CN110447536B - Tissue culture method of short-lived plant Xinjiang arabidopsis thaliana - Google Patents
Tissue culture method of short-lived plant Xinjiang arabidopsis thaliana Download PDFInfo
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Abstract
The invention belongs to the field of plant tissue culture methods, and particularly relates to a tissue culture method of a short-lived plant Xinjiang small Arabidopsis. The Xinjiang small arabidopsis tissue culture method comprises the following steps: 1) sterilizing Xinjiang small arabidopsis seeds, and 2) dibbling and culturing the sterilized seeds on a culture medium; 3) selecting an explant from the small arabidopsis seedlings cultured in the step 2); 4) performing callus induction culture on the explant in an adventitious bud culture medium to obtain callus; further performing adventitious bud induction culture to obtain adventitious buds; 5) and (4) carrying out rooting induction culture on the adventitious bud in a rooting culture medium to obtain a complete plant. The invention provides tissue culture of different explants of small Arabidopsis, initially establishes tissue culture technology of different explants of small Arabidopsis, and provides a certain research basis for subsequent research on gene transformation and function identification of small Arabidopsis. Provides a theoretical basis for further research on the stress tolerance resources and the environmental adaptability of the small arabidopsis thaliana.
Description
Technical Field
The invention belongs to the field of plant tissue culture methods, and particularly relates to a tissue culture method of a short-lived plant Xinjiang small Arabidopsis.
Background
Sinkiang Arabidopsis thaliana (Arabidopsis pumila) is a short-lived plant in early spring, and is mainly distributed in extreme environments in Sinkiang. Small Arabidopsis thaliana is a closely related species of the model plant Arabidopsis thaliana (Arabidopsis thaliana), which belongs to the family Brassicaceae. Compared with Arabidopsis thaliana, it has stronger salt-resistant drought resistance, stronger photosynthetic efficiency and larger fruit quantity, and the plant type is larger than that of Arabidopsis thaliana. It has been reported that small arabidopsis thaliana, compared to arabidopsis thaliana, can adapt to drought environments and tolerate some degree of salt stress. Is a potential model material for researching biological and environmental adaptive mechanisms, and is also a model plant for researching the rapid growth and development mechanism of short-lived plants. Many researchers use plant callus to perform transgenic related research, and the tissue culture technology of small arabidopsis thaliana has not been reported yet.
Disclosure of Invention
The technical problem solved by the invention is that: provides a tissue culture method of Xinjiang small arabidopsis thaliana.
In order to solve the technical problems, the invention provides a tissue culture method of a short-lived plant Xinjiang small Arabidopsis, which comprises the following steps:
1) sterilizing seeds of Xinjiang small arabidopsis;
2) dibbling and culturing the disinfected seeds on a culture medium;
3) Selecting an explant from the small arabidopsis seedlings cultured in the step 2);
4) performing callus induction culture on the explant in an adventitious bud culture medium to obtain compact callus; further performing adventitious bud induction culture to obtain adventitious buds;
5) and (4) carrying out rooting induction culture on the adventitious bud in a rooting culture medium to obtain a complete plant.
Wherein the step 1) comprises: washing Sinkiang arabidopsis thaliana seed with 70% ethanol for 1min, washing with 20% sodium hypochlorite solution for 8-10min, and washing with sterile water for 6-7 times, shaking thoroughly each time, and mixing.
Further, the explant is a root or hypocotyl.
When the explant is a root or a hypocotyl, in the step 2, the culture step of the seeds is vernalization treatment for 3d under the condition of 4 ℃, and vertical culture is carried out for 7-8 days under the conditions of 16h of illumination/8 h of darkness and 22 ℃ to obtain a week-old small arabidopsis seedling.
Further, the explant is a wide and thick fresh green leaf or a thick and strong petiole.
When the explant is a wide and thick fresh green leaf or a thick and strong petiole, in the step 2), the culture step of the seeds comprises the steps of vernalization treatment for 3 days at 4 ℃, vertical culture for 7-8 days at 16h of illumination/8 h of darkness and at 22 ℃ to obtain a small arabidopsis thaliana seedling with the seedling age of one week, transplanting the small arabidopsis thaliana seedling into a sterile wide-mouth bottle containing an MS culture medium, sealing to ensure sterility, and culturing for 4 weeks at 16h of illumination/8 h of darkness and at 22 ℃ to obtain a small arabidopsis thaliana seedling with the seedling age of five weeks and with a large number of rosette leaves.
Wherein, the conditions of the callus induction culture in the step 4 are as follows: 16h of illumination/8 h of darkness, the temperature is 22 ℃, and the conditions for adventitious bud induction culture are as follows: 16h light/8 h dark, temperature 22 ℃.
Wherein, the rooting induction culture conditions in the step 5 are as follows: 16h light/8 h dark, temperature 22 ℃.
After the step 5, a step 6 of hardening off the whole plant and transplanting the plant to obtain a mature seedling is further included, wherein the hardening off conditions in the step 6 are as follows: 16h light/8 h dark, temperature 22 ℃.
In the above method, each liter of said MS medium comprises: 30g of sucrose, 0.5g of 2-morpholine ethanesulfonic acid and 4.4g of a basal medium, wherein the pH value of the MS medium is 5.8; the adventitious bud induction culture comprises MS +0.5 mg/L6-BA and 0.1mg/L NAA, the MS +0.5 mg/L6-BA and 0.1mg/L NAA are culture media obtained by adding 6-BA and NAA into an MS culture medium, in the MS +0.5 mg/L6-BA and 0.1mg/L NAA, the content of 6-BA is 0.5mg/L, and the content of NAA is 0.1 mg/L; the rooting culture medium is MS +0.1mg/L NAA, the MS +0.1mg/L NAA is a culture medium obtained by adding NAA into the MS culture medium, and the NAA content in the MS +0.1mg/L NAA is 0.1 mg/L.
Therefore, the invention provides tissue culture of different explants of the small arabidopsis thaliana, establishes tissue culture technology of different explants of the small arabidopsis thaliana, provides a certain research basis for subsequent research on gene transformation and function identification of the small arabidopsis thaliana, and provides a theoretical basis for further research on the development of stress-tolerant resources of the small arabidopsis thaliana and environmental adaptability.
Drawings
FIG. 1 is a flow chart of tissue culture of different explants of Arabidopsis;
FIG. 2 is a process of tissue culture of a small Arabidopsis root explant, wherein in FIG. 2, A is the formation of compact callus by a small Arabidopsis root; b is the adventitious bud formed by the callus of the root; c is a large number of adventitious buds formed by the callus of the roots; d, transferring the small arabidopsis tissue culture seedlings into a rooting culture medium; e and F are that a large number of roots grow out of the tissue culture seedlings, and the scale in the figure represents 1 cm;
FIG. 3 shows the tissue culture process of the hypocotyl explant of small Arabidopsis thaliana, wherein A in FIG. 3 shows the formation of a large number of calli from the hypocotyl of small Arabidopsis thaliana; b is the adventitious bud formed by the callus of hypocotyl; c, transferring the adventitious bud into a rooting culture medium; d is that a large number of roots grow out from the tissue culture seedlings, and the scale in the figure represents 1 cm;
FIG. 4 shows the process of tissue culture of a petiole explant of Arabidopsis thaliana, wherein A in FIG. 4 shows the formation of a large number of calli from the petiole of Arabidopsis thaliana; b, callus of petiole begins to form adventitious bud; c, transferring the adventitious bud into a rooting culture medium; d is that a large number of roots grow out from the tissue culture seedlings, and the scale in the figure represents 1 cm;
FIG. 5 shows the process of tissue culture of a small Arabidopsis leaf explant, wherein A in FIG. 5 shows the formation of a large number of calli from the small Arabidopsis leaf; b, leaf callus begins to form adventitious buds; c, transferring the adventitious bud into a rooting culture medium; d is that a large number of roots grow out from the tissue culture seedlings, and the scale bar in the figure represents 1 cm.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, and the examples are given only for illustrating the present invention and not for limiting the scope of the present invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The components of the culture medium:
MS medium per liter contained: 30g sucrose +0.5g 2-morpholinoethanesulfonic acid (2- (N-Morpholino) ethanesulfonic acid, Germany) +4.4g Basal Medium (powdered MS Basal Medium from Murashige & Skoog basic Medium with Vitamins, America), pH 5.8.
Adventitious bud culture medium: MS culture medium +6-BA (0.5mg/L) + NAA (0.1 mg/L);
rooting culture medium: MS medium + NAA (0.1 mg/L).
Different explants of the root, hypocotyl, leaf and petiole of the small arabidopsis can form a large amount of callus within a certain time, and finally the callus can be induced to form a large amount of adventitious buds, and the adventitious buds can be induced to form a large amount of roots after being transferred into a rooting culture medium for rooting induction. The general flow of tissue culture of different explants of small Arabidopsis is shown in FIG. 1.
Example 1
Washing the seeds of Xinjiang small Arabidopsis thaliana in an ultra-clean workbench for 1min by using 70% ethanol, and then washing the seeds for 8-10min by shaking by using a 20% sodium hypochlorite solution. And finally, washing the mixture for 6 to 7 times by using sterile water, and fully oscillating and uniformly mixing the mixture each time. And (2) dibbling the small arabidopsis seeds in a culture dish containing MS, performing vernalization treatment for 3d at 4 ℃, then vertically culturing for 7-8 days in an illumination culture box with the conditions of 16h illumination/8 h darkness and 22 ℃ to obtain small arabidopsis seedlings, and taking roots of the small arabidopsis seedlings as explants.
The root (about 1cm) of a young Arabidopsis thaliana seedling grown for 8 days was cut out as an explant. The cut explants are placed on adventitious bud culture medium, a plurality of explants are randomly placed on each dish, the explants are transferred to fresh culture medium every week, the callus induction and adventitious bud induction conditions are observed, and the explants are photographed and counted well.
The small arabidopsis root explants are cultured in a light incubator with 16h light/8 h dark and 22 ℃ for about 7 days to start callus formation, and a large amount of compact callus tissue is formed after about 20 days of culture (as shown in figure 2-A). The 343 root explants are subjected to callus induction culture on the adventitious bud culture medium for about 20 days and counted, 341 explants successfully induce callus, and the total callus induction rate is 99.42%. After further culturing for about 30 days under the conditions of 16h light/8 h dark and 22 ℃ temperature, the callus of the root begins to form adventitious buds (as shown in FIG. 2-B), the number of explants which can induce the adventitious buds to be generated is 335, and the percentage of all explants is 97.7% (see Table 1). Cutting when adventitious bud grows to 1-2cm and has 4 or more strong leaves (as shown in figure 2-C), transferring into sterile triangular flask containing rooting medium, inducing and culturing to root, sealing and culturing at 22 deg.C for about 20 days under 16h light/8 h dark condition to induce root formation (as shown in figure 2-D), and continuing culturing until large amount of strong root is cultured from regenerated seedling and complete plant is grown (as shown in figures 2-E and 2-F). Then opening the sealing film of the aseptic seedling, adding double distilled water into the bottle, taking the water amount just submerging the culture medium as the standard, not sealing the opening during the seedling hardening period of the seedling, frequently supplementing water, transplanting after 5 days, and obtaining the complete small arabidopsis thaliana plant after survival.
Example 2
Washing seeds of Xinjiang small Arabidopsis seeds in an ultra-clean workbench for 1min by using 70% ethanol, and then washing the seeds for 8-10min by shaking with 20% sodium hypochlorite solution. And finally, washing the mixture for 6 to 7 times by using sterile water, and fully oscillating and uniformly mixing the mixture each time. And (2) dibbling the small arabidopsis seeds in a culture dish containing MS, performing vernalization treatment for 3d at 4 ℃, then vertically culturing for 7-8 days in an illumination culture box with the conditions of 16h illumination/8 h darkness and 22 ℃ to obtain small arabidopsis seedlings, and taking hypocotyls of the small arabidopsis seedlings as explants.
Hypocotyls (about 1cm) of 8-day-old young Arabidopsis seedlings were cut out as explants. The cut explants are placed on adventitious bud culture medium (MS culture medium +6-BA (0.5mg/L) + NAA (0.1mg/L)), a plurality of explants are randomly placed on each dish, the explants are transferred to fresh culture medium every week, the callus induction and adventitious bud induction conditions are observed, and the explants are photographed and counted well.
The small Arabidopsis hypocotyl explants were cultured at 16h light/8 h dark at 22 ℃ for about 10 days to begin callus formation (as shown in FIG. 3-A), and of the 232 hypocotyl explants, a total of 217 explants were induced to callus, with a total callus induction rate of 93.53% (see Table 1). The hypocotyl explant is cultured for about 2 months under the conditions of 16h of light/8 h of dark at 22 ℃ to start to form adventitious buds, and the adventitious bud induction rate is 89.7% (as shown in figure 3-B). Cutting when adventitious bud grows to 1-2cm and has 4 or more strong leaves (as shown in figure 3-C), transferring into triangular flask containing rooting medium (MS culture medium + NAA (0.1mg/L)) for inducing and culturing to root, sealing and culturing at 22 deg.C under 16h light/8 h dark condition until large amount of strong root is cultured from the regenerated seedling, and growing into complete plant (figure 3-D). Then opening the sealing film of the aseptic seedling, adding double distilled water into the bottle, taking the water amount just submerging the culture medium as the standard, not sealing the opening during the seedling hardening period of the seedling, frequently supplementing water, transplanting after 5 days, and obtaining the complete small arabidopsis thaliana plant after survival.
Example 3
Washing seeds of Xinjiang small Arabidopsis seeds in an ultra-clean workbench for 1min by using 70% ethanol, and then washing the seeds for 8-10min by shaking with 20% sodium hypochlorite solution. Finally, washing the mixture for 6 to 7 times by sterile water, fully oscillating and uniformly mixing the mixture each time. Dibbling small arabidopsis seeds in a culture dish containing MS, performing vernalization treatment for 3d at 4 ℃, then placing the small arabidopsis seeds in a light culture box with 16h light/8 h dark and 22 ℃ for vertical culture for 7-8 days to obtain small arabidopsis seedlings, transplanting the small arabidopsis seedlings with good growth condition into a wide-mouth bottle containing an MS culture medium, then placing the small arabidopsis seedlings in the culture box under the same culture condition for about 4 weeks, and taking thick and strong petioles as explants.
The petiole (about 1cm) of a small Arabidopsis thaliana was excised as an explant for about 4 weeks. The cut explants are placed on adventitious bud induction culture medium (MS culture medium +6-BA (0.5mg/L) + NAA (0.1mg/L)), a plurality of explants are randomly placed on each dish, the explants are transferred to fresh culture medium every week, the callus induction and adventitious bud induction conditions are observed, and the explants are photographed and made to be statistical.
The petiole explants of arabidopsis thaliana were callus-forming slightly later and callus formation began when they were cultured for 12-13 days under 16h light/8 h dark at 22 ℃. The petiole tissue of arabidopsis thaliana is cultured for about 20 days under the conditions of 16h of light/8 h of dark and 22 ℃ to form a large amount of green healthy calluses (as shown in figure 4-A), 393 explants are successfully induced to generate adventitious buds in a total of 405 petiole explants, and the callus induction rate is 97.04% (see table 1). Culturing under 16h light/8 h dark at 22 deg.C for about 44 days to form adventitious bud with adventitious bud induction rate of 87.9% (as shown in FIG. 4-B). Transferring the adventitious bud into a rooting medium for induced rooting (as shown in figure 4-C), until a large amount of strong roots are cultured from the regenerated seedling and grow into a complete plant (as shown in figure 4-D), hardening and transplanting can be carried out according to the method, and the complete small Arabidopsis plant can be obtained after survival.
Example 4
Washing seeds of Xinjiang small Arabidopsis seeds in an ultra-clean workbench for 1min by using 70% ethanol, and then washing the seeds for 8-10min by shaking with 20% sodium hypochlorite solution. Finally, washing the mixture for 6 to 7 times by sterile water, fully oscillating and uniformly mixing the mixture each time. Dibbling small arabidopsis seeds in a culture dish containing MS, performing vernalization treatment for 3d at 4 ℃, then placing the small arabidopsis seeds in a light culture box with 16h light/8 h dark and 22 ℃ temperature for vertical culture for 7-8 days to obtain small arabidopsis seedlings, transplanting the small arabidopsis seedlings with good growth condition into a sterile wide-mouth bottle containing an MS culture medium, sealing the bottle to ensure sterility, then placing the bottle in the culture box under the same culture condition for about 4 weeks, and taking wide and thick fresh green leaves as explants.
Cutting leaves of small Arabidopsis thaliana about 4 weeks (about 1 cm)2) As an explant. The cut explants are placed on a germination induction medium (MS medium +6-BA (0.5mg/L) + NAA (0.1mg/L)), a plurality of explants are randomly placed on each dish, the explants are transferred to a fresh medium every week, the callus induction and adventitious bud induction conditions are observed, and the explants are photographed and counted well.
The leaf tissue of the arabidopsis thaliana is cultured under the conditions of 16h light/8 h dark and 22 ℃ for about 12 days to start to form callus, 311 explants in the total 322 leaf explants are successfully induced to form callus, the callus induction rate is 96.58 percent (see table 1), the leaf tissue of the arabidopsis thaliana is cultured under the conditions of 16h light/8 h dark and 22 ℃ for about 12-13 days to form a large amount of callus (as shown in figure 5-A), the callus is continuously subjected to adventitious bud induction, the adventitious bud induction rate is 92.5 percent and is slightly earlier than the appearance time of adventitious buds of a petiole, and the adventitious bud induction rate is higher than the adventitious bud induction rate of the petiole (see table 1) after the callus is cultured under the conditions of 16h light/8 h dark and 22 ℃ for about 42 days to induce the adventitious buds (as shown in figure 5-B). Cutting when adventitious bud grows to 1-2cm and has 4 or more strong leaves (shown in figure 5-C), transferring into rooting culture medium, inducing and culturing to root until regenerated seedling has a large amount of strong roots and grows into complete plant (shown in figure 5-D), hardening seedling and transplanting according to the method, and obtaining complete small Arabidopsis plant after survival.
TABLE 1 statistics of tissue culture data for different explants of Small Arabidopsis
Lower case letters represent statistically significant differences
The experimental data were statistically analyzed using SPSS 19.0 software. Significance analysis statistical data were analyzed using one-way anova and Duncan's multiple range test to determine significance differences between groups of data, with P <0.05 considered significant differences.
In combination with FIG. 2-FIG. 5 and Table 1, it can be seen that there is a significant difference in callus induction time and adventitious bud induction time between the different explants compared to the four different explants of Small Arabidopsis. The callus formation time of the radicles was about 7 days, and the adventitious bud formation time was about 32 days (table 1); the callus formation time of hypocotyl is about 10 days, and the adventitious bud appears for about 2 months. By comparison of the data, the time for the callus formation of hypocotyls and the formation of adventitious buds was found to be significantly longer than that of roots (Table 1). The callus formation time of the leaf blade and petiole explants of the arabidopsis thaliana is obviously longer than that of the radicle and hypocotyl, wherein the longest callus formation time of the petiole is about 2 weeks; the induction forming time of the adventitious buds of the leaf blade and the leaf stalk is obviously longer than that of the adventitious buds of the radicle and shorter than that of the hypocotyl. Combining the above results, among the four explants, the callus and adventitious bud formation time of the young root of Arabidopsis was the shortest, and secondly, the callus formation time of the hypocotyl was shorter, but the adventitious bud formation time was the longest. Leaves and petioles are longer in callus formation, but adventitious buds are induced for a shorter time than hypocotyls.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific examples, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is made possible within the scope of the claims attached below.
Claims (9)
1. A Sinkiang arabidopsis tissue culture method is characterized by comprising the following steps:
1) the disinfection of the seeds of Xinjiang small Arabidopsis,
2) dibbling and culturing the disinfected seeds on a culture dish containing MS;
3) selecting an explant from the small arabidopsis seedlings cultured in the step 2);
4) performing callus induction culture on the explant in an adventitious bud culture medium to obtain callus; further performing adventitious bud induction culture to obtain adventitious buds;
5) Carrying out rooting induction culture on the adventitious bud in a rooting culture medium to obtain a complete plant;
the adventitious bud culture medium is MS +0.5 mg/L6-BA +0.1 mg/L NAA; the rooting culture medium is MS +0.1 mg/L NAA; each liter of the MS culture medium comprises: 30 g of sucrose, 0.5 g of 2-morpholinoethanesulfonic acid and 4.4 g of basal medium, the pH value of the MS medium being 5.8.
2. The method for tissue culture of arabidopsis thaliana of claim 1, wherein the step 1) comprises: washing Sinkiang arabidopsis thaliana seed with 70% ethanol for 1 min, washing with 20% sodium hypochlorite solution for 8-10 min, and washing with sterile water for 6-7 times, shaking thoroughly each time, and mixing.
3. The method for tissue culture of arabidopsis thaliana of claim 1 or 2, wherein the explant is a root or hypocotyl.
4. The method for tissue culture of arabidopsis thaliana of claim 3, wherein in step 2), the seeds are cultured by vernalization at 4 ℃ for 3 d, and are cultured for 7-8 days under 16 h light/8 h dark and 22 ℃ to obtain one week-old arabidopsis thaliana seedlings.
5. The tissue culture method of Xinjiang small Arabidopsis according to claim 1 or 2, wherein the explant is a wide and fertile fresh green leaf or a thick and strong petiole.
6. The Xinjiang small Arabidopsis tissue culture method according to claim 5, wherein in the step 2), the seed culture step is vernalization treatment at 4 ℃ for 3 d, vertical culture is performed for 7-8 days under the conditions of 16 h light/8 h dark and 22 ℃ to obtain small Arabidopsis seedlings, the small Arabidopsis seedlings are transplanted into a culture medium containing MS, the culture medium is sealed to ensure sterility, and the small Arabidopsis seedlings are cultured for 4 weeks under the conditions of 16 h light/8 h dark and 22 ℃ to obtain five-week-old small Arabidopsis seedlings.
7. The method for culturing Xinjiang small Arabidopsis tissue according to claim 1, wherein the callus induction culture conditions in step 4) are as follows: 16 h of illumination/8 h of darkness, the temperature is 22 ℃, and the conditions for adventitious bud induction culture are as follows: 16 h light/8 h dark, temperature 22 ℃.
8. The method for tissue culture of arabidopsis thaliana of claim 1, wherein the rooting induction culture in step 5) is performed under the following conditions: 16 h light/8 h dark, temperature 22 ℃.
9. The Xinjiang small Arabidopsis tissue culture method according to claim 1, wherein after the step 5), the whole plant is acclimatized and transplanted to obtain a mature seedling, and the acclimatization conditions are as follows: 16 h light/8 h dark, temperature 22 ℃.
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| WO2002010344A2 (en) * | 2000-07-27 | 2002-02-07 | Cambridge University Technical Services Ltd | Synchronised arabidopsis cell suspensions and uses thereof |
| CN106256196A (en) * | 2015-11-20 | 2016-12-28 | 沈阳善达生物科技有限公司 | Efficiently induce method and the inducing culture of Colombia's type Arabidopsis callus |
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| WO2002010344A2 (en) * | 2000-07-27 | 2002-02-07 | Cambridge University Technical Services Ltd | Synchronised arabidopsis cell suspensions and uses thereof |
| CN106256196A (en) * | 2015-11-20 | 2016-12-28 | 沈阳善达生物科技有限公司 | Efficiently induce method and the inducing culture of Colombia's type Arabidopsis callus |
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| 新疆荒漠环境典型短命植物小鼠耳芥（Arabidopsis pumila）快速生长与耐逆性机制;金玉环;《CNKI》;20210601;第1-143页 * |
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