CN103858757A - Stevia rebaudiana bertoni tissue culture method adopting leaf as explant, and special culture medium thereof - Google Patents
Stevia rebaudiana bertoni tissue culture method adopting leaf as explant, and special culture medium thereof Download PDFInfo
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Abstract
The present invention discloses a stevia rebaudiana bertoni tissue culture method adopting leaf as explant, and a special culture medium thereof. The stevia rebaudiana bertoni tissue culture method comprises: inoculating isolated stevia rebaudiana bertoni leaf into a callus induction and differentiation culture medium to culture to obtain stevia rebaudiana bertoni regeneration bud, wherein the callus induction and differentiation culture medium is a solid culture medium prepared by adding a plant growth substance, a carbon source and a gel to a plant tissue culture base culture medium, and the plant growth substance contains NAA and TDZ. With the stevia rebaudiana bertoni tissue culture method adopting the leaf as the explans, the regeneration frequency and the regeneration proliferation coefficient of the stevia rebaudiana bertoni can be increased, the regeneration period can be shortened, and the stable and efficient regeneration system is established for the transgene research of the stevia rebaudiana bertoni.
Description
Technical field
The present invention relates to STEVIA REBAUDIANA method for tissue culture and its special culture media, more particularly to a kind of STEVIA REBAUDIANA method for tissue culture and its special culture media using blade as explant.
Background technology
STEVIA REBAUDIANA (Stevia rebaudiana Bertoni) also known as " stevia rebaudianum ", " Herba Hedyotis cantonensis ", originate in South America Paraguay, drunk always by local resident as sweet drink for centuries, it is a kind of small-sized perennial composite family (Compositae) Si Taiwei Subgenus, perennial root, fibrous root type, perennial, short-day, herbaceous plant.Japan introduces STEVIA REBAUDIANA from Brazil within 1970, starts to tame, cultivates, prepares glucosides, while carrying out the experiment such as toxicity, food inspection, and develops STEVIA REBAUDIANA product-stevioside first；China started successively to plant experimentally success from Japan's introduction STEVIA REBAUDIANA by R＆D institutions such as Nanjing Botanical Garden Mem. Sun Yat-Sen, the Chinese Academy of Agricultural Sciences in 1976.There are a large amount of plantations on the ground such as present Jiangsu, Fujian, Shandong, Xinjiang, Henan, Anhui, and the gross area is up to more than 1,000,000 mu, and China has turned into the most country of plantation STEVIA REBAUDIANA area in the world, is also the producing country and exported country of stevioside maximum in the world.
Steviol glycoside（Stevioside）Be extracted from the sweetleaf of STEVIA REBAUDIANA the high sugariness of a class, natural products that is low in calories, being had no side effect to human body, also have certain auxiliary therapeutic action to obesity, diabetes, high blood pressure, heart disease, carious tooth etc..The sugariness of steviol glycoside is 300 times of sucrose, and its calorific value is the 1/300 of sucrose, so many producers substitute sucrose using steviol glycoside as a kind of sweetener at present.Steviol glycoside is preferable confectionery, human body can not decompose stevia rebaudianum glycocide and convert it to glucose, also therefore it will not be absorbed by blood vessel, it can be excreted in vitro with fibre morphology after digestion, therefore fat or increase diabetic glucose level is led to without unnecessary calorie is left, STEVIA REBAUDIANA is favored by obesity patient and diabetes patient deeply.Steviol glycoside is closest to the natural low caloric value sweetener of sucrose taste, and it is the natural sucrose substitute that the third has that Development volue and health are praised highly outside sugarcane beet sugar, is described as in the world " third place in the world sucrose ".Steviol glycoside replaces sucrose processed food beverage in part to substantially reduce with sugared cost.As people increasingly weigh to the degree of concern of health, stevioside will have good market prospects as a kind of feature carbohydrate.The Ministry of Public Health of China have approved stevioside for the quantity-unlimiting natural sweetener used and pharmaceutical sweetener auxiliary material respectively in 1985 and nineteen ninety, U.S. FDA can be used as sweetener in December, 2008 to steviol glycoside formally to be examined, and European Union also had been approved by steviol glycoside and used as a kind of sweetener in European Union on November 14th, 2011.STEVIA REBAUDIANA is increasingly becoming the focus of food and medicine area research exploitation, and this will drive the fast development of STEVIA REBAUDIANA industry.
STEVIA REBAUDIANA is the not affine cross-pollinatd plant of selfing, and genetic stability is poor, is unfavorable for the homozygosis of cenospecies and the anti-miscellaneous pure keeping of kind, moreover, Stevia seed is tiny, germination percentage is low, only can not meet demand of the in the market to STEVIA REBAUDIANA by seminal propagation.The good characteristic of kind can not only be kept using tissue culture technique, can not also be influenceed to breed throughout the year by external condition.1970s, domestic many research units have just carried out the research of STEVIA REBAUDIANA tissue cultures.But be all mostly by it is fast it is numerous for the purpose of experiment, using the stem apex with bud point, lateral bud etc., it expands, and numerous coefficient is limited, and growth coefficient is low.Using explants such as the blades without bud point, general report is only 20-60%, regeneration growth coefficient 3-10.Shen Xiuli etc.（1996）By explant of blade on addition BA 0.5mg/L and NAA0.5mg/LMS culture medium, adventitious shoot regeneration rate is 20%, and regeneration growth coefficient is 3；Ni Dexiang etc.（1985）STEVIA REBAUDIANA blade is used for explant, adventitious shoot regeneration rate is 37% on MS addition BA 2.0mg/L and NAA 2.0mg/L culture mediums, and regeneration growth coefficient is 9.3/10 piece；Jain etc.（2009）On addition BAP 2.2uM and IAA 2.8uM MS culture mediums, the adventitious shoot regeneration rate of STEVIA REBAUDIANA blade explant is 45%, regenerates growth coefficient 9.2.General STEVIA REBAUDIANA blade explant need to could regenerate by 3,4 generation callus squamous subcultures, and the recovery time is long, and regeneration growth coefficient is low.
The content of the invention
The technical problems to be solved by the invention are to provide regeneration rate height and/or the regeneration high three kinds of STEVIA REBAUDIANA method for tissue culture and its special culture media using blade as explant of growth coefficient.
STEVIA REBAUDIANA method for tissue culture one provided by the present invention using blade as explant, including the step of obtaining STEVIA REBAUDIANA regeneration bud is cultivated into the excised leaf access callus induction of STEVIA REBAUDIANA and progress in differential medium；The callus induction and the solid medium that differential medium is that addition plant growth substance, carbon source and gel are made in plant tissue culture media basal culture medium；The plant growth substance contains NAA and TDZ.
In above-mentioned STEVIA REBAUDIANA method for tissue culture one, the plant growth substance can also contain other plant hormones or plant growth regulator, such as 6-BA, KT, IBA, 2,4-D, IAA.
In above-mentioned STEVIA REBAUDIANA method for tissue culture one, in the callus induction and differential medium, NAA concentration can be 0.1mg/L-0.2mg/L, such as 0.1mg/L, 0.15mg/L, 0.2mg/L, 0.1-0.15mg/L, 0.15-0.2mg/L, TDZ concentration can be 1.0mg/L-3.0mg/L, such as 1.0mg/L, 2.0mg/L, 3.0mg/L, 1.0-1.5mg/L, 1.5-2.0mg/L, 2.0-2.5mg/L, 2.5-3.0mg/L.
STEVIA REBAUDIANA method for tissue culture two provided by the present invention using blade as explant, including the step of obtaining STEVIA REBAUDIANA regeneration bud is cultivated into the excised leaf access callus induction of STEVIA REBAUDIANA and progress in differential medium；The callus induction and the solid medium that differential medium is that addition plant growth substance, carbon source and gel are made in plant tissue culture media basal culture medium；The plant growth substance contains NAA and 6-BA.
In above-mentioned STEVIA REBAUDIANA method for tissue culture two, the plant growth substance can also contain other plant hormones or plant growth regulator, such as IBA, 2,4-D, IAA.
In the STEVIA REBAUDIANA method for tissue culture two, the callus induction and differential medium can be A, B, C or D；
In A, the callus induction and differential medium, the concentration of the NAA is 0.1mg/L-0.2mg/L, and 6-BA concentration is 1.0mg/L-3.0mg/L；
B, the plant growth substance contain NAA, 6-BA and KT；Specifically, the concentration of the NAA is 0.1mg/L-0.2mg/L, and 6-BA concentration is 1.0mg/L-3.0mg/L, and KT concentration is 1.0mg/L-2.0mg/L；
C, the plant growth substance are NAA and 6-BA；In the callus induction and differential medium, NAA concentration is 0.1mg/L-0.2mg/L, and 6-BA concentration is 1.0mg/L-4.0mg/L；
D, the plant growth substance are NAA, 6-BA and KT；In the callus induction and differential medium, NAA concentration is 0.1mg/L-0.2mg/L, and 6-BA concentration is 1.0mg/L-3.0mg/L, and KT concentration is 1.0mg/L-2.0mg/L.
In above-mentioned STEVIA REBAUDIANA method for tissue culture one and method two, the culture can carry out normal illumination culture again first to carry out dark culturing.
STEVIA REBAUDIANA method for tissue culture three provided by the present invention using blade as explant, including the step of obtaining STEVIA REBAUDIANA regeneration bud is cultivated into the excised leaf access callus induction of STEVIA REBAUDIANA and progress in differential medium；The callus induction and the solid medium that differential medium is that addition plant growth substance, carbon source and gel are made in plant tissue culture media basal culture medium；The culture carries out normal illumination culture again first to carry out dark culturing.
In above-mentioned STEVIA REBAUDIANA method for tissue culture three, the callus induction contains NAA and 6-BA with plant growth substance described in differential medium.
In above-mentioned STEVIA REBAUDIANA method for tissue culture three, the plant growth substance can also contain other plant hormones or plant growth regulator, such as IBA, 2,4-D, IAA.
Specifically, in above-mentioned STEVIA REBAUDIANA method for tissue culture three, the callus induction and differential medium can be above-mentioned A, B, C or D.
In above-mentioned STEVIA REBAUDIANA method for tissue culture one, method two and method three, the plant tissue culture media basal culture medium can be MS culture mediums, White culture mediums, Miller culture mediums, Heller culture mediums, LS culture mediums, ER culture mediums, MT culture mediums, NN culture mediums, NLN culture mediums, Nitsh cultures, H culture mediums, B5 medium, N6 culture mediums, SH culture mediums, DKW culture mediums or WPM culture mediums.
Wherein, the solvent of the MS culture mediums is that water, solute and its concentration are as shown in table 1.
The solute and its concentration of table 1.MS culture mediums（mg/L）
In above-mentioned STEVIA REBAUDIANA method for tissue culture one, method two and method three, the time of the dark culturing can be 1-6 days, 2-6 days, 3-6 days, 4-6 days, 5-6 days, 1 day, 2 days, 3 days, 4 days, 5 days or 6 days.
In above-mentioned STEVIA REBAUDIANA method for tissue culture one, method two and method three, the normal illumination can be daily 12-14 hours（Such as 12 hours, 14 hours, 13 hours）Remaining time of illumination is dark.
In above-mentioned STEVIA REBAUDIANA method for tissue culture one, method two and method three, the intensity of illumination of the normal illumination is 2000-3000lux（Such as 2000lux, 2100lux, 2200lux, 2300lux, 2400lux, 2500lux, 2600lux, 2700lux, 2800lux, 2900lux or 3000lux）.
In above-mentioned STEVIA REBAUDIANA method for tissue culture one, method two and method three, the dark culturing and the normal illumination culture are at 22-25 DEG C（Such as 22-23 DEG C, 22-24 DEG C, 23-25 DEG C, 23-24 DEG C or 24-25 DEG C）It is lower to carry out.
In above-mentioned STEVIA REBAUDIANA method for tissue culture one, method two and method three, in addition to the STEVIA REBAUDIANA regeneration bud carry out squamous subculture and/or to the STEVIA REBAUDIANA regeneration bud carry out culture of rootage the step of.
In above-mentioned STEVIA REBAUDIANA method for tissue culture one, method two and method three, the subculture medium that the squamous subculture is used can be the solid medium that adds NAA in the plant tissue culture media basal culture medium, 6-BA, carbon source and gel are made；Specifically, in the subculture medium, NAA concentration is 0.1mg/L-0.2mg/L, and 6-BA concentration is 0.2mg/L-1.0mg/L；
And/or,
The root media that the culture of rootage is used can be to add the solid medium that NAA, carbon source and gel are made in the plant tissue culture media basal culture medium；Specifically, concentration of the NAA in the root media is 0.1mg/L-0.4mg/L.
In above-mentioned STEVIA REBAUDIANA method for tissue culture one, method two and method three, all gels can be agar, carragheen or curing agent（Gelrite）；Specifically, concentration of the agar in all culture mediums is 7-11g/L；
And/or,
The carbon source is sucrose, glucose and/or maltose；Specifically concentration of the sucrose in the culture medium is 15-30g/L.
In above-mentioned STEVIA REBAUDIANA method for tissue culture one, method two and method three, the blade may be from aseptic seedling.The aseptic seedling can be prepared as follows：By on the STEVIA REBAUDIANA sterilizing seed access germination solid medium of budding, 22-25 DEG C is subsequently placed in（Such as 22-23 DEG C, 22-24 DEG C, 23-25 DEG C, 23-24 DEG C or 24-25 DEG C）, it is daily 12-14 hours（Such as 12-14 hours, 14 hours, 13 hours）Culture obtains aseptic seedling under remaining time dark condition of illumination；The germination solid medium is the solid medium that addition NAA, the carbon source and the gel are made in the plant tissue culture media basal culture medium, NAA concentration is 0.1mg/L-0.2mg/L in the germination solid medium, the concentration of the carbon source is 15-30g/L, and the concentration of the gel is 7-11g/L.
STEVIA REBAUDIANA tissue culture medium (TCM) provided by the present invention using blade as explant, including culture medium a and culture medium b, the culture medium a are callus induction and differential medium, the culture medium b is root media and/or subculture medium；The callus induction is callus induction and differential medium described in above-mentioned STEVIA REBAUDIANA method for tissue culture one and method two with differential medium, and the subculture medium is the subculture medium in above-mentioned STEVIA REBAUDIANA method for tissue culture one and method two；The root media is the root media in above-mentioned STEVIA REBAUDIANA method for tissue culture one and method two.
Certainly, the above-mentioned STEVIA REBAUDIANA tissue culture medium (TCM) using blade as explant only can be also made up of the culture medium a and the culture medium b.
The culture medium a and the equal independent packagings of culture medium b in the above-mentioned STEVIA REBAUDIANA tissue culture medium (TCM) using blade as explant.
Callus induction described in above-mentioned STEVIA REBAUDIANA method for tissue culture one and method two falls within protection scope of the present invention with differential medium.
The pH of above-mentioned callus induction and differential medium, subculture medium, root media, the solid medium that germinates can be 5.8-5.9.
The method of the present invention is suitable for the kinds of all STEVIA REBAUDIANAs, such as Huinong No. 1, Huinong No. 2, rich No. 1 of Anhui, greenstone 131, Nanjing 158, keeps field 1, keeps field 2.
Experiment is proved, the STEVIA REBAUDIANA method for tissue culture using blade as explant of the present invention, it is 100% that blade, which accesses 14 days Callus formation rates of callus induction and differential medium, illumination cultivation forms regeneration bud after 20-30 days, the regeneration rate of adventitious bud reaches 50-90% after 50 days, and regeneration growth coefficient reaches 10-30, and regeneration bud subculture cycle is 14 days, the rooting rate of subculture regeneration bud is 100%, and survival rate is 100% after the regeneration transplantation of seedlings taken root.The STEVIA REBAUDIANA method for tissue culture using blade as explant of the present invention, can improve the regeneration frequency and regeneration growth coefficient of STEVIA REBAUDIANA, shorten the regeneration period, be the regenerating system that STEVIA REBAUDIANA transgenic research establishes stability and high efficiency.
Brief description of the drawings
Fig. 1 is the STEVIA REBAUDIANA regeneration flow photo using blade as explant.
A, seed asepsis germination；B, aseptic seedling；C, blade explant；D, callus；E, callus regeneration bud；F, regrowth；G, culture of rootage；H, transplanted seedling.
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experimental method in following embodiments, is conventional method unless otherwise specified.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
NAA in following embodiments is 1- methyl α-naphthyl acetates, and purchased from SIGMA companies, 6-BA is 6-benzyl aminopurine, purchased from SIGMA companies, and TDZ is phenyl thiadiazolyl group urea, and purchased from SIGMA companies, KT is kinetin（6-Furfurylaminopurine）, purchased from SIGMA companies.
The plant tissue culture media basal culture medium used in following embodiments of the present invention is MS culture mediums, and its solvent is that water, solute and its concentration are as shown in table 2.
The solute and its concentration of table 2.MS culture mediums（mg/L）
Embodiment 1, the STEVIA REBAUDIANA tissue cultures by explant of blade
1st, the acquisition of aseptic seedling：
Pick out full seed STEVIA REBAUDIANA kind " Huinong No. 1 " seed（Purchased from the perfectly sound rich agricultural producers' cooperative in Linquan County）, first wash 30s with 75% alcohol, then with 1% hypochlorite disinfectant 15 minutes, finally with after aseptic water washing 4-6 times.Sterilizing seed is seeded on sterile moistening filter paper（A in Fig. 1）, germination solid medium is transferred to after budding（MS+0.1mg/l NAA+30g/L sucrose+8g/L agar）On, it is subsequently placed in 22 DEG C, daily 14h illumination（Intensity of illumination is 2000lux）40 days or so seedlings are cultivated under 10h dark conditions（B in Fig. 1）, aseptic seedling is taken out and cuts blade explant.Above-mentioned germination solid medium is that the concentration of NAA in the solid medium that addition NAA, sucrose and agar are obtained in MS culture mediums, the germination solid medium is 0.1mg/L, and the concentration of sucrose is 30g/L, and the concentration of agar is 8g/L.The pH of the germination solid medium is 5.8.
2nd, callus induction and differentiation：
Young leaflet tablet at the top of the aseptic seedling of 40 days seedling ages is taken to be cut into 0.5 × 0.5cm2Size, is inoculated in callus induction and differential medium Mb2 respectively（MS+2.0mg/L 6-BA+0.1mg/L NAA）、Mc32（MS+0.1mg/LNAA+2.0mg/L TDZ）、Me1（MS+2.0mg/L 6-BA+0.2mg/L NAA+1.0mg/L KT）On, respectively in 22 DEG C of dark culturings 0, the dark normal illuminations of 14h illumination 10h are transferred to after 4,6 or 10 days（Intensity of illumination 2000-3000lux）Cultivated under the conditions of 22 DEG C, every bottle of 11 blades, each 10 bottles of processing is repeated 3 times, observes and record Callus formation and adventitious buds differentiation situation on different culture media under the same conditions.Meter is cultivated from being transferred under normal lighting conditions, 50 days statistics regeneration rates and regeneration growth coefficient.Regeneration rate（%）=regenerated adventitious bud explant number/inoculation explant sum × 100%；Regenerate growth coefficient=regenerated adventitious bud sum/inoculation explant sum.
Wherein, Mb2 is that the concentration of NAA in the solid medium that addition 6-BA, NAA, sucrose and agar are obtained in MS culture mediums, Mb2 is 0.1mg/L, and 6-BA concentration is 2.0mg/L, and the concentration of sucrose is 30g/L, and the concentration of agar is 8g/L；Mb2 pH is 5.8.
Mc32（MS+0.1mg/L NAA+2.0mg/L TDZ）NAA concentration is 0.1mg/L in the solid medium that addition TDZ, NAA, sucrose and agar are obtained in MS culture mediums, Mc32, and TDZ concentration is 2.0mg/L, and the concentration of sucrose is 30g/L, and the concentration of agar is 8g/L；Mc32 pH is 5.8.
Me1（MS+2.0mg/L 6-BA+0.2mg/L NAA+1.0mg/L KT）6-BA concentration is 2.0mg/L in the solid medium that addition 6-BA, KT, NAA, sucrose and agar are obtained in MS culture mediums, Me1, and NAA concentration is 0.2mg/L, and KT concentration is 1.0mg/L, and the concentration of sucrose is 30g/L, and the concentration of agar is 8g/L；Me1 pH is 5.8.
As a result show, regeneration rate of the explant on Mc32 culture mediums and regeneration growth coefficient such as table 3 and table 4.Wherein, explant on Mc32 culture mediums without dark culturing directly in normal illumination dark 14h illumination 10h（Intensity of illumination 2000lux）Cultivated 14 days under the conditions of 22 DEG C, 100% blade the callus of yellow green occurs in notching edge, meter is cultivated from being transferred under normal lighting conditions, start within 30 days regeneration budlet occur, meter is cultivated from being transferred under normal lighting conditions, the regeneration rate of 50 days is 51.0%, and regeneration growth coefficient is 10.Regeneration bud form is normal.The entitled Mc320 of the processing.
Explant is on Mc32 culture mediums, 22 DEG C of dark culturings 4 days（Such as C in Fig. 1）The dark normal illuminations of 14h illumination 10h are transferred to afterwards（Intensity of illumination 2000lux）Cultivated 10 days under the conditions of 22 DEG C, all blades the callus of yellow green occur in notching edge（D in Fig. 1）, meter is cultivated from being transferred under normal lighting conditions, starts within 20 days regeneration budlet occur（E in Fig. 1）.Meter is cultivated from being transferred under normal lighting conditions, the regeneration rate of 50 days is 89.6, and regeneration growth coefficient is 30.Regeneration bud form is normal.The entitled Mc324 of the processing.
Explant is on Mc32 culture mediums, and 22 DEG C of dark culturings are transferred to the dark normal illuminations of 14h illumination 10h after 6 days（Intensity of illumination 2000lux）Cultivated 10 days under the conditions of 22 DEG C, 100% blade the callus of yellow green occurs in notching edge, meter is cultivated from being transferred under normal lighting conditions, start within 20 days regeneration budlet occur.Meter is cultivated from being transferred under normal lighting conditions, the regeneration rate of 50 days is 49.1, and regeneration growth coefficient is 20.Regeneration bud form is normal.The entitled Mc326 of the processing.
Regeneration rate of the explant on Me1 culture mediums such as table 3.Wherein, explant on Me1 culture mediums without dark culturing directly in normal illumination dark 14h illumination 10h（Intensity of illumination 2000lux）Cultivated 10 days under the conditions of 22 DEG C, 100% blade the callus of yellow green occurs in notching edge, meter is cultivated from being transferred under normal lighting conditions, started within 30 days regeneration budlet occur, meter is cultivated from being transferred under normal lighting conditions, the regeneration rate of 50 days is 56.0.The entitled Me10 of the processing.
Explant is on Me1 culture mediums, and 22 DEG C of dark culturings are transferred to the dark normal illuminations of 14h illumination 10h after 4 days（Intensity of illumination 2000lux）Cultivated 10 days under the conditions of 22 DEG C, 100% blade the callus of yellow green occurs in notching edge, meter is cultivated from being transferred under normal lighting conditions, start within 30 days regeneration budlet occur.Meter is cultivated from being transferred under normal lighting conditions, the regeneration rate of 50 days is 78.2.The entitled Me14 of the processing.
Explant is on Me1 culture mediums, and 22 DEG C of dark culturings are transferred to the dark normal illuminations of 14h illumination 10h after 6 days（Intensity of illumination 2000lux）Cultivated 10 days under the conditions of 22 DEG C, 100% blade the callus of yellow green occurs in notching edge, meter is cultivated from being transferred under normal lighting conditions, start within 30 days regeneration budlet occur.Meter is cultivated from being transferred under normal lighting conditions, the regeneration rate of 50 days is 54.5.The entitled Me16 of the processing.
Regeneration rate of the explant on Mb2 culture mediums such as table 3.Wherein, explant on Mb2 culture mediums without dark culturing directly in normal illumination dark 14h illumination 10h（Intensity of illumination 2000lux）Cultivated 10 days under the conditions of 22 DEG C, 100% blade the callus of yellow green occurs in notching edge, meter is cultivated from being transferred under normal lighting conditions, started within 30 days regeneration budlet occur, meter is cultivated from being transferred under normal lighting conditions, the regeneration rate of 50 days is 12.2.The entitled Mb20 of the processing.
Explant is on Mb2 culture mediums, and 22 DEG C of dark culturings are transferred to the dark normal illuminations of 14h illumination 10h after 4 days（Intensity of illumination 2000lux）Cultivated 10 days under the conditions of 22 DEG C, 100% blade the callus of yellow green occurs in notching edge, meter is cultivated from being transferred under normal lighting conditions, start within 30 days regeneration budlet occur.Meter is cultivated from being transferred under normal lighting conditions, the regeneration rate of 50 days is 81.9.The entitled Mb24 of the processing.
Explant is on Mb2 culture mediums, and 22 DEG C of dark culturings are transferred to the dark normal illuminations of 14h illumination 10h after 6 days（Intensity of illumination 2000lux）Cultivated 10 days under the conditions of 22 DEG C, 100% blade the callus of yellow green occurs in notching edge, meter is cultivated from being transferred under normal lighting conditions, start within 30 days regeneration budlet occur.Meter is cultivated from being transferred under normal lighting conditions, the regeneration rate of 50 days is 78.2.The entitled Mb26 of the processing.
The explant of table 3. different callus inductions and differential medium the different dark culturing times regeneration rate
The regeneration growth coefficient of the explant of table 4. different dark culturing times in Mc32
| Dark culturing 0 day | Dark culturing 4 days | Dark culturing 6 days |
| 10±3.2 | 30±5.6 | 20±5.2 |
3rd, the squamous subculture of regeneration bud
The regeneration bud obtained to step 2, carry out sterile cutting, budlet is transferred on subculture medium, at 22-25 DEG C, daily illumination in 12-14 hours, 10-12 hours are dark, cultivated under the conditions of intensity of illumination 2000-3000lux, regeneration budlet is set quickly normally to grow up, it is to avoid regeneration bud vitrifying and death.Every bottle of 8 regeneration buds, each 10 bottles of processing, are repeated 3 times under the same conditions.
The squamous subculture uses following subculture medium：The solid medium that addition NAA, 6-BA, sucrose and agar are obtained in MS culture mediums, concentration of the NAA in subculture medium is 0.1mg/L, concentration of the 6-BA in subculture medium is 1.0mg/L, concentration of the sucrose in subculture medium is 30g/L, and concentration of the agar in subculture medium is 8g/L.The pH of the subculture medium is 5.8.
As a result show that Mc324 regeneration bud grows regrowth in 20 days in subculture medium（F in Fig. 1）, the regrowth grown carries out successive propagation according still further to same procedure, and every 14 days subcultures once, after subculture 3 times, carry out the culture of rootage of the regeneration bud of step 4.
Mc320, Mc326, Mb24, Mb26, Me10, Me14 and Me16 regeneration bud grow regrowth in 20 days in subculture medium, the regrowth grown carries out successive propagation according still further to same procedure, every 14 days subcultures once, after subculture 3 times, carry out the culture of rootage of the regeneration bud of step 4.
4th, the culture of rootage of regeneration bud
The STEVIA REBAUDIANA regrowth that step 3 squamous subculture is obtained is transferred to root media, at 22-25 DEG C, daily 12-14 hours illumination, and 10-12 hours dark, and culture of rootage is carried out under the conditions of intensity of illumination 2000-3000lux, obtains intact plant（Rooted seedling）Transplanting culture is carried out afterwards.Take out the rooted seedling in blake bottle, the culture medium of root is rinsed out with clear water, it is transplanted into Nutrition Soil, preservative film is carried out after the moisturizing hardening culture of 7 days in covering, preservative film is opened at 25-28 DEG C, daily illumination in 12 hours, 12 hours dark, cultivate, finally normally blossom and bear fruit under the conditions of intensity of illumination 2000-3000lux.
The culture of rootage uses following root media：In the solid medium that addition NAA, sucrose and agar are obtained in MS culture mediums, the root media, NAA concentration is 0.1mg/L, and the concentration of sucrose is 30g/L, and the concentration of agar is 8g/L, and pH is 5.8.
As a result show, Mc320, Mc324, Mc326, Mb24, Mb26, Me10, Me14 and Me16 regeneration bud start to take root after being transferred to root media 14 days, the rooting rate of regeneration bud is 100%.Wherein, Mc324 regeneration bud is taken root 20 days（G in Fig. 1）Height of seedling reaches that 3cm is transplanted, rooted seedling（H in Fig. 1）Transplanting survival rate be 100%.
Claims (15)
1. the method for tissue culture of STEVIA REBAUDIANA, including the step of obtaining STEVIA REBAUDIANA regeneration bud is cultivated into the excised leaf access callus induction of STEVIA REBAUDIANA and progress in differential medium；The callus induction and the solid medium that differential medium is that addition plant growth substance, carbon source and gel are made in plant tissue culture media basal culture medium；The plant growth substance contains NAA and TDZ.
2. according to the method described in claim 1, it is characterised in that：The plant growth substance is NAA and TDZ；In the callus induction and differential medium, NAA concentration is 0.1mg/L-0.2mg/L, and TDZ concentration is 1.0mg/L-3.0mg/L.
3. the method for tissue culture of STEVIA REBAUDIANA, including the step of obtaining STEVIA REBAUDIANA regeneration bud is cultivated into the excised leaf access callus induction of STEVIA REBAUDIANA and progress in differential medium；The callus induction and the solid medium that differential medium is that addition plant growth substance, carbon source and gel are made in plant tissue culture media basal culture medium；The plant growth substance contains NAA and 6-BA.
4. method according to claim 3, it is characterised in that：The callus induction is A, B, C or D with differential medium；
In A, the callus induction and differential medium, the concentration of the NAA is 0.1mg/L-0.2mg/L, and 6-BA concentration is 1.0mg/L-3.0mg/L；
B, the plant growth substance contain NAA, 6-BA and KT；Specifically, the concentration of the NAA is 0.1mg/L-0.2mg/L, and 6-BA concentration is 1.0mg/L-3.0mg/L, and KT concentration is 1.0mg/L-2.0mg/L；
C, the plant growth substance are NAA and 6-BA；In the callus induction and differential medium, NAA concentration is 0.1mg/L-0.2mg/L, and 6-BA concentration is 1.0mg/L-4.0mg/L；
D, the plant growth substance are NAA, 6-BA and KT；In the callus induction and differential medium, NAA concentration is 0.1mg/L-0.2mg/L, and 6-BA concentration is 1.0mg/L-3.0mg/L, and KT concentration is 1.0mg/L-2.0mg/L.
5. according to any described method in claim 1-4, it is characterised in that：The culture carries out normal illumination culture again first to carry out dark culturing.
6. the method for tissue culture of STEVIA REBAUDIANA, including the step of obtaining STEVIA REBAUDIANA regeneration bud is cultivated into the excised leaf access callus induction of STEVIA REBAUDIANA and progress in differential medium；The callus induction and the solid medium that differential medium is that addition plant growth substance, carbon source and gel are made in plant tissue culture media basal culture medium；The culture carries out normal illumination culture again first to carry out dark culturing.
7. the method according to claim 5 or 6, it is characterised in that：The time of the dark culturing is 1-6 days, 4-6 days, 4 days or 6 days.
8. according to any described method in claim 5-7, it is characterised in that：The normal illumination is that daily remaining time of 12-14 hours illumination is dark.
9. according to any described method in claim 5-8, it is characterised in that：The intensity of illumination of the normal illumination is 2000-3000lux.
10. according to any described method in claim 5-9, it is characterised in that：The dark culturing and the normal illumination culture are carried out at 22-25 DEG C.
11. according to any described method in claim 1 to 10, it is characterised in that：The step of methods described also includes carrying out the STEVIA REBAUDIANA regeneration bud squamous subculture and/or carrying out culture of rootage to the STEVIA REBAUDIANA regeneration bud.
12. method according to claim 11, it is characterised in that：The subculture medium that the squamous subculture is used is the solid medium that addition NAA, 6-BA, carbon source and gel are made in the plant tissue culture media basal culture medium；Specifically, in the subculture medium, NAA concentration is 0.1mg/L-0.2mg/L, and 6-BA concentration is 0.2mg/L-1.0mg/L；
And/or,
The root media that the culture of rootage is used is the solid medium that addition NAA, carbon source and gel are made in the plant tissue culture media basal culture medium；Specifically, concentration of the NAA in the root media is 0.1mg/L-0.4mg/L.
13. according to any described method in claim 1-12, it is characterised in that：All gels are agar, carragheen or curing agent（Gelrite）；Specifically, concentration of the agar in all culture mediums is 7-11g/L；
And/or,
The carbon source is sucrose, glucose and/or maltose；Specifically concentration of the sucrose in the culture medium is 15-30g/L.
14. the tissue culture medium (TCM) of STEVIA REBAUDIANA, including culture medium a and culture medium b, the culture medium a are callus induction and differential medium, the culture medium b is root media and/or subculture medium；The callus induction and callus induction and differential medium that differential medium is described in any one of claim 1-4,6 and 13 claim methods described, the subculture medium is the subculture medium in the methods described of claim 12 or 13；The root media is the root media in the methods described of claim 12 or 13.
15. the callus induction and differential medium of STEVIA REBAUDIANA blade, are the callus induction and differential medium described in the claim methods described of any one of claim 1-4,6 and 13.
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| CN114836466A (en) * | 2022-05-13 | 2022-08-02 | 宁夏大学 | Agrobacterium-mediated genetic transformation method for stevia rebaudiana |
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|---|---|---|---|---|
| CN104304003A (en) * | 2014-09-09 | 2015-01-28 | 何克勤 | Culture medium formula capable of delaying growth of stevia rebaudiana germplasm tissue cultured seedling |
| CN105104207A (en) * | 2015-09-22 | 2015-12-02 | 安徽科技学院 | Method for obtaining regenerated plants of stevia rebaudiana Bertoni |
| CN105918119A (en) * | 2016-04-22 | 2016-09-07 | 中国科学院合肥物质科学研究院 | Method for in-vitro high-efficiency regeneration of leaf of Chuzhou chrysanthemum |
| CN106613953A (en) * | 2016-11-03 | 2017-05-10 | 明光市大全甜叶菊专业合作社 | Method for tissue culture of stevia rebaudiana |
| CN106613956A (en) * | 2016-11-03 | 2017-05-10 | 明光市大全甜叶菊专业合作社 | Bacteriostatic stevia rebaudiana culture medium |
| CN108112474A (en) * | 2016-11-28 | 2018-06-05 | 山东农业大学 | A kind of method that cultured in vitro improves STEVIA REBAUDIANA RA contents |
| CN107494260A (en) * | 2017-07-04 | 2017-12-22 | 浦江县合洪园艺研发有限公司 | STEVIA REBAUDIANA bud breaks up the preparation method of nutrient solution |
| CN107980630A (en) * | 2017-10-25 | 2018-05-04 | 北京农业生物技术研究中心 | A kind of preparation method of STEVIA REBAUDIANA callus |
| 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 |
| 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 |
| CN114836466A (en) * | 2022-05-13 | 2022-08-02 | 宁夏大学 | Agrobacterium-mediated genetic transformation method for stevia rebaudiana |
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