CN116622767A - Rapid and efficient culture method for induction and proliferation of hairy roots of mare - Google Patents

Rapid and efficient culture method for induction and proliferation of hairy roots of mare Download PDF

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CN116622767A
CN116622767A CN202310718843.4A CN202310718843A CN116622767A CN 116622767 A CN116622767 A CN 116622767A CN 202310718843 A CN202310718843 A CN 202310718843A CN 116622767 A CN116622767 A CN 116622767A
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陈红锋
王文涛
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South China Botanical Garden of CAS
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Abstract

The invention discloses a rapid and efficient culture method for inducing and proliferating hairy roots of mare. The method comprises the steps of preparing an explant of the marshy embryo, activating agrobacterium rhizogenes, preparing an aggressive solution, impregnating the agrobacterium rhizogenes, co-culturing, and carrying out secondary and expansion culture. The method for inducing the mare embryo is simple to operate, high in efficiency and high in induction rate, provides a new way for converting the hairy roots of the mare embryo into secondary metabolites such as triterpenoid saponins of the mare embryo by utilizing agrobacterium rhizogenes, and can be used for producing alternative resources of pharmaceutical components of the industrial mare embryo on a large scale. Meanwhile, the method has important significance for the subsequent establishment of a genetic transformation system of the mare's fetus.

Description

Rapid and efficient culture method for induction and proliferation of hairy roots of mare
Technical field:
the invention belongs to the technical field of biology, and particularly relates to a rapid and efficient method for inducing and proliferating hairy roots of a mare embryo (Ardisia kteniophylla A.DC).
The background technology is as follows:
the Caesalpinia cristata (Ardisia kteniophylla A.DC) is a Primachiaceae plant, evergreen shrubs, mainly distributed in Guangxi, guangdong, jiangxi, fujian and other places in China, and also in North Vietnam, indonesia, malaysia and Thailand, often grown in woods and in the shade and wet places in the stream. As the traditional ethnic medicine commonly used in China, the Caima embryo is called as Ling nan Yaowang, the root or rhizome thereof is the traumatic medicine in the Ling nan minority ethnic area of China, and the Chinese medicine has the expression of 'two feet are not separated from each other and not separated from the Caima embryo'. The earliest words of medicinal efficacy of Zaoma embryo are recorded in Qing Qianlong annual "Lu Chuan Ben Cao" and "Ben Cao gang mu Shi Yi". At present, the trojan horse embryo is recorded in 100 kinds of Zhuang medicine commonly used, and plays an important role in the theory of traditional Yao medicine, seventy-two wind. In addition, the vats are also used for medicines, have the effects of detoxification, removing necrotic tissue, promoting granulation and activating blood circulation, and are also common health-care raw materials for traditional medicated bath and foot bath in minority nationality areas.
Pharmacological studies now show that the main chemical components of the marmalade fetus comprise triterpene saponins, coumarone extracts, benzoquinone, phenols, sterols, volatile oils and the like, wherein the triterpene saponins are main pharmacodynamic active components, and various triterpene saponins have remarkable anti-tumor effects. The structural types of the triterpene sapogenins in the marmalade found so far are mainly pentacyclic triterpene oleanane derivatives, and the triterpene sapogenins mainly based on the Sickalamin A are modified by hydroxylation, glycosylation and the like to form various and rich triterpene saponins of the marmalade. In addition, the maroon tire leaves in the seedling stage are purple red, and the maroon tire has the main side veins with silvery white bright lines, so that the maroon tire is a ground cover plant with great ornamental value. Is a national medicinal plant with great research value.
In recent years, with the increasing development and utilization of natural plant medicines in the world, the market of traditional Chinese medicine health products and food therapy rapidly develops, the demand of the marshy tire is also rapidly increased, so that wild plants are greatly excavated, wild resources are greatly reduced, the trace of the marshy tire is difficult to see in the wild, and raw materials are seriously supplied and not required. In 2018, the mare's embryo was also listed in the "major protection wild plant directory in Guangdong province" (first lot). At present, large-scale and standardized planting of the mare embryo is not available. Moreover, the marshy embryo used as the shrub has long growth cycle, difficult formation yield, long time consumption for breeding or raising seedlings by the conventional method, low development speed and increased production cost due to small propagation coefficient and large seed consumption. And moreover, the yield is reduced due to serious damage of diseases and insect pests, and the yield and quality of medicinal materials are seriously influenced. Therefore, the horse-riding embryo is required to be subjected to intensive protection research. Meanwhile, the compound in the marmalade has complex components, low content, unstable property, easy decomposition and the like, so that important metabolites such as triterpene saponin and bergenin of the marmalade are difficult to obtain. Wild trojan embryo resources are important protection plants in the field, and are difficult to be used for extracting related medicinal components on a large scale, and a tissue culture technology cannot establish a high-efficiency trojan embryo bioreactor meeting production requirements. Hairy roots (Hair roots) formed by infecting plants with agrobacterium rhizogenes are malignant proliferation organs, and compared with conventional cell culture, the hairy roots have the characteristics of hormone autotrophy, rapid growth, short period and the like, can be subjected to mass proliferation culture under laboratory conditions, originate from single cells, have stable genetic characters and have the characteristic secondary metabolic pathway of parent plants. The growth of the plant is rapid, the hereditary character is stable, the development and the utilization of natural medicines are facilitated, the plant is an important raw material for producing secondary metabolites of medicinal plants, and at present, a hairy root induction and culture system is established for various medicinal plants such as ginseng, astragalus root, belladonna, red sage root, licorice root, cinnabar root and the like, and the hairy roots of hundreds of medicinal plants are successfully induced. By combining genetic transformation and taking hairy roots as carriers, the effective medicinal ingredients with higher yield can be obtained after the optimized conditions are obtained through screening. For some wild medicinal plant resources with limited extraction, the culture and development of hairy roots are led by factors, and become a new technology combining genetic engineering and cell engineering. At present, no report on success of transforming the mare hair roots is yet found at home and abroad.
The method establishes a technology system for inducing the hairy roots of the marmalade, utilizes the root system of the marmalade to possess a parent characteristic secondary metabolism way, can be used as a bioreactor for the enlarged production of important secondary metabolites of the marmalade, and breaks the limitation of plant raw materials for further excavating the high-value metabolites in the marmalade of the valuable ethnic medicinal plant.
The invention comprises the following steps:
in view of the above, the present invention provides a rapid and efficient method for culturing the hairy roots and proliferation of the mare embryo by using agrobacterium rhizogenes to induce the mare embryo to generate hairy roots and proliferation. Provides a new way for producing secondary metabolites such as the triterpene saponin of the mare embryo and bergenin by utilizing agrobacterium-mediated transformation of the hairy roots of the mare embryo, and has important significance for establishing a genetic transformation system of the mare embryo.
The invention provides a rapid and efficient method for culturing the induction and proliferation of hairy roots of mare, which comprises the following steps:
step one, preparation of an explant of a mare's fetus
Soaking mature seeds of the marmalade embryo in water, selecting full seeds, placing the full seeds in a container, flushing the seeds with running water, sterilizing the seeds, and culturing the sterilized seeds on a 1/2MS solid culture medium to obtain aseptic seedlings of the marmalade embryo;
step two, the activation of agrobacterium rhizogenes and the preparation of an intrusion dye solution
Inoculating agrobacterium rhizogenes into TY liquid culture medium containing kanamycin for culturing until OD600 = 0.6-0.9, centrifuging bacterial liquid to remove supernatant, adding 1/2MS liquid culture medium with equal volume, and re-suspending to obtain dyeing liquid;
step three, agrobacterium rhizogenes dip dyeing and co-cultivation
(1) Pre-culture of explants
Selecting aseptic seedlings with good growth vigor obtained in the first step, cutting leaves or stem sections into sections by using an aseptic scalpel on an ultra-clean workbench, inoculating the treated explants on a 1/2MS solid culture medium for culture, and completing a pre-culture stage of the explants;
(2) Co-culture of bacterial liquid
Taking out the pre-cultured explant, placing the explant in the dyeing liquid activated in the second step, adding acetosyringone, placing the explant in a shaking table for shake culture, taking out the impregnated explant, sucking the residual bacterial liquid on the surface by using sterile filter paper, inoculating the bacterial liquid to a 1/2MS solid culture medium, and placing the solid culture medium in a climatic chamber for culturing for 2-3 d;
(3) Degerming culture
Transferring the explant subjected to co-culture to a 1/2MS solid culture medium containing 300-500 mg/L of cefotaxime sodium under aseptic conditions, culturing in a dark manner in an artificial climatic chamber, transferring every 7-10d, and reducing the concentration of cefotaxime sodium until the explant is cultured in a culture medium without cefotaxime sodium and no colony is generated;
step four, subculturing and expanding culture
Cutting and culturing the root strain of the induced hairy root in a sterile environment, placing in a 1/2MS solid culture medium for a period of time, selecting hairy root with strong root system, more branches, rapid growth and no geotropic property, transferring into a B5 liquid culture medium for expansion culture, shake culturing in the dark, and growing the hairy root in large quantity.
Preferably, the explant is a stem segment; inoculating the treated explant on a 1/2MS solid culture medium for culture, wherein the pre-culture time is 2 days; inoculating agrobacterium rhizogenes into TY liquid culture medium containing kanamycin, and culturing until the concentration OD600 of the bacteria liquid for dip dyeing is 0.7, wherein OD 600=0.7; the step (2) is placed in a shaking table for shake culture, the culture time is 30min, and the culture is inoculated onto a 1/2MS solid culture medium and placed in a climatic chamber for 2d; the B5 liquid culture medium is a vitamin-containing B5 liquid culture medium, wherein the vitamin-containing B5 liquid culture medium is prepared from 25g/L of B5+sucrose+vitamin, the pH value is 5.8-6.0, the vitamins are inositol 100mg/L, hydrochloric acid 1mg/L, pyridoxine hydrochloride 1mg/L and thiamine hydrochloride 10mg/L, and the culture condition is that shake culture is performed in the dark at 25 ℃ and 100rpm.
Preferably, the sterilization in the first step is performed after the medical alcohol with the volume fraction of 75% is sterilized for 20-30 s; and then placing the seeds in a sodium hypochlorite solution with the mass fraction of 10% for disinfection for 3-5 min, and culturing the sterilized seeds on a 1/2MS solid culture medium under the culture conditions that the temperature is 25+/-2 ℃ and the photoperiod is 10-16 h.
Preferably, the cutting of the leaf blade or the stem segment in the first step is a segment with the leaf blade size of 1cm multiplied by 1cm, and the stem segment is beveled to a length of 0.5-1 cm.
Preferably, step two is characterized in that Agrobacterium rhizogenes is inoculated into TY liquid medium containing kanamycin and cultured until the OD600 = 0.6-0.9, and Agrobacterium rhizogenes ArA4 is inoculated into TY liquid medium containing 50mg/l kanamycin and cultured with shaking at 28 ℃ and 160rpm for 24 hours until the OD600 = 0.7.
Preferably, the step two of taking the bacterial liquid and centrifuging to remove the supernatant is to centrifuge at 6000rpm for 10min, and removing the supernatant.
Preferably, the formula of the TY liquid medium is as follows: tryptone (Tryptone) 5 g+yeast extract (Yeast extract) 3g, pH 7.2.
Preferably, the 1/2MS solid culture medium in the first to third steps is 1/2 MS+30 g/l of sucrose+6 g/l of agar, the pH is 5.8-6.0, and the 1/2MS liquid culture medium is 1/2MS solid culture medium lacking agar.
Preferably, the acetosyringone is added in the third step, 100 mu mol/L acetosyringone is added after shaking culture in a shaking table, and the mixture is placed at 28 ℃ and shaking culture is carried out for 30min in a shaking table at 90-100 rpm. Further preferably 100rpm.
Preferably, the transferring is performed every 7-10d, which is performed every 10d, and the concentration of cefotaxime sodium Cef in the sterilization culture medium is 100 mg.L -1 Decreasing in sequence.
According to the technical scheme, the rapid and efficient method for cultivating the hairy roots of the mare has the outstanding advantages that:
1. the method for inducing the hairy roots of the mare is adopted, the hairy roots have high induction rate, are not easy to be infected by bacteria, reach more than 50 percent, and simultaneously the required materials are easy to obtain, the operation is simple and easy to implement, and the cost is low.
2. The hairy root of the mare is induced by the method, the obtained hairy root is easy to grow in a culture medium without any exogenous hormone, and the hairy root has the advantages of high growth speed, good genetic stability, strong and stable synthesis capability, and is an ideal carrier for producing secondary metabolites.
3. The hairy roots obtained by the invention are easy to carry out mass propagation culture in a B5 (vitamin-containing) liquid culture medium, and can be produced in a large scale to be used as a substitute resource of industrial mare embryo for extracting secondary metabolites.
The invention successfully induces the mare embryo to generate hairy roots by utilizing agrobacterium rhizogenes, and has stronger potential of generating secondary metabolites. The hairy roots can generate secondary metabolites in the culture medium, can induce the secondary metabolites to be continuously generated by artificially changing the growth environment, are secreted in the culture medium, are convenient to collect and apply, are an effective way for replacing artificial planting and extracting the secondary metabolites of the mare embryo, and also lay a good foundation for the construction of a genetic transformation system of the species and further application of the genetic transformation system.
The method for inducing the mare embryo is simple to operate, high in efficiency and high in induction rate, provides a new way for converting the hairy roots of the mare embryo into secondary metabolites such as triterpenoid saponins of the mare embryo by utilizing agrobacterium rhizogenes, and can be used for producing alternative resources of pharmaceutical components of the industrial mare embryo on a large scale. Meanwhile, the method has important significance for the subsequent establishment of a genetic transformation system of the mare's fetus.
Description of the drawings:
FIG. 1 is a picture of co-cultivation according to the invention.
FIG. 2A picture of the co-cultured Agrobacterium rhizogenes ArA4 dip-stained roots of the invention, (A) induced leaf blades to produce hairy roots; (B) inducing the stem segments to produce hairy roots.
FIG. 3 proliferation-cultured Caesalpinia spicata roots in 1/2MS solid medium after the sterilization culture according to the present invention.
FIG. 4 expanded culture of the liquid culture medium of the hairy roots of the mare according to the invention.
FIG. 5 shows the result of PCR detection of the lanugo root roller B gene of the present invention.
The specific embodiment is as follows:
the following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The present invention will be further specifically illustrated by the following examples, which are not to be construed as limiting the invention, but rather as falling within the scope of the present invention, for some non-essential modifications and adaptations of the invention that are apparent to those skilled in the art based on the foregoing disclosure.
Example 1:
a high-efficiency tissue culture method for inducing and proliferating the hairy roots of a mare comprises the following steps:
step one, preparation of an explant of a mare's fetus
Soaking mature seeds of the mare embryo in water for 16h, selecting full seeds, placing the seeds in a container, flushing the seeds with running water, and sterilizing the seeds with 75% medical alcohol for 20s on an ultra-clean workbench; then placing the seeds in 10% sodium hypochlorite solution for sterilization for 5min, shaking for several times with sterile water, and then sucking the water with sterilized filter paper. Transferring the sterilized seeds to a 1/2MS solid culture medium, and culturing for 60 days in a climatic chamber with a photoperiod of 16h at 25+/-2 ℃ to obtain the aseptic seedlings of the marshy.
Step two, the activation of agrobacterium rhizogenes and the preparation of an intrusion dye solution
The Agrobacterium rhizogenes ArA4 stored at low temperature was removed, and 10. Mu.L of the activated bacterial liquid was extracted into 50mL TY liquid medium (containing 50mg/L kanamycin) under aseptic conditions on an ultra-clean bench, and shake-cultured at 28℃and 160rpm for 24 hours until the OD 600=0.7 or so. Centrifuging the bacterial liquid at 6000rpm for 10min, removing supernatant, adding an equal volume of 1/2MS liquid culture medium, and re-suspending to obtain the dyeing liquid. The formula of the TY liquid culture medium is as follows: tryptone (Tryptone) 5 g+3 g of Yeast extract (Yeast extract), pH 7.2, kanamycin 50mg/l was added.
Step three, agrobacterium rhizogenes dip dyeing and co-cultivation
(1) Pre-culture of explants
Selecting aseptic seedlings with good growth vigor obtained in the first step, and shearing the leaves to 1cm by using an aseptic scalpel on an ultra-clean workbench 2 And cutting the stem into small sections with the length of 0.5-1 cm. Inoculating the treated explant on a 1/2MS solid culture medium for 2d, and completing the pre-culture stage of the explant;
(2) Co-culture of bacterial liquid
Taking out the pre-cultured explant, placing the explant in the agrobacterium rhizogenes bacteria liquid (infection liquid) activated in the step two, adding 100 mu mol/L acetosyringone, placing the mixture at 28 ℃ and shaking culture for 30min at 100rpm (figure 1), taking out the impregnated explant, sucking the residual bacteria liquid on the surface by using sterile filter paper, inoculating the bacteria liquid onto a 1/2MS solid culture medium, and placing the solid culture medium in a climatic chamber for culturing for 2-3 d.
(3) Degerming culture
Transferring the explant subjected to co-culture into 1/2MS solid medium containing 300mg/L cefotaxime sodium under aseptic condition, culturing in artificial climatic chamber (25+ -1deg.C) in dark, transferring every 10d, and adjusting the concentration of cefotaxime sodium to 100 mg.L -1 Sequentially decreasing until the culture medium is cultured in the culture medium without the cefotaxime sodium and no colony is generated.
Step four, subculturing and expanding culture
Root strains which are induced to grow to about 5cm hairy roots are cut off by scissors in a sterile environment for culture, and are independently placed in a new 1/2MS solid culture medium for culture for a period of time, hairy roots (figure 3) which are thick in root system, more in branches, rapid in growth and free of geotropism are selected, transferred into a B5 (vitamin-containing) liquid culture medium for expansion culture, shake-cultured in the dark at 25 ℃ and at 100rpm, and the hairy roots grow in a large quantity (figure 4). The B5 (vitamin-containing) liquid culture medium is B5 (vitamin-containing) +sucrose 25g/l, and the pH value is 5.8-6.0, and the specific table is shown in the attached table.
The 1/2MS solid culture medium is 1/2 MS+30 g/l sucrose+6 g/l agar, and the liquid culture medium with pH of 5.8-6.0,1/2MS is agar-removed.
The inventor obtains the optimal preparation process parameters of the horse-walking fetal hair root through creative tests: the explant is a stem segment; the preculture time is 2 days; the concentration OD600 of the bacteria liquid for dip dyeing is 0.7; the bacteria liquid dip-dyeing time is 30min; the co-cultivation time is 2 days; the most suitable expansion medium is B5 (vitamin-containing) liquid medium.
The specific optimization test implementation process is as follows:
example 2: effect of explant type on Caulophyllum reesei Induction
The agrobacteria Ara4, C58C1, MSU440 and K599 stored at low temperature were removed, 10. Mu.L of the activated bacterial liquid was aspirated in 50mL TY liquid medium (containing 50mg/L kanamycin) under aseptic conditions on an ultra clean bench, and shake-cultured at 28℃and 160rpm for 24 hours until OD600 = 0.7 or so. Centrifuging the bacterial liquid at 6000rpm for 10min, removing supernatant, adding an equal volume of 1/2MS liquid culture medium, and re-suspending to obtain the dyeing liquid. The formula of the TY liquid culture medium is as follows: tryptone (Tryptone) 5 g+3 g of Yeast extract (Yeast extract), pH 7.2, kanamycin 50mg/l was added.
Pre-culture of explants
Selecting sterile seedling with good growth obtained in step one of example 1, cutting leaf to 1cm with sterile scalpel on ultra clean bench 2 And cutting the stem into small sections with the length of 0.5-1 cm. Inoculating the treated explant on a 1/2MS solid culture medium for 2d, and completing the pre-culture stage of the explant;
co-culture of bacterial liquid
Taking out the pre-cultured explant, placing the explant in an activated agrobacterium rhizogenes bacterial solution, adding 100 mu mol/L acetosyringone, placing the mixture in a shaking table at 28 ℃ and 100rpm for shake culture for 30min, taking out the impregnated explant, sucking the residual bacterial solution on the surface by using sterile filter paper, inoculating the bacterial solution onto a 1/2MS solid culture medium, and placing the solid culture medium in a climatic chamber for culturing for 2-3 d. The following procedure is the same as in example 1.
In this example, the stems and leaves of the aseptic seedlings of the mare's embryo are respectively infected with Agrobacterium rhizogenes ArA4, C58C1, MSU440 and K599, and the influence of different types of explants on the induction rate of hairy roots is examined. The results are shown in table 1 and fig. 2:
TABLE 1 Effect of explants on hairy root induction rate
As shown in Table 1, arA4 has the highest induction rate of the hairy root of the stem segment, reaching 46.91% + -0.04%, while the induction rate of the leaf is slightly lower, being 37.07% + -0.09%; whereas the induction rate of K599 on the mare embryo explant was lowest, the induction rates on the stem and leaf were 31.65% + -4.43% and 26.85% + -5.45%, respectively. Therefore, agrobacterium rhizogenes ArA4 is the most suitable root inducing bacteria for the marmalade, and the stem segments of the marmalade are the best explant material. Inductivity= (number of root explants/total number of explants) ×100%.
Example 3: effect of Pre-incubation time on the induction of hairy roots in mare
Pre-culture of explants
The sterile seedling with good growth condition obtained in the step one of the example 1 is selected, and the stem is beveled into small sections with the length of 0.5-1 cm by using a sterile scalpel on an ultra-clean workbench. The treated explants were inoculated on 1/2MS solid medium for 0-3d to complete the pre-culture stage of the explants, followed by co-culture of the bacterial liquid, sterilization culture and subculture and expansion culture as in example 1. The results are shown in Table 2.
TABLE 2 Effect of Pre-incubation time on the induction of hairy roots by mare's embryo
As shown in Table 2, experiments were carried out using the stem segments of the mare embryo as explants, and when the preculture time was 2 days, a higher induction rooting rate was obtained and the sterilization difficulty was easy.
Example 4 Effect of Agrobacterium rhizogenes concentration on the induction of hairy roots in horse
This example is the same as example 1, except that the Agrobacterium rhizogenes ArA4 strain solution concentration is different, as shown in Table 3.
TABLE 3 Effect of bacterial liquid concentration on the induction of hairy roots in mare
As shown in Table 3, the induction rate of hairy roots is obviously different when the stem segments of the mare embryo are immersed and dyed under the condition of different bacterial liquid concentrations. When the concentration of bacterial liquid is lower, the induction rate of hairy roots is low, and the browning rate and the bacterial infection rate are relatively low. With the increase of the concentration of the bacterial liquid, the induction rate of hairy roots becomes high, but the browning rate and the bacterial infection rate are also relatively improved. In summary, when the OD600 value is 0.7, the induction rate of the transfer of the mare hair is highest and reaches 56.67%, and the browning rate and the bacterial infection rate are also lower. Therefore, the concentration of the bacterial liquid OD600 = 0.7 is suitable for dandelion hairy root induction experiments.
Example 5: influence of infection time on the induction rate of hairy roots of mare
This example is the same as example 1, except that the infection time of Agrobacterium rhizogenes ArA4 strain liquid is different, and the details are shown in Table 4.
TABLE 4 Effect of the padding time on the induction of hairy roots in mare
The proper infection time can enable agrobacterium rhizogenes to fully contact with the explant, and the explant is effectively infected under the action of acetosyringone. However, when the time is insufficient, the induction rate is low because the explant is not sufficiently contacted with Agrobacterium to perform effective infection. As shown in Table 4, the induction rate of the hairy roots of mare was highest and reached 46.67% at a time of 30min, and the bacterial contamination rate and browning rate were low. When the infection time is more than 30min, both the bacterial infection rate and the browning rate are increased, so that the induction rate is also reduced. Therefore, when the infection time is 30min, the method is suitable for rooting of the mare embryo explants.
Example 6: effect of Co-culture time on the rate of induction of hairy roots in Caesalpinia
This example is the same as example 1, except that the co-cultivation time of Agrobacterium rhizogenes ArA4 strain solution is different, as shown in Table 5
TABLE 5 Effect of Co-culture time on the induction of hairy roots in mare
Co-cultivation is the process by which the explant is contacted with Agrobacterium rhizogenes in a solid medium to integrate the T-DNA sequence of the Agrobacterium into the plant's nuclear genome. The increase of the co-cultivation time, the contact of the explant with the agrobacterium rhizogenes and the prolonged infection time are beneficial to the improvement of the induction rate. However, too long a co-cultivation time can lead to overgrowth of Agrobacterium rhizogenes, leading to increased infection of the explants and browning and ultimately death. As shown in Table 5, it was found that the induction rate of hairy roots of mare reached the highest level at 2 days of co-culture time, but the infection rate of explants increased significantly and the induction rate also tended to decrease as the co-culture time increased to 3 days, as compared with the control group without co-culture. Therefore, the co-culture time is 2 days, and the method is suitable for rooting of the mare embryo explants.
Example 7: influence of cefotaxime sodium concentration in degerming medium on induction rate of hairy roots of Caesalpinia crista
This example is the same as example 1 except that the initial concentration of cefotaxime sodium in the sterile medium is different, as shown in Table 6
TABLE 6 Effect of cefotaxime sodium concentration on Caulophyllum vaginalis root Induction
As shown in table 6, as the concentration of cefotaxime sodium increases, the bacterial contamination rate is continuously reduced, and the induction rate of hairy roots tends to be increased and then decreased; at 300mg/L, the induction rate is highest and reaches 56.67%, and the bacterial contamination rate is low and is only 1.67%. Meanwhile, we also find that when the cefotaxime sodium is not added, the induction rate of the mare embryo explant is also higher, 46.67%, and the bacterial infection rate is also better controlled. The method mainly comprises the steps of centrifugally precipitating strains, discarding a supernatant TY culture medium, and re-suspending by using an equal volume of 1/2MS liquid culture medium in the dip-dyeing process, so that the growth of agrobacterium in the induction process can be well controlled, and the bacteria can be effectively inhibited and hairy roots can be rapidly induced. Therefore, agrobacterium rhizogenes is subjected to centrifugal precipitation and re-suspension before dip-dyeing, and meanwhile, the concentration of cefotaxime sodium is controlled to be 300mg/L, so that the rooting of the runny embryo explants can be most effectively induced, and the growth of colonies can be inhibited.
Example 8: effect of liquid expansion Medium on growth of Ma Taifa roots
This example is the same as example 1 except that the liquid expansion medium is different, as shown in Table 7
TABLE 7 Effect of different media on Liriophyllum spicatum liquid culture
As shown in Table 7, as the cultivation time increases, ma Taifa roots all show a continuous increasing trend in 6 kinds of culture media, and the B5 (vitamin added) culture media are most beneficial to the growth of hairy roots, the growth speed of the obtained hairy roots is fastest, the biomass accumulation is maximum, and the fresh weight reaches 23.57+/-0.83 g at 60 days. The second was N6 medium, 1/2MS medium, with the worst growth in MS medium. Thus, the optimal liquid medium for growth of the hairy roots of mare is vitamin-enriched B5 medium.
Example 9 identification of the hairy root of Caesalpinia
The root-causing gene in the T-DNA region of Ri plasmid in Agrobacterium rhizogenes is closely related to hairy root formation, and in order to confirm whether the induced root is hairy root, it is necessary to detect the molecular level of hairy root and determine whether the induced hairy root is inserted with rol gene.
We identified the rolB gene of the T-DNA region on Ri plasmids in ArA4, C58C1, MSU440 and K599, and the DNA of the young horse embryo and test tube seedling without Agrobacterium rhizogenes infection was used as a negative control, and the rolB gene amplification was performed on the extracted Ma Taifa pieces of DNA. As shown in FIG. 5, all of the hairy roots induced by ArA4, C58C1, MSU440 and K599 amplified the target fragment of the rolB gene, while the negative control group did not show that the 4 Agrobacterium rhizogenes rolB genes were integrated into the genome of the mare embryo, and the induced roots were hairy roots of the mare embryo.
In summary, the present invention provides a method for culturing the induction and proliferation of the hairy roots of mare, and the inventor obtains the best experimental conditions through creative experiments: the stem segments of the marmalade are used as explants, the preculture is carried out for 2 days, the concentration OD600 of the ArA4 bacterial liquid of the agrobacterium rhizogenes is 0.7, the infection time is 30min, the co-culture is carried out for 2 days, the concentration of cefotaxime sodium in a culture medium is 300mg/L, and the liquid expansion culture medium is B5 (added with vitamins).
List of Ma Taifa liquid culture medium formulas
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. A rapid and efficient culture method for inducing and proliferating hairy roots of mare is characterized by comprising the following steps:
step one, preparation of an explant of a mare's fetus
Soaking mature seeds of the marmalade embryo in water, selecting full seeds, placing the full seeds in a container, flushing the seeds with running water, sterilizing the seeds, and culturing the sterilized seeds on a 1/2MS solid culture medium to obtain aseptic seedlings of the marmalade embryo;
step two, the activation of agrobacterium rhizogenes and the preparation of an intrusion dye solution
Inoculating agrobacterium rhizogenes into TY liquid culture medium containing kanamycin for culturing until OD600 = 0.6-0.9, centrifuging bacterial liquid to remove supernatant, adding 1/2MS liquid culture medium with equal volume, and re-suspending to obtain dyeing liquid;
step three, agrobacterium rhizogenes dip dyeing and co-cultivation
(1) Pre-culture of explants
Selecting aseptic seedlings with good growth vigor obtained in the first step, cutting leaves or stem sections into sections by using an aseptic scalpel on an ultra-clean workbench, inoculating the treated explants on a 1/2MS solid culture medium for culture, and completing a pre-culture stage of the explants;
(2) Co-culture of bacterial liquid
Taking out the pre-cultured explant, placing the explant in the dyeing liquid activated in the second step, adding acetosyringone, placing the explant in a shaking table for shake culture, taking out the impregnated explant, sucking the residual bacterial liquid on the surface by using sterile filter paper, inoculating the bacterial liquid to a 1/2MS solid culture medium, and placing the solid culture medium in a climatic chamber for culturing for 2-3 d;
(3) Degerming culture
Transferring the explant subjected to co-culture to a 1/2MS solid culture medium containing 300-500 mg/L of cefotaxime sodium under aseptic conditions, culturing in a dark manner in an artificial climatic chamber, transferring every 7-10d, and reducing the concentration of cefotaxime sodium until the explant is cultured in a culture medium without cefotaxime sodium and no colony is generated;
step four, subculturing and expanding culture
Cutting and culturing the root strain of the induced hairy root in a sterile environment, placing in a 1/2MS solid culture medium for a period of time, selecting hairy root with strong root system, more branches, rapid growth and no geotropic property, transferring into a B5 liquid culture medium for expansion culture, shake culturing in the dark, and growing the hairy root in large quantity.
2. The method according to claim 1, wherein the explant is a stem segment; inoculating the treated explant on a 1/2MS solid culture medium for culture, wherein the pre-culture time is 2 days; inoculating agrobacterium rhizogenes into TY liquid culture medium containing kanamycin, and culturing until the concentration OD600 of the bacteria liquid for dip dyeing is 0.7, wherein OD 600=0.7; the step (2) is placed in a shaking table for shake culture, the culture time is 30min, and the culture is inoculated onto a 1/2MS solid culture medium and placed in a climatic chamber for 2d; the B5 liquid culture medium is a vitamin-containing B5 liquid culture medium, wherein the vitamin-containing B5 liquid culture medium is prepared from 25g/L of B5+sucrose+vitamin, the pH value is 5.8-6.0, the vitamins are inositol 100mg/L, hydrochloric acid 1mg/L, pyridoxine hydrochloride 1mg/L and thiamine hydrochloride 10mg/L, and the culture condition is that shake culture is performed in the dark at 25 ℃ and 100rpm.
3. The method according to claim 1, wherein the sterilization in the first step is performed after the sterilization of 75% by volume medical alcohol for 20 to 30 seconds; and then placing the seeds in a sodium hypochlorite solution with the mass fraction of 10% for disinfection for 3-5 min, and culturing the sterilized seeds on a 1/2MS solid culture medium under the culture conditions that the temperature is 25+/-2 ℃ and the photoperiod is 10-16 h.
4. The method according to claim 1, wherein the cutting of the leaf or stem into small segments in the first step is a cutting of leaf size of 1cm x 1cm and stem is beveled to a length of 0.5-1 cm.
5. The method according to claim 1, wherein the step two of inoculating Agrobacterium rhizogenes to TY liquid medium containing kanamycin for cultivation until OD600 = 0.6-0.9 is that Agrobacterium rhizogenes ArA4 is inoculated to TY liquid medium containing 50mg/l kanamycin, and shake-cultivated at 28 ℃ at 160rpm for 24 hours until OD600 = 0.7;
and step two, taking bacterial liquid, centrifuging to remove supernatant, namely centrifuging at 6000rpm for 10min, and removing supernatant.
6. The culture method according to claim 1, wherein the TY liquid medium has a formula of: tryptone 5 g+yeast extract 3g, pH 7.2.
7. The method according to claim 1, wherein the 1/2MS solid medium in the first to third steps is 1/2 MS+30 g/l sucrose+6 g/l agar, and the pH is 5.8-6.0, and the 1/2MS liquid medium is 1/2MS solid medium lacking agar.
8. The method according to claim 1, wherein the step three is performed by adding acetosyringone, shaking culture at 28℃and shaking culture at 90-100 rpm for 30 min.
9. The method according to claim 8, wherein the shaking culture is performed at a rotation speed of 100rpm.
10. The method according to claim 1, wherein the transfer in the third step is performed every 7-10d by replacing every 10d, and the concentration of cefotaxime sodium Cef in the sterilized medium is 100 mg.L -1 Decreasing in sequence.
CN202310718843.4A 2023-06-16 2023-06-16 Rapid and efficient culture method for induction and proliferation of hairy roots of mare Pending CN116622767A (en)

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