does not catch up with and problem such as labour's costliness.Be applied to improve energetically in the agrotechnique of the output of the farm crop on the existing land area and quality, also exist the serious gesture that supply falls short of demand.
The object of the present invention is to provide a kind of cape jasmine cell tissue to cultivate the method for natural rooting agent, it can realize industrial production on a large scale, is not subjected to the restriction of season and land area.
The invention provides a kind of cape jasmine cell tissue and cultivate the method for producing natural rooting agent, in the root-growing agent of being produced except that containing partial pigment and sugar, mainly contain gardenoside 1～9 ‰, gardenoside 1～9 ‰, genipin-1-0-gentiobioside 1～9 ‰, this method comprises foundation, cultivation and the gardenoside extraction step of cape jasmine cell tissue, it is characterized in that:
(1) the cape jasmine cell tissue of Cai Yonging is the callus of being induced generation by leaf of Capa Jasmine;
(2) employed substratum is the MS substratum of improvement, and component is as follows:
Macroelement: NH 4NO 31000～2000 (mg/L)
KNO 3?????????????????????????1500～2500
CaCl 2·2H 2O?????????????????300～500
MgSO 4·7H 2O?????????????????150～200
Trace element: KI 1.5～1.7
H 3BO 3???????????????????????5.5～7.0
MnSO 4·7H 2O?????????????????20～25
ZnSO 4·7H 2O?????????????????5～15
Na 2MoO 4·2H 2O??????????????0.1～0.5
CuSO 4·5H 2O???0.01～0.03
CoCl 2·6H 2????0.01～0.03
Na 2EDTA????????35～40
FeSO 4·7H 2O???20～30
Growth regulatory substance: inositol 80～120
Nicotinic acid 0.3～0.7
Pyridoxine hydrochloride B 10.3～0.7
Vitamin B 60.05～0.2
Glycine 1.0～3.0
α-Nai Yisuan (NAA) 0.5～5
Kinetin (KT) 0.05～0.5
Sucrose 1.0～5.0
Agar 0～5.0
PH value 5.0～6.5
(3) culture condition of cape jasmine cell
(a) solid culture
The MS solid medium of improvement, inoculum size 1～5g/100ml, 20～30 ℃ of temperature, humidity 60～85%, light intensity 1000～3000lx;
(b) liquid culture
The MS liquid nutrient medium of improvement, inoculum size 1～5g/100ml, 20～30 ℃ of temperature, rotating speed 50～150rpm, light intensity 1000～3000lx.
The process of inducing of cape jasmine callus is among the present invention: get the blade on the cape jasmine plant, clean, sterilization, section is tiled on the solid MS substratum of improvement, and temperature is 20～30 ℃, humidity 60～85%, cultivated 20～40 days under light intensity 1000～3000lx condition, paddle cutout is partly swelled, and a faint yellow round and round cell mass that grows is the cape jasmine callus.
Gardenoside is extracted as the methyl alcohol lixiviate of cape jasmine cell among the present invention.
The natural rooting agent that cape jasmine cell tissue provided by the invention is cultivated needs to dilute 100～1000 times in use, and being higher than this concentration has restraining effect to plant-growth, generally seed soaking: dilute 500～1000 times; Seed dressing: dilute 500～1000 times; Foliage-spray: dilute 300～600 times.
The present invention is owing to adopt the cell tissue cultured method to produce the gardenoside natural rooting agent, thereby be not subject to seasonal restrictions (all can produce throughout the year), floor space is little, be easy to screen outstanding kind, the gardenoside of being produced is soluble in water, and the content height can promote plant establishment, be convenient to suitability for industrialized production, have vast market prospect.Below by embodiment in detail the present invention is described in detail.
Accompanying drawing 1 is the seed selection flow process of cape jasmine cell elite plant strain.
Embodiment 1
1. the cape jasmine callus induces
1) gets blade from the cape jasmine plant and clean (following steps are all carried out at the sterilisable chamber Bechtop) with clear water;
2) the blade 8%NaClO of Xi Jinging 3Use aseptic water washing 3～5 times behind the sterilization 10min, water is blotted with aseptic paper;
3) blade being cut into the dice of about 1cm, being tiled on the MS substratum of improvement, is 25 ℃ in temperature, and humidity is 75%, cultivates in the illumination box of light intensity 2000lx:
4) about one month blade of cultivation, its edge notches partly swells, and grows a flaxen round and round cell mass, is the cape jasmine callus.
2. Gai Liang MS substratum macroelement: NH 4NO 31650 (mg/L)
KNO 3??????????????1900
CaCl 2·2H 2O??????400
MgSO 4·7H 2O??????190
KH 2PO 4???????????170
Trace element: KI 1.66
H 3BO 3????????????6.20
MnSO 4·7H 2O??????22.3
ZnSO 4·7H 2O??????8.60
Na 2MoO 4·2H 2O???0.25
CuSO 4·5H 2O??????0.025
CoCl 2·6H 2O??????0.025
Na 2EDTA???????????37.3
FeSO 4·7H 2O??????27.8
Inositol 100
Nicotinic acid 0.5
Pyridoxine hydrochloride B 10.5
Vitamin B 60.1
Glycine 2.0
α-Nai Yisuan (NAA) 1.0
Kinetin (KT) 0.1
Sucrose 3.0 (%)
Agar 0.8
PH value 5.8
3. the foundation of cape jasmine solid cell and suspension cell culture
1) above eugonic faint yellow cell mass is taken off, change in the new improvement MS solid medium of having prepared, inoculum size 1g/100ml is 25 ℃ in temperature, and humidity is 75%, and light intensity is to continue to cultivate in the illumination box of 2000lx.Through repeatedly repeating, get final product the relatively more consistent cell strain system of acquired character, be the cell solid culture.
2) with the solid culture cell of subculture 3～5 times, crushing is transferred in the improvement MS liquid nutrient medium, and inoculum size 2g/100ml is 25 ℃ in temperature, and rotating speed is 80rpm, and light intensity is to cultivate on the shaking table of 2000lx.
3) cultivate after about one month, the cape jasmine cell conditioned medium nutrient solution elimination with being cultivated adds new liquid modified MS medium, continues to cultivate to be the cape jasmine suspension culture.
4. the biomass of cape jasmine callus and suspension cell
Training method Container ml Substratum ml Inoculum size g Cultivate fate d Final weight g Increase multiple b Speed of growth g/L, d Turnout g/L
Solid ??100△ ??30 ??1 ?30 ?25 ?25 ?26.6 ??798.0
Liquid ??250△ ??80 ??5 ?30 ?150 ?30 ?60.4 ??1812.3
The general solid culture of the cell growth rate of plant tissue culture is 10g/L.d, and suspension culture is 20g/L.d.
5. the preparation method of thick gardenoside in the cape jasmine cell
1) the cape jasmine suspension cell is centrifugal, removes moisture;
2) sedimentary cell adds behind the 400ml methyl alcohol cold soaking 48h centrifugally, and supernatant is stand-by;
3) post precipitation add again behind the 300ml methyl alcohol cold soaking 2h centrifugal, this process triplicate, supernatant is stand-by;
4) the whole supernatant liquors of centrifugal gained are merged, concentrate, remove methyl alcohol with distillation under vacuum;
5) dry product after concentrating adds 1～3 times dissolved in distilled water, is the gardenoside crude extract, and except that containing partial pigment and sugar, its main component is in the thick gardenoside liquid:
Gardenoside 0.297%
Iridoid glycosides: gardenoside 0.260%
Genipin-1-0-gentiobioside 0.212%
The influence of hormone pair cell biomass in the embodiment 2 cape jasmine cell modified MS medium:
Hormone (mg/L) Cell enlargement amount (g/ bottle) Cell enlargement rate (%)
MS substratum IAA 2.0 ????2.1 ????210
????????????KT???0.1 ????2.4 ????240
Modified MS medium NAA 1.0 ????3.1 ????310
????????????KT??0.1 ????2.9 ????290
The thick gardenoside that embodiment 3 different training methods are produced:
Training method Substratum Cell yield (fresh weight g/L) Thick gardenoside content (accounting for fresh weight %) Total thick gardenoside turnout (g/L)
Solid culture Modified MS medium ??798.0 ????2.0 ????1.16
Suspension culture Modified MS medium ??1812.0 ????4.5 ????2.50
Embodiment 4 thick gardenosides are used for the rooting effect of wheat:
Thick gardenoside concentration (ppm) Seedling long (cm) Miao Chong (g) Radical (root) Root long (cm) Root heavy (g)
????0 ??13.9 ??0.70 ????5.6 ??8.30 ??0.30
????0.1 ??15.4 ??0.80 ????7.1 ??10.0 ??0.40
????10.0 ??16.5 ??0.95 ????7.0 ??11.0 ??0.40
????100.0 ??16.9 ??1.05 ????7.4 ??11.6 ??0.70
????1000.0 ??9.6 ??5.00 ????3.7 ??1.10 ??0.12
Gardenoside of the present invention can be used as the root-growing agent that general plant such as grain, vegetables, fruit etc. promote its root growth.Use seed soaking, be stained with methods such as root, seed dressing and foliage-spray, all can impel the root system of plant prosperity, improve output, the working concentration scope is: dilute 100～1000 times.

Claims (3)

1. a cape jasmine cell tissue is cultivated the method for producing natural rooting agent, in the root-growing agent of being produced except that containing partial pigment and sugar, mainly contain gardenoside 1～9 ‰, gardenoside 1～9 ‰, genipin-1-0-gentiobioside 1～9 ‰, this method comprises foundation, cultivation and the gardenoside extraction step of cape jasmine cell tissue, it is characterized in that:
(1) the cape jasmine cell tissue of Cai Yonging is the callus of being induced generation by leaf of Capa Jasmine;
(2) employed substratum is the MS substratum of improvement, and component is as follows:
Macroelement: NH 4NO 31000～2000 (mg/L)
KNO 3?????????????????????????1500～2500
CaCl 2·2H 2O?????????????????300～500
MgSO 4·7H 2O?????????????????150～200
Trace element: KI 1.5～1.7
H 3BO 3???????????????????????5.5～7.0
MnSO 4·7H 2O?????????????????20～25
ZnSO 4·7H 2O?????????????????5～15
Na 2MoO 4·2H 2O??????????????0.1～0.5
CuSO 4·5H 2O?????????????????0.01～0.03
CoCl 2·6H 2??????????????????0.01～0.03
Na 2EDTA??????????????????????35～40
FeSO 4·7H 2O?????????????????20～30
Growth regulatory substance: inositol 80～120
Nicotinic acid 0.3～0.7
Pyridoxine hydrochloride B 10.3～0.7
Vitamin B e0.05～0.2
Glycine 1.0～3.0
α-Nai Yisuan (NAA) 0.5～5
Kinetin (KT) 0.05～0.5
Sucrose 1.0～5.0
Agar 0～5.0
PH value 5.0～6.5
(3) culture condition of cape jasmine cell
(a) solid culture
The MS solid medium of improvement, inoculum size 1～5g/100ml, 20～30 ℃ of temperature, humidity 60～85%, light intensity 1000～3000lx;
(b) liquid culture
The MS liquid nutrient medium of improvement, inoculum size 1～5g/100ml, 20～30 ℃ of temperature, rotating speed 50～150rpm, light intensity 1000～3000lx.
2. cultivate the method for producing natural rooting agent according to the described cape jasmine cell tissue of claim 1, it is characterized in that: the process of inducing of cape jasmine callus is: get the blade on the cape jasmine plant, clean sterilization, section, be tiled on the solid MS substratum of improvement, temperature is 20～30 ℃, and humidity 60～85% was cultivated 20～40 days under light intensity 1000～3000lx condition, paddle cutout is partly swelled, and a faint yellow round and round cell mass that grows is the cape jasmine callus.
3. cultivate the method for producing natural rooting agent according to the described cape jasmine cell tissue of claim 1, it is characterized in that: being extracted as of gardenoside the methyl alcohol lixiviate of cape jasmine cell.
CN 99112969 1999-06-02 1999-06-02 Process for generating natural rooting agent by tissue culture of cape jasmine cells Expired - Fee Related CN1120886C (en)

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CN1120886C CN1120886C (en) 2003-09-10

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109287482A (en) * 2018-10-30 2019-02-01 江西智汇中药材种植有限公司 A kind of Yellow Fructus Gardeniae tissue-culturing rapid propagation special culture media
CN109468264A (en) * 2018-12-20 2019-03-15 长沙学院 A kind of production method of Gardenoside
CN109554328A (en) * 2018-12-20 2019-04-02 长沙学院 The method for improving Determination of Gardenoside in gardenia florida cell
CN109644869A (en) * 2018-12-20 2019-04-19 长沙学院 The method for obtaining Gardenoside by tissue cultures

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109287482A (en) * 2018-10-30 2019-02-01 江西智汇中药材种植有限公司 A kind of Yellow Fructus Gardeniae tissue-culturing rapid propagation special culture media
CN109287482B (en) * 2018-10-30 2022-03-01 江西智汇中药材种植有限公司 Special culture medium for tissue culture and rapid propagation of gardenia jasminoides
CN109468264A (en) * 2018-12-20 2019-03-15 长沙学院 A kind of production method of Gardenoside
CN109554328A (en) * 2018-12-20 2019-04-02 长沙学院 The method for improving Determination of Gardenoside in gardenia florida cell
CN109644869A (en) * 2018-12-20 2019-04-19 长沙学院 The method for obtaining Gardenoside by tissue cultures
CN109644869B (en) * 2018-12-20 2021-08-10 长沙学院 Method for obtaining geniposide by tissue culture

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