CN106718934B - A kind of Rhizoma Atractylodis Macrocephalae regenerating system directly breaking up adventitious bud using plumular axis and radicle - Google Patents
A kind of Rhizoma Atractylodis Macrocephalae regenerating system directly breaking up adventitious bud using plumular axis and radicle Download PDFInfo
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- CN106718934B CN106718934B CN201710070674.2A CN201710070674A CN106718934B CN 106718934 B CN106718934 B CN 106718934B CN 201710070674 A CN201710070674 A CN 201710070674A CN 106718934 B CN106718934 B CN 106718934B
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- 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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Abstract
The invention discloses a kind of Rhizoma Atractylodis Macrocephalae regenerating systems for directly breaking up adventitious bud using plumular axis and radicle, it include: that Atractylodes macrocephala seed is cleaned, is sterilized, Atractylodes macrocephala seed after disinfection is inoculated in MS culture medium, cultivates in dark place to seed and sprouts, be transferred under light and continue to cultivate；Radicle and plumular axis are cut, is cut into small pieces as explant, Fiber differentiation on bud inducement cultivation base is seeded to, its differentiation is made to grow green bud；It cuts green bud and is forwarded in root media and cultivate, root induction；By the Transplantation of Regenerated Plantlets taken root to get Rhizoma Atractylodis Macrocephalae plant.Regenerating system provided by the invention substantially increases the proliferation frequency of Rhizoma Atractylodis Macrocephalae, has saved production cost, improves reproductive efficiency, and the large-scale production for medicinal Rhizoma Atractylodis Macrocephalae provides technical support；And due to the organ generation without callus, the genetic stability of plant is higher, this provides quality guarantee to improve Rhizoma Atractylodis Macrocephalae quality, industrial seedling rearing and virus-free using plant gene engineering technology.
Description
Technical field
The present invention relates to the reproduction techniques of medicinal material, specifically a kind of directly to break up adventitious bud using plumular axis and radicle
Rhizoma Atractylodis Macrocephalae regenerating system.
Background technique
Rhizoma Atractylodis Macrocephalae ( Atractylodes macrocephalaKoidz.) belong to composite family [WTBX herbaceos perennial, do
Dry rhizome, gas faint scent, sweet-bitter flavor is warm-natured, mainly originates in the ground such as Zhejiang, Hunan, Hubei and the Anhui in China (Chinese Pharmacopoeia
2010), in latent produced performance optimal, spy claims " in art ".The medical value of Rhizoma Atractylodis Macrocephalae is loaded in Shennong's Herbal for the first time, is arranged
For top grade.Rhizoma Atractylodis Macrocephalae is traditional one of the Chinese medicine in China, and active chemical includes volatile oil and polysaccharide etc..It is main in volatile oil
Contain atractylol, atractylone, sesquiterpenoids etc..The compounds such as volatile oil, lactone have stronger bioactivity.Rhizoma Atractylodis Macrocephalae
With antitumor, promote gastrointestinal motility, adjust the effect (Chen Xiaoping etc., 2011) of gastrointestinal dysfunction, there is strengthening the spleen and replenishing qi,
Anti-senile dementia anti-aging is anti-oxidant, improves immunity, and anticoagulant effect of anti-inflammatory diuresis (5 happy celerys etc., 2011) is " strong
The main component of the Chinese patent drugs such as spleen ball ", " shenling baizhu powder ", " stomach pill of aucklandia and amomum fruit ".In addition, Rhizoma Atractylodis Macrocephalae still contains, there are many plant other
Have pharmacological activity compound (full river of dragon etc., 2004;Chen Bingbing, 2012).Rhizoma Atractylodis Macrocephalae is good Qi-tonifying drug (poplar simultaneously
Pretty young woman etc., 2012).
Currently, Wild Plant Atractylodes macrocephala Koidz resource is endangered, the most of Rhizoma Atractylodis Macrocephalae from artificial growth of raw medicinal herbs used in the market,
And artificial cultivation Rhizoma Atractylodis Macrocephalae need to pass through cumbersome, prolonged seedling raising process (Wang Liuzhu, 2009 years).In recent years, people start to make
With tissue culture rapid propagating technology, this can obtain a large amount of Rhizoma Atractylodis Macrocephalae test tube seedlings within a short period of time to meet the standardized production kind of Rhizoma Atractylodis Macrocephalae
It plants.The country is less about the report of the in vitro plant regeneration of Rhizoma Atractylodis Macrocephalae, mainly using Rhizoma Atractylodis Macrocephalae lateral bud (Shen Yinzhu etc., 1988;Peng Fei
Deng 2001), blade (Chen Juan etc., 2006), stem apex or rhizomes (beam is small quick etc., 2009) sprout young shoot, young shoot conduct
Explant carries out its in vitro breeding.The technology greatly accelerates the artificial propagation speed of Rhizoma Atractylodis Macrocephalae compared with traditional seedling-raising technique,
But its proliferation frequency is lower, and so that it is also easy to produce body cell hereditary variation by the organ of callus, to influence
The stay in grade of regeneration plant.
Summary of the invention
The object of the present invention is to provide a kind of Rhizoma Atractylodis Macrocephalae regenerating systems for directly breaking up adventitious bud using plumular axis and radicle, with solution
The certainly existing Rhizoma Atractylodis Macrocephalae artificial propagation time is long, and frequency is low, the poor problem of the quality stability of regeneration plant.
The purpose of the present invention is what is be achieved through the following technical solutions: a kind of directly to break up adventitious bud using plumular axis and radicle
Rhizoma Atractylodis Macrocephalae regenerating system, comprising the following steps:
(a) Atractylodes macrocephala seed is cleaned, disinfection, the Atractylodes macrocephala seed after disinfection is inoculated in MS culture medium, it is black in 25 ± 2 DEG C
Dark place, which is cultivated to seed, sprouts, and is transferred under light and continues to cultivate；
(b) culture to plant height 2-3cm when take out, cut radicle and plumular axis, be cut into the fritter that length is 0.3-0.5 cm, make
It for explant, is seeded on bud inducement cultivation base, is 25 ± 2 DEG C, light application time 14h/d, intensity of illumination 3000- in temperature
4000 lx Fiber differentiations make the green bud that 2-3cm long is grown on its differentiation to explant；The bud inducement cultivation base refers to
The culture that the sucrose of TDZ, 0-0.5mgNAA, 30g and the agar of 8g that 0.5-1.5 mg is added in the MS culture medium of 1L obtain
Base, the pH value of the bud inducement cultivation base are 5.8-6.2；
(c) it cuts the green bud to be forwarded in the culture bottle for filling root media, is 25 ± 2 DEG C, illumination 14 in temperature
H/d, intensity of illumination 3000-4000 lx continue to cultivate, and root induction obtains regeneration plant；The root media refers to 1/
The culture medium that the agar of the NAA of 0.5mg or the sucrose of IBA, 30g and 8g obtain, the culture of rootage are added in 2MS culture medium
The pH value of base is 5.8-6.2；
(d) it will take root and the good regeneration plant of growth conditions take out, transplanting is to by vermiculite: sand: the matter of Nutrition Soil
Amount is than continuing cultivation in the sterilization matrix for 1: 1: 1 composition to get Rhizoma Atractylodis Macrocephalae plant.
The cultivation temperature for continuing culture under light described in step (a) is 25 ± 2 DEG C, light application time 14h/d, intensity of illumination
For 3000-4000lx.
Disinfection described in step (a) refers to that it is 0.1% that the Atractylodes macrocephala seed after cleaning is used to mass concentration ratio before removal kind skin
Mercuric chloride impregnate 10 min of seed, aseptic water washing 3-5 times after impregnating 6 h in sterile water, peels off kind of a skin；Mass ratio is used again
Concentration be 1.5% NaClO sterilize 10 min, aseptic water washing 3-5 times.
Preferably, bud inducement cultivation base described in step (b), which refers to, is added to 1.5 mg's in the MS culture medium of 1L
The culture medium that the sucrose of TDZ, 0.2mgNAA, 30g and the agar of 8g obtain.
MS culture medium described in step (a) and step (b) is to be prepared by the following method: by MS a great number of elements, MS
Microelement, MS organic matter, 30g sucrose, 8g agar are dissolved in 1L distilled water, and adjusting pH value is 5.8-6.2,121 DEG C of sterilizing 15-
20min to obtain the final product.
1/2MS culture medium described in step (c) is to be prepared by the following method: 1/2MS a great number of elements, MS is micro
Element, MS organic matter, 30g sucrose, 8g agar are dissolved in 1L distilled water, and adjusting pH value is 5.8-6.2,121 DEG C of sterilizing 15-
20min to obtain the final product.
The present invention using Rhizoma Atractylodis Macrocephalae different explants carry out adventitious bud induction break up test, establish Rhizoma Atractylodis Macrocephalae it is efficient from
Body rapid propagation method obtains aseptic seedling using sterilization method, chooses plumular axis and radicle both specific explants, pass through spy
Fixed bud inducement cultivation base and root media carry out directly induction differentiation, establish the efficient rapid regeneration breeding system of Rhizoma Atractylodis Macrocephalae.
The genetic transformation and quickly in vitro breeding that regenerating system provided by the invention can be used for Rhizoma Atractylodis Macrocephalae, substantially increase the proliferation frequency of Rhizoma Atractylodis Macrocephalae
Rate shortens repoductive time, has saved production cost, improves production efficiency, is the large-scale production and quality of medicinal Rhizoma Atractylodis Macrocephalae
Improvement provides technical support；And due to the organ generation without callus, the genetic stability of plant is higher, this
To improve Rhizoma Atractylodis Macrocephalae quality using plant gene engineering technology, being provided using Vitro Quick Reproduction technology industrial seedling rearing and virus-free
Quality guarantee.
Detailed description of the invention
The growth conditions figure of radicle in the step of Fig. 1 is embodiment 1 (2).
The growth conditions figure of the step of Fig. 2 is embodiment 1 (2) mesocotyl.
The growth conditions figure of the step of Fig. 3 is embodiment 1 (3) regeneration plant.
The step of Fig. 4 is embodiment 1 (4) is by the growth conditions figure after Transplantation of Regenerated Plantlets to Nutrition Soil matrix.
Specific embodiment
Following example is for present invention be described in more detail, but embodiment does not do any type of limit to the present invention
It is fixed.Unless stated otherwise, the present invention uses reagent, method and apparatus is the art conventional reagents, method and apparatus.But
The invention is not limited in any way.
The preparation of MS culture medium: MS a great number of elements, MS microelement, MS organic matter, 30g sucrose, 8g agar are dissolved in 1L
In distilled water, adjusting pH value is 5.8-6.2, and 121 DEG C of sterilizing 15-20min to obtain the final product.
The preparation of 1/2 MS culture medium: by 1/2MS a great number of elements, MS microelement, MS organic matter, 30g sucrose, 8g agar
It is dissolved in 1L distilled water, adjusting pH value is 5.8-6.2, and 121 DEG C of sterilizing 15-20min to obtain the final product.
Embodiment 1
(1) acquisition of aseptic seedling.Big, the full Atractylodes macrocephala seed of grain is chosen, is rinsed well with flowing water, is with mass concentration ratio
After 75% alcohol impregnates 1 min, carry out disinfection.Use two-step method to sterilize: before removal kind of skin with mass concentration ratio for 0.1% liter
Mercury impregnates 10 min of seed, and aseptic water washing 3-5 times after impregnating 6 h in sterile water, peels off kind of a skin；Mass concentration ratio is used again
For 1.5% NaClO sterilize 10 min, aseptic water washing 3-5 times.Seed after disinfection is inoculated in and is not added with any growth regulating
In the MS culture medium of agent, cultivates at 25 ± 2 DEG C of dark to seed after sprouting, be transferred under light and continue to cultivate, cultivation temperature: 25
± 2 DEG C, light application time: 14h/d, intensity of illumination are as follows: 3500 lx.
(2) continue culture 1 week after Atractylodes macrocephala seed sprouting, taken out when plant height is 2-3cm, cut radicle and plumular axis, make
For explant, it is cut into the fritter of 0.3-0.5 cm, is seeded to the differentiation of evoking adventive bud on bud inducement cultivation base, cultivation temperature:
25 ± 2 DEG C, light application time: 14h/ days, intensity of illumination: 3000-4000lx；Culture 30 days, has differentiated green bud, has counted each of which
Explant Bud Differentiation number is 13.13 ± 2.75(plumular axis) and 7.95 ± 1.72(radicle), Differentiation ration of adventitious buds is respectively
91.18%(plumular axis) and 68.30%(radicle).Wherein bud inducement cultivation base is to be added to 1.5 mg in the MS culture medium of 1L
The agar mixing of the sucrose and 8g of TDZ, 0.2mgNAA, 30g, adjusting pH value is the training after 5.8,121 DEG C of 15 min of high pressure sterilization
Support base.
(3) the green bud of the 2-3 cm long of robust growth on explant is cut, is forwarded to the culture for filling root media
In bottle, vegetable material illumination cultivation condition is 25 ± 2 DEG C, 14 h/d of illumination, 3500 lx of intensity of illumination, and root induction must regenerate
Plant；Wherein root media refers to the fine jade of sucrose and 8g that NAA, 30g of 0.5mg are added in the 1/2MS culture medium of 1L
Rouge mixing, adjusting pH value is the culture medium after 5.8,121 DEG C of 15 min of high pressure sterilization.
(4) it will take root and the good regeneration plant of growth conditions, opening cultivate bottle cap, middle hardening 2-3 days between culture
Plant is carefully taken out afterwards, cleans culture medium, is transplanted to the vermiculite, sand and Nutrition Soil in mass ratio for 1: 1: 1 and is mixed sterilization matrix
In, it cultivates in small alms bowl, sprinkles profoundly water, film covers moisturizing, pays attention to divulging information and 25 DEG C or so of environment temperature is kept to be continued
It cultivates, obtains Rhizoma Atractylodis Macrocephalae plant.
Embodiment 2
Method only varies by bud inducement cultivation base in step (2) with embodiment 1: being added to 1.0 in the MS culture medium of 1L
The agar mixing of the sucrose and 8g of TDZ, 30g of mg, adjusting pH value is 5.8,121 DEG C of 15 min of high pressure sterilization, is prepared
Culture medium.
Embodiment 3
Method only varies by bud inducement cultivation base in step (2) with embodiment 1: being added in the MS culture medium of 1L
The agar mixing of the sucrose and 8g of TDZ, 0.2mgNAA, 30g of 0.5mg, adjusting pH value is 5.8,121 DEG C of high pressure sterilizations 15
Min, the culture medium being prepared.
Embodiment 4
Method only varies by bud inducement cultivation base in step (2) with embodiment 1: being added to 1.0 in the MS culture medium of 1L
The agar mixing of the sucrose and 8g of TDZ, 0.2mgNAA, 30g of mg, adjusting pH value are 5.8,121 DEG C of 15 min of high pressure sterilization, system
Culture medium made of standby.
Embodiment 5
Method only varies by bud inducement cultivation base in step (2) with embodiment 1: being added to 1.5 in the MS culture medium of 1L
The agar mixing of the sucrose and 8g of TDZ, 0.5mgNAA, 30g of mg, adjusting pH value are 5.8,121 DEG C of 15 min of high pressure sterilization, system
Culture medium made of standby.
Embodiment 6
Method only varies by bud inducement cultivation base in step (2) with embodiment 1: being added to 1.5 in the MS culture medium of 1L
The agar mixing of the sucrose and 8g of TDZ, 30g of mg, adjusting pH value is 5.8,121 DEG C of 15 min of high pressure sterilization, is prepared
Culture medium.
Comparative example 1
Method only varies by bud inducement cultivation base in step (2) with embodiment 1: being added in the MS culture medium of 1L
The mixing of the agar of the sucrose of 6-BA, 30g of 1.0mg and 8g, adjustings pH value are 5.8,121 DEG C of 15 min of high pressure sterilization, prepare and
At culture medium.
Comparative example 2
Method is changed to " cutting true leaf and cotyledon " with embodiment 1, by " radicle and plumular axis is cut " in step (2)；It is induced
The formula of culture medium is the same as embodiment 2.
Comparative example 3
Method is changed to " cutting true leaf and cotyledon " with embodiment 1, by " radicle and plumular axis bud is cut " in step (2)；It is lured
The formula of culture medium is led with comparative example 1.
Comparative example 4
Method only varies by bud inducement cultivation base in step (2) with embodiment 1: being added in the MS culture medium of 1L
2.0mg TDZ, 0.2mgNAA, 30g sucrose and 8g agar mixing, adjustings pH value be 5.8,121 DEG C of high pressure sterilizations 15
Min, the culture medium being prepared.
Comparative example 5
Method only varies by bud inducement cultivation base in step (2) with embodiment 1: being added in the MS culture medium of 1L
The agar mixing of the sucrose and 8g of TDZ, 1.2mgNAA, 30g of 1.5mg, adjusting pH value is 5.8,121 DEG C of high pressure sterilizations 15
Min, the culture medium being prepared.
Embodiment 7
Under identical breeding condition and time, inductivity/% of step (2) and each in Statistics Implementation example and comparative example
Explant is averaged bud number.Statistical result is shown in Tables 1 and 2.
1 embodiment and comparative example of table breaks up the statistics of adventitious bud and callus induction rate
* different lowercase letter indication differences are significant (P < 0.01)
2 comparative example 2 and 3 of table breaks up the statistics of adventitious bud and callus induction rate
Influence of the different bud inducement cultivation bases to Differentiation ration of adventitious buds be very it can be seen from the statistical data of Tables 1 and 2
Greatly, wherein adventitious buds differentiation number of the radicle in different culture mediums has extremely significant difference (P < 0.01)；It is induced in bud
6-BA is added in culture medium to compare, either plumular axis explant or radicle explant, add bud in the bud inducement cultivation base of TDZ
Inductivity is higher, it is more to generate adventitious bud number；Show that the effect of TDZ induction Rhizoma Atractylodis Macrocephalae explant generation adventitious bud is more preferable, but TDZ
To true leaf and cotyledon, but effect is excessively poor, it is seen that TDZ only has suitability for differentiation plumular axis and radicle.In addition, we are from reality
Apply example and comparative example statistics data it was found that, radicle and Epicotyl Explants of Phaseolus Differentiation ration of adventitious buds and adventitious bud number by
(P < 0.01) is significantly affected to different TDZ concentration；When two kinds of explant Differentiation ration of adventitious buds are 1.5 in TDZ concentration
mg·L-1When highest, and when TDZ concentration be 2.0mgL-1When but show it is very unsatisfactory；Under identical TDZ concentration conditions, upper embryo
The Differentiation ration of adventitious buds of axis explant and each explant average division bud number are above radicle explant；And it can see
When NAA concentration is higher, the differentiation of Rhizoma Atractylodis Macrocephalae radicle and Epicotyl Explants of Phaseolus adventitious bud can be inhibited.
From the aspect of callus induction rate, radicle and the induction in 1 step of embodiment (2) of plumular axis explant are indefinite
Bud, when culture was to 4 weeks, radicle and plumular axis explant have directly differentiated adventitious bud, no callus production from explant
Raw, the growth conditions of radicle are as shown in Figure 1, the growth conditions of plumular axis are as shown in Figure 2；Green bud (adventitious bud) is cut to turn to plant to life
Root induction in root culture medium cultivates 20 days or so, root restriction occurs in the part of the base in contact culture medium of bud, 25 days start
It takes root, for root long up to 2-3 cm, growth conditions are as shown in Figure 3 after cultivating 40 d；To Rhizoma Atractylodis Macrocephalae regeneration plant root long to 2-3 cm
When left and right, after hardening 2-3 days, transplant into soil matrix, growth conditions are as shown in Figure 4.
In addition, " NAA " in embodiment 1 in the root media of (4) step can be used " IBA " replace, actual effect with
Embodiment 1 is suitable.
Embodiment 8
Comparative example 6 is " the Rhizoma Atractylodis Macrocephalae stem tip tissue culture that Tao Yuan scape in 2010 etc. is delivered on " medical biotechnology " periodical
And fast breeding technique optimizing research ".
Comparative example 7 be Chen Juan in 2006 etc. delivered on " Chinese agronomy notification " periodical " Rhizoma Atractylodis Macrocephalae leaf regeneration plant and
The fast numerous research of Multiple Buds ".
For method provided by the invention compared with documents 6 and documents 7, experiment effect is shown in Table 3.
The comparing result of table 3 present invention and documents reproductive frequency and repoductive time
It can be seen that the propagation method of the more existing some explants of propagation method of the invention from the data of table 3, it is numerous
Grow frequency faster, repoductive time it is shorter, the quality of no callus, product is more stable.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by the embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (2)
1. a kind of Rhizoma Atractylodis Macrocephalae regenerating system for directly breaking up adventitious bud using radicle, which comprises the following steps:
(a) Atractylodes macrocephala seed is cleaned, sterilizes, the Atractylodes macrocephala seed after disinfection is inoculated in MS culture medium, at 25 ± 2 DEG C of dark
Culture to seed is sprouted, and is transferred under light and is continued to cultivate；
(b) culture cuts radicle, is cut into the fritter of a length of 0.3-0.5 cm as explant, connects to plant height to take out when 2-3cm
Kind on bud inducement cultivation base, temperature is 25 ± 2 DEG C, light application time 14h/d, intensity of illumination are 3000-4000lx induction
Culture makes the green bud that 2-3cm long is grown on its differentiation to explant；The bud inducement cultivation base refers to the MS culture medium in 1L
In be added to the sucrose of TDZ, 0-0.5mgNAA, 30g of 0.5-1.5 mg and culture medium that the agar of 8g obtains；
(c) the green bud is cut, is forwarded in the culture bottle for filling root media, is 25 ± 2 DEG C, 14 h/ of illumination in temperature
D, intensity of illumination 3000-4000 lx continues to cultivate, and root induction obtains regeneration plant；The root media refers to the 1/ of 1L
The culture medium that the agar of the NAA of 0.5 mg or the sucrose of IBA, 30g and 8g obtain is added in 2MS culture medium；
(d) it will take root and the good regeneration plant of growth conditions take out, transplanting is to by vermiculite: sand: the mass ratio of Nutrition Soil
Continue cultivation in sterilization matrix for 1: 1: 1 composition to get Rhizoma Atractylodis Macrocephalae plant.
2. the Rhizoma Atractylodis Macrocephalae regenerating system according to claim 1 for directly breaking up adventitious bud using radicle, which is characterized in that step
(a) cultivation temperature for continuing culture under the light described in is 25 ± 2 DEG C, light application time 14h/d, intensity of illumination 3000-
4000lx。
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| CN110250009B (en) * | 2019-07-25 | 2022-07-26 | 恩施土家族苗族自治州农业科学院(恩施土家族苗族自治州硒应用技术与产品开发研究院) | Method for expanding propagation of bighead atractylodes rhizome seedling by utilizing cluster buds of bighead atractylodes rhizome and application of method |
| CN113383707B (en) * | 2021-06-24 | 2022-04-19 | 中国科学院合肥物质科学研究院 | Method for establishing high-efficiency in-vitro regeneration system of Qishu mature embryos |
| CN113475395B (en) * | 2021-07-06 | 2022-05-03 | 中国科学院合肥物质科学研究院 | Method for direct regeneration and in-vitro rooting of hypocotyls in Qishu |
| CN115843682B (en) * | 2022-10-28 | 2024-02-27 | 华中农业大学 | Method for inducing and regenerating tulip hypocotyl callus |
| CN117016394B (en) * | 2023-09-19 | 2024-04-26 | 中国中医科学院中药研究所 | Test-tube plantlet of bighead atractylodes rhizome, culture method thereof and method for culturing seedlings to be transplanted of bighead atractylodes rhizome |
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