CN115885852A - Tissue culture method for leaf of chicory - Google Patents
Tissue culture method for leaf of chicory Download PDFInfo
- Publication number
- CN115885852A CN115885852A CN202211650540.5A CN202211650540A CN115885852A CN 115885852 A CN115885852 A CN 115885852A CN 202211650540 A CN202211650540 A CN 202211650540A CN 115885852 A CN115885852 A CN 115885852A
- Authority
- CN
- China
- Prior art keywords
- culture
- chicory
- medium
- rooting
- culture medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/40—Afforestation or reforestation
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention belongs to the field of tissue culture, and relates to a tissue culture method of chicory leaves, which comprises the following steps: 1) Carrying out induction culture on sterile leaf blades of the chicory in an induction culture medium to generate adventitious buds; the induction medium comprises: MS culture medium, 6-BA 0.5-1 mg/L, NAA 0.1-0.2 mg/L, GA 3 1.0mg/L; 2) Transferring the adventitious bud into a proliferation culture medium for proliferation culture to obtain a proliferation seedling; the multiplication medium comprises: MS culture medium, 6-BA0.5mg/L, NAA 0.02-0.06 mg/L, adenine 5.0mg/L; transferring the proliferated seedlings into a rooting culture medium for rooting culture to obtain seedlings, wherein the rooting culture medium comprises a 1/2MS culture medium, 0.8-1 mg/L IBA and 3g/L active carbon; and (3) hardening the seedlings, transplanting the seedlings to a substrate, and culturing to obtain the seedling of the nodulated red chicory. The tissue culture method provided by the invention can obviously improve the rooting rate and the rooting amount of the seedling of the chicory.
Description
Technical Field
The invention belongs to the technical field of tissue culture, and particularly relates to a tissue culture method of nodulation red chicory leaves.
Background
The chicory of the Compositae family is a perennial herb, originates from coastal Zhongya and North Africa of the Mediterranean region, is generally planted in European and American countries, is bright in color, rich in nutrition and much bitter in taste compared with softened chicory, and is widely applied to seedling culture of horticultural plants due to the advantages of no season limitation, detoxification, rapidness and mass propagation along with the development of tissue culture technology, while the seeds of the chicory of the Compositae family mainly depend on foreign import and have higher price in the market, which brings certain difficulty for the practical production, popularization and cultivation of the chicory.
For example, patent CN104604689a discloses a method for rapidly obtaining chicory explants and increasing the callus rate thereof, compared with the traditional inoculation and culture method, the yield of chicory explants in unit time is increased by more than 50% compared with the common inoculation method, the callus rate of explants can be increased to 100%, but the final rooting rate and rooting amount of explants which are actually grown in large-scale production and cultivation cannot meet the industrial requirements.
Therefore, how to improve the rooting effect of the chicory, especially the rooting rate and the rooting amount of the chicory become problems which need to be solved urgently at present.
Disclosure of Invention
The invention aims to provide a tissue culture method of the leaf of the chicory with excellent rooting effect, which can obviously improve the rooting rate and the rooting amount of seedlings.
In order to realize the aim, the invention provides a tissue culture method of the leaf of the chicory, which comprises the following steps:
1) Carrying out induction culture on sterile leaf blades of the chicory in an induction culture medium to generate adventitious buds; the induction medium comprises: MS culture medium, 6-BA 0.5-1 mg/L, NAA 0.1-0.2 mg/L, GA 3 1.0mg/L；
2) Transferring the adventitious bud into a proliferation culture medium for proliferation culture to obtain a proliferation seedling; the multiplication medium comprises: MS culture medium, 6-BA0.5mg/L, NAA 0.02-0.06 mg/L, adenine 5.0mg/L;
3) Transferring the proliferated seedlings into a rooting culture medium for rooting culture to obtain seedlings, wherein the rooting culture medium comprises a 1/2MS culture medium, 0.8-1 mg/L IBA and 3g/L active carbon;
4) And (3) hardening the seedlings, transplanting the seedlings to a substrate, and culturing to obtain the seedling of the nodulated red chicory.
Preferably, the sterile chicory leaf has a size of 0.2-0.3 mm.
Preferably, the aseptic processing of the nodulated chicory leaves comprises the following steps: the leaves are disinfected by 84 disinfectant and alcohol in sequence and washed by distilled water for later use.
Preferably, the MS culture medium further comprises sucrose and agar, wherein the concentration of the sucrose is 25-35 g/L, and the concentration of the agar is 5-8 g/L.
Preferably, the induction medium comprises MS medium, 6-BA0.5mg/L, NAA0.1mg/L, GA 3 1.0mg/L。
Preferably, the induction culture conditions are: the illumination intensity is 1500-2000 Lx, and the temperature is 24-26 ℃; the induction culture time is 30d.
Preferably, the proliferation medium comprises MS medium, 6-BA0.5mg/L, NAA0.04mg/L, adenine 5.0mg/L.
Preferably, the propagation culture conditions are: the illumination intensity is 2000 to 3500Lx, and the illumination time is 12h/d; the temperature is 24-26 ℃, and the humidity is 80%; the proliferation culture time is 20-23 d.
Preferably, the rooting medium comprises 1/2MS medium, IBA0.8mg/L and activated carbon 3g/L.
Preferably, the rooting culture conditions are: the illumination intensity is 2000 to 3500Lx, and the illumination time is 12h/d; the temperature is 24-26 ℃, and the humidity is 80%; the rooting culture time is 10-25 days.
Advantageous effects
The invention provides a tissue culture method of nodulation chicory leaves, which comprises the following steps: a tissue culture method of leaf of chicory of nodulation red includes the following steps: 1) Carrying out induction culture on sterile leaf blades of the chicory in an induction culture medium to generate adventitious buds; the induction medium comprises: MS culture medium, 6-BA 0.5-1 mg/L, NAA 0.1-0.2 mg/L, GA 3 1.0mg/L; 2) Transferring the adventitious bud into a multiplication culture medium for multiplication culture to obtain a multiplication seedling; the multiplication medium comprises: MS culture medium, 6-BA0.5mg/L, NAA 0.02-0.06 mg/L, adenine 5.0mg/L; 3) Transferring the proliferated seedling into a rooting culture medium for rooting culture to obtain a seedling, wherein the rooting culture medium comprises a 1/2MS culture medium, 0.8-1 mg/L IBA and 3g/L active carbon; 4) Hardening off the seedlings, transplanting the seedlings to a substrate for culturing to obtain knotsChicory seedlings of globe amaranth.
According to the tissue culture method of the leaf of the chicory, provided by the invention, the leaf of the chicory is subjected to tissue culture through induction, proliferation and rooting culture to obtain a seedling, and the seedling is obtained after hardening and transplanting; the induction culture medium can differentiate a small amount of callus, so that the callus forms a large amount of bud points, and the optimal germination effect is realized; the multiplication culture medium can form callus on the base of the adventitious bud small segment successively, and then bud points are formed on the callus to differentiate cluster buds to form more tender stems; the rooting culture medium can improve the whole rooting rate and the rooting amount of the tender stem, and finally, the excellent chicory seedling with the nodulation can be quickly and efficiently produced through hardening and transplanting. The experimental results show that: the tissue culture method of the nodulation red chicory leaves provided by the invention has the advantages that the rooting rate can reach 100%, the rooting time is relatively centralized, and the rooting amount of each section of tender shoot can also reach 3-6.
Drawings
FIG. 1 is a graph showing the rooting effect of the nursery stock after rooting culture in example 3-1;
FIG. 2 is a graph showing the growth of seedlings of the nodulated chicory after transplantation in example 3-1.
Detailed Description
The invention provides a tissue culture method of nodulation chicory leaves, which comprises the following steps:
1) Carrying out induction culture on the sterile cichorium intybus leaves in an induction culture medium to generate adventitious buds; the induction medium comprises: MS culture medium, 6-BA 0.5-1 mg/L, NAA 0.1-0.2 mg/L, GA 3 1.0mg/L；
2) Transferring the adventitious bud into a multiplication culture medium for multiplication culture to obtain a multiplication seedling; the multiplication medium comprises: MS culture medium, 6-BA0.5mg/L, NAA 0.02-0.06 mg/L, adenine 5.0mg/L;
3) Transferring the proliferated seedling into a rooting culture medium for rooting culture to obtain the seedling, wherein the rooting culture medium comprises a 1/2MS culture medium, 0.8-1 mg/L IBA and 3g/L active carbon.
4) And (3) hardening the seedlings, transplanting the seedlings to a substrate, and culturing to obtain the seedling of the nodulated red chicory.
The invention carries out induction culture on sterile chicory leaf blades in an induction culture medium to generate adventitious buds.
The chicory leaves are not particularly limited in the invention, and the size of the sterile chicory leaves is preferably 0.2-0.3 mm. In the present invention, the preparation of said chicory leaves with nodulation preferably comprises the following steps: collecting leaves near the growing point from robust growing and disease and insect pest free chicory seedlings, washing the collected leaves with distilled water, cutting off the periphery and stem skin of the leaves, putting into a glass bottle, and sealing with gauze for later use.
In the present invention, the aseptic processing of the aseptic chicory leaves preferably comprises the following steps: the leaves are disinfected by 84 disinfectant and alcohol in sequence and washed by distilled water for later use. In the invention, the concentration of the 84 disinfectant is preferably 0.3-0.5%, and the disinfection time of the 84 disinfectant is preferably 5-20 min; the alcohol is preferably 70% alcohol, and the alcohol disinfection time is preferably 5-10 min; the times of washing with distilled water are 3-4 times. In the present invention, the aseptic processing is preferably performed in a clean bench.
In the present invention, the induction medium includes: MS culture medium, 6-BA 0.5-1 mg/L, NAA 0.1-0.2 mg/L, GA 3 1.0mg/L; in the present invention, the MS culture medium preferably further comprises sucrose and agar, and the sucrose concentration is preferably 25 to 35g/L, and more preferably 30g/L; the concentration of the agar is preferably 5 to 8g/L, and more preferably 6 to 7g/L; the pH of the induction medium is preferably 5.6. The invention takes MS culture medium as basic culture medium, and adds 6-BA, NAA and GA 3 An induction culture medium is formed, the dosage of the induction culture medium is regulated and controlled to induce the chicory leaf blades of the nodulation red chicory, so that a small amount of callus can be differentiated, a large number of bud points are formed on the callus, and a high germination effect is realized. In a specific embodiment of the present invention, the induction medium is more preferably MS medium, 6-BA0.5mg/L, NAA0.1mg/L, GA 3 1.0mg/L, the preferable scheme of the invention is mainly because the NAA content can influence the formation of the bud points, the low content can promote the formation of the bud points, the high content is unfavorable for the formation of the bud points, and the adventitious buds with more bud pointsIn the future, more tender stems are germinated, and the germination rate is relatively higher.
In the present invention, the induction culture conditions are preferably: the illumination intensity is 1500-2000 Lx, and the temperature is 24-26 ℃; in the present invention, the induction culture time is preferably 25 to 30 days, and more preferably 30 days.
After the adventitious bud is generated, the adventitious bud is transferred into a multiplication culture medium for multiplication culture to obtain a multiplication seedling.
In the present invention, the proliferation medium comprises: MS culture medium, 6-BA0.5mg/L, NAA 0.02-0.06 mg/L, adenine 5.0mg/L; in the present invention, the MS culture medium preferably further comprises sucrose and agar, and the concentration of sucrose is preferably 25 to 35g/L, and more preferably 30g/L; the agar concentration is preferably 5 to 8g/L, more preferably 6 to 7g/L, and the pH of the growth medium is preferably 5.6. According to the invention, after adventitious buds are transferred into a multiplication culture medium, callus is formed on the small-section base of the adventitious buds in succession, bud points are formed on the callus, cluster buds are differentiated, NAA auxin in the multiplication culture medium has a promoting effect on the callus, and meanwhile, the research shows that the too large or too small callus formed in the multiplication process has an influence on the differentiation and growth of the cluster buds on the callus, and the optimal area for forming the callus is 0.7-1.4 cm 2 Compared with other auxin (such as IAA), 0.02-0.06 mg/LNAA is selected to control the area of the callus in the optimal area, which is more beneficial to differentiation and amplification of the cluster buds. In a specific embodiment of the invention, the multiplication culture medium further preferably comprises an MS culture medium, 6-BA0.5mg/L, NAA0.04mg/L and adenine 5.0mg/L, because the multiplication culture medium with the optimal proportion has a large growth coefficient of tender stems in the multiplication culture process, the growth condition of bud seedlings is higher than that of other proportion culture media, the higher plant interception segment number in the transfer process is increased, and 3-4 segments can be generally intercepted, so that more tissue culture quantity of the chicory tender stems can be provided.
The present invention preferably cuts the germinated adventitious bud into small segments and then performs propagation culture. In the present invention, the number of the propagation cultures is preferably 2 to 3.
In the present invention, the proliferation culture conditions are preferably: the illumination intensity is 2000 to 3500Lx, and the illumination time is 12h/d; the temperature is 24-26 ℃, and the humidity is 80%; in the present invention, the growth culture time is preferably 20 to 23d, and more preferably 23d.
After the proliferated seedling is obtained, the proliferated seedling is transferred into a rooting culture medium for rooting culture to obtain the seedling.
In the invention, the rooting culture medium comprises 1/2MS culture medium, 0.8-1 mg/L IBA and 3g/L active carbon; in the present invention, the 1/2MS medium preferably further comprises sucrose and agar, and the concentration of sucrose is preferably 25 to 35g/L, and more preferably 30g/L; the concentration of the agar is preferably 5 to 8g/L, more preferably 6 to 7g/L, and the pH of the rooting medium is preferably 5.6. The invention transfers the proliferated seedling into the rooting culture medium for rooting culture, thus promoting the proliferated seedling to rapidly root. In the specific embodiment of the invention, the rooting medium preferably comprises 1/2MS medium, IBA0.8mg/L and 3g/L of activated carbon. The preferable composition of the rooting culture medium can improve the rooting number and rooting rate of axillary buds or young shoots of the seedlings, and the rooting amount (strips) on each root is also obviously improved.
In the present invention, the rooting culture conditions are preferably: the illumination intensity is 2000 to 3500Lx, and the illumination time is 12h/d; the temperature is 24-26 ℃, the humidity is 80 percent, and the shading degree is 50 percent; in the present invention, the rooting culture time is preferably 10 to 25 days, and more preferably 25 days.
After the seedlings are obtained, the seedlings are trained and transplanted to a substrate for culture, and the seedling of the chicory with the head red is obtained.
In the invention, the standard of the nursery stock before transplanting is preferably 3-4 cm high and has 2-3 1cm roots; in the invention, the seedling exercising time is preferably 2-3 d, and in the invention, the seedling exercising preferably comprises the following steps: after the refined seedlings reach the standard, opening a culture bottle cap to destroy the sterile environment, and adding 6-10 mL of sterile double-distilled water into the bottle every 12h during seedling refining.
In the invention, the substrate is preferably vermiculite, the culture time is preferably 20d, and the culture process after transplanting comprises the following steps: after the hardening, the culture medium at the root is carefully washed clean and transplanted into a nutrition pot containing vermiculite matrix.
The equipment and the device used in the induction culture, the proliferation culture and the rooting culture are not particularly limited, and the conventional equipment in the field can be adopted.
The tissue culture method of the leaf of chicory nodulation according to the present invention is described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.
Examples 1 to 1
1) Leaf selection and aseptic processing: collecting leaves near a growing point from robust growing and disease and insect pest-free chicory seedlings, washing the collected leaves with distilled water, cutting off the periphery and stem skin of the leaves, putting the leaves into a glass bottle, and sealing the glass bottle with gauze for later use; washing the glass bottle with the blades for 20min by running water, then disinfecting the glass bottle in 0.3% 84 disinfectant for 10min, washing the glass bottle with distilled water for 3-4 times, then bringing the glass bottle into an inoculation chamber and putting the glass bottle into a super-clean workbench; and (3) disinfecting the leaves in 70% alcohol for 5min, washing with distilled water for 3-4 times to obtain sterile leaf blades of the chicory, cutting the stem skin around the leaves, moving the leaves to a dissecting mirror, and cutting the leaves into squares with the size of 0.2-0.3 mm by using a blade for later use.
2) And (3) induction culture: inoculating sterile leaf of chicory with nodulation ball into an induction culture medium for 30d of induction culture to generate adventitious buds, wherein the pH value of the induction culture medium is 5.6, and the method comprises the following steps: MS culture medium, sucrose 30g/L, agar 6g/L, 6-BA0.5mg/L, NAA0.1mg/L, GA 3 1.0mg/L; the induction culture conditions are that the illumination intensity is 1500Lx and the temperature is 24-26 ℃.
3) And (3) proliferation culture: transferring the adventitious bud into a proliferation culture medium for proliferation culture for 20d to obtain a proliferation seedling; the pH value of the proliferation culture medium of 5.6 comprises: MS culture medium, sucrose 30g/L, agar 6g/L, 6-BA0.5mg/L, NAA0.04mg/L, adenine 5.0mg/L; the proliferation culture condition is illumination intensity 2000Lx, and the illumination time is 12h/d; the temperature is 24-26 ℃, and the humidity is 80%; the number of propagation cultures was two.
4) Transferring the proliferated seedlings with axillary buds or young tip segments into a rooting culture medium for rooting culture for 25 days to obtain seedlings; the rooting culture medium comprises a 1/2MS culture medium, 30g/L of sucrose, 6g/L of agar, 0.8mg/L of IBA0 and 3g/L of activated carbon; the rooting culture conditions are as follows: the illumination intensity is 2000Lx, and the illumination time is 12h/d; the temperature is 24-26 ℃, the humidity is 80%, and the shading degree is 50%.
5) And (3) hardening seedlings with root systems which grow to 3-4 cm in height and are about lcm for 2-3 d, transplanting the seedlings to a vermiculite matrix, culturing for 20d, and planting the seedlings in a nutrition pot after the seedlings completely survive to obtain the seedlings of the cichorium intybus.
Examples 1 to 2
The induction medium of examples 1-2 had a pH of 5.6 and included: MS culture medium, sucrose 30g/L, agar 6g/L, 6-BA1mg/L, NAA0.1mg/L, GA 3 1.0mg/L, and the remaining treatment was the same as in example 1-1.
Examples 1 to 3
The induction media of examples 1-3 had a pH of 5.6 and included: MS culture medium, sucrose 30g/L, agar 6g/L, 6-BA1mg/L, NAA0.2mg/L, GA 3 1.0mg/L, and the remaining treatment was the same as in example 1-1.
Examples 1 to 4
The induction media of examples 1-4 had a pH of 5.6 and included: MS culture medium, sucrose 30g/L, agar 6g/L, 6-BA0.5mg/L, NAA0.2mg/L, GA 3 1.0mg/L, and the remaining treatment was the same as in example 1-1.
Table 1 shows the germination of leaves of examples 1-1 to 1-4 on different induction media
As can be seen from the data and the germination conditions in Table 1, the germination rates of examples 1-1 and 1-2 are higher than those of examples 1-3 and 1-4, and the germination effect is better, which indicates that the low NAA content is beneficial to the formation of the bud points, the high NAA content is unfavorable to the formation of the bud points, the number of tender stems germinated from the leaves with more bud points is large, and the number of subsequent proliferation and rooting can be further ensured under the condition of high germination rate. Compared with the two culture medium formulas of the examples 1-1 and 1-2, the germination conditions are the same, but the germination rates are different, namely the germination rate of the example 1-2 is 72%, while the germination rate of the example 1-1 reaches 90%, which is obviously higher than that of the example 1-2.
Example 2-1
1) Leaf selection and aseptic processing: collecting leaves near a growing point from strong and disease and pest free chicory seedlings, washing the collected leaves with distilled water, cutting off the periphery and stem skin of the leaves, putting the leaves into a glass bottle, and sealing the glass bottle with gauze for later use; washing the glass bottle with the blades for 20min by running water, then disinfecting the glass bottle in 84 disinfectant with the concentration of 0.5% for 15min, washing the glass bottle with distilled water for 3-4 times, then bringing the glass bottle into an inoculation chamber, and putting the glass bottle into a super-clean workbench; and (3) disinfecting the leaves in 70% alcohol for 10min, washing with distilled water for 3-4 times to obtain sterile cichorium intybus leaves, cutting the stem skin around the leaves, moving the leaves under a dissecting mirror, and cutting the leaves into squares with the size of 0.2-0.3 mm by using a blade for later use.
2) And (3) induction culture: inoculating sterile leaf of chicory with nodulation ball into an induction culture medium for 30d of induction culture to generate adventitious buds, wherein the pH value of the induction culture medium is 5.6, and the method comprises the following steps: MS culture medium, sucrose 30g/L, agar 6g/L, 6-BA0.5mg/L, NAA 0.1.1 mg/L, GA 3 1.0mg/L; the induction culture conditions are that the illumination intensity is 2000Lx and the temperature is 24-26 ℃.
3) And (3) proliferation culture: transferring the adventitious bud into a multiplication culture medium for multiplication culture for 20d to obtain a multiplication seedling; the proliferation medium has a pH of 5.6 and comprises: MS culture medium, sucrose 30g/L, agar 6g/L, 6-BA0.5mg/L, NAA 0.04.04 mg/L and adenine 5.0mg/L; the proliferation culture condition is illumination intensity 2500Lx, and the illumination time is 12h/d; the temperature is 24-26 ℃, and the humidity is 80%; the number of propagation cultures was two.
4) Transferring the proliferated seedlings with axillary buds or young tip segments into a rooting culture medium for rooting culture for 25 days to obtain seedlings; the rooting culture medium comprises a 1/2MS culture medium, 30g/L of sucrose, 6g/L of agar, 0.8mg/L of IBA0 and 3g/L of activated carbon; the rooting culture conditions are as follows: the illumination intensity is 2500Lx, and the illumination time is 12h/d; the temperature is 24-26 ℃, the humidity is 80%, and the shading degree is 50%.
5) Carrying out seedling hardening for 2-3 d on seedlings with roots growing to 3-4 cm high and about l cm long, transplanting the seedlings to a vermiculite matrix for culturing for 20d, and planting the seedlings in a nutrition pot after the seedlings completely survive to obtain the seedling of the chicory.
Examples 2 to 2
The proliferation medium of example 2-2 had a pH of 5.6 and included: MS medium, sucrose 30g/L, agar 6g/L, 6-BA0.5mg/L, NAA0.02mg/L, adenine 5.0mg/L, and the rest of the treatment was the same as in example 2-1.
Examples 2 to 3
The proliferation medium of examples 2-3 had a pH of 5.6 and included: MS medium, sucrose 30g/L, agar 6g/L, 6-BA0.5mg/L, NAA0.06mg/L, adenine 5.0mg/L, the rest of the treatment was the same as in example 2-1.
Comparative example 1
Comparative example 1-1 differs from example 2-1 in that no auxin NAA was added to the propagation medium.
Comparative example 2 to 1
Comparative example 2-1 differs from example 2-1 in that NAA0.04mg/L in the propagation medium was replaced with IAA0.04mg/L, and the rest of the treatment was the same as in example 2-1.
Comparative examples 2 to 2
Comparative example 2-2 differs from example 2-2 in that NAA0.02mg/L in the proliferation medium was replaced with IAA0.02mg/L, and the rest of the treatment was the same as in example 2-2.
Comparative examples 2 to 3
Comparative example 2-3 differs from example 2-3 in that NAA0.06mg/L in the multiplication medium was replaced with IAA0.06mg/L, and the rest of the treatment was the same as in example 2-3.
The results of examining the proliferation growth of the young stem during the proliferation culture in examples 2-1 to 2-3 were as follows:
TABLE 2 growth of shoots on multiplication Medium in examples 2-1 to 2-3
Note: calculating the height of the nursery stock above the culture medium; growth factor = average total amount of growth/average length of primary inoculated stem segments.
As can be seen from the data in Table 2, the growth coefficients of the young stems in examples 2-1 to 2-3 are all greater than 2.00, which indicates that the proliferation effect of the 3 groups of culture media is superior, wherein the growth coefficient of the stem section of the culture medium in example 2-1 is the largest, followed by example 2-2 and example 2-3 is the smallest. The larger the growth coefficient is, the higher the seedling growth is, the higher the number of segments of the plant is cut during the transfer is increased, and 3 to 4 segments can be cut usually. Therefore, the culture medium MS +6-BA0.5 mg/L + NAA0.04mg/L in example 2-1 is most desirable, and the effect for amplification propagation is most excellent.
The study of the growth of callus during the propagation culture in examples 2-1 to 2-3 and comparative example 1-2 led to the following conclusion:
TABLE 3 callus growth on proliferation Medium in example 2 and comparative examples 1-2
Note: relative number = number per group/total number; the area of the large-callus is 1.4cm 2 The above; the area of the medium-callus is 0.7-1.4 cm 2 (ii) a The area of the small-callus is 0.7cm 2 The following.
As is apparent from Table 3, although the callus formed in the culture media in the examples and comparative examples were different in size, callus was formed, and the addition of auxin to the culture media had a callus-promoting effect, and the callus formed was too large or too small, which had an effect on the differentiation and growth of multiple shoots on the callus, and the optimal callus formation area was 0.7 to 1.4cm 2 . The culture media of examples 2-1 to 2-3The medium size callus area pieces to which NAA auxin was added were higher than in comparative examples 1-1 and comparative examples 2-1 to 2-3, indicating that the addition of NAA auxin was more favorable for the differentiation and growth of clumpy buds.
Example 3-1
1) Leaf selection and aseptic processing: collecting leaves near a growing point from robust growing and disease and insect pest-free chicory seedlings, washing the collected leaves with distilled water, cutting off the periphery and stem skin of the leaves, putting the leaves into a glass bottle, and sealing the glass bottle with gauze for later use; washing the glass bottle with the blades for 20min by running water, then disinfecting the glass bottle in 84 disinfectant with the concentration of 0.5% for 15min, washing the glass bottle with distilled water for 3-4 times, then bringing the glass bottle into an inoculation chamber, and putting the glass bottle into a super-clean workbench; and (3) sterilizing the leaves in 70% alcohol for 10min, washing with distilled water for 3-4 times to obtain sterile leaf of chicory, cutting the stem skin around the leaves, moving the leaves to a dissecting mirror, and cutting the leaves into squares with the size of 0.2-0.3 mm by using a blade for later use.
2) And (3) induction culture: inoculating sterile leaf of chicory with nodulation ball into an induction culture medium for 30d of induction culture to generate adventitious buds, wherein the pH value of the induction culture medium is 5.6, and the method comprises the following steps: MS culture medium, sucrose 30g/L, agar 6g/L, 6-BA0.5mg/L, NAA0.1mg/L, GA 3 1.0mg/L; the induction culture conditions are that the illumination intensity is 2000Lx and the temperature is 24-26 ℃.
3) And (3) proliferation culture: transferring the adventitious bud into a proliferation culture medium for proliferation culture for 20d to obtain a proliferation seedling; the proliferation medium has a pH of 5.6 and comprises: MS culture medium, sucrose 30g/L, agar 6g/L, 6-BA0.5mg/L, NAA0.04mg/L, adenine 5.0mg/L; the proliferation culture condition is 3500Lx of illumination intensity and 12h/d of illumination time; the temperature is 24-26 ℃, and the humidity is 80%; the number of propagation cultures was two.
4) Transferring the proliferated seedlings with axillary buds or young tip segments into a rooting culture medium for rooting culture for 25 days to obtain seedlings; the rooting medium has pH of 5.6, and comprises 1/2MS medium, sucrose 30g/L, agar 6g/L, IBA0.8mg/L and active carbon 3g/L; the rooting culture conditions are as follows: the illumination intensity is 3000Lx, and the illumination time is 12h/d; the temperature is 24-26 ℃, the humidity is 80%, and the shading degree is 50%.
5) And (3) hardening seedlings with root systems which grow to be 3-4 cm high and have lengths of about lcm for 2-3 d, transplanting the seedlings to a vermiculite matrix for culturing for 20d, and planting the seedlings in a nutrition pot after the seedlings completely survive to obtain the seedling of the chicory.
Examples 3 to 2
The rooting medium of example 3-2 had a pH of 5.6 and comprised of 1/2MS medium, sucrose 30g/L, agar 6g/L, IBA mg/L, activated carbon 3g/L, and the rest of the treatment was the same as in example 3-1.
Comparative example 3
The rooting medium of comparative example 3 had a pH of 5.6 and comprised of 1/2MS medium, sucrose 30g/L, agar 6g/L, IBA0.5mg/L, activated carbon 3g/L, and the rest of the treatment was the same as in example 3-1.
After the proliferated seedlings with axillary buds or young shoot segments are transferred into a rooting culture medium, the rooting condition of the seedlings is regularly observed, the number of rooting plants is counted when the seedlings are transferred to 10d and 15d, the rooting rate and the rooting amount are counted when the seedlings reach 25d, and the experimental results are shown in table 4:
TABLE 4 rooting conditions of rooting medium
As can be seen from the data in Table 4, the number of roots in the culture medium of example 3-1 was always the highest at 10d and 15d, the statistical rooting rate at 25d was the highest in example 3-1 and reached 100%. Meanwhile, the rooting time of the embodiment 3-1 is relatively centralized, and 96 percent of roots are already rooted at the 15 th day; as can be seen from the rooting effect chart in figure 1, the rooting amount is relatively high, the average value is 3-6, and the rooting rate and the rooting amount are 2-3 times higher than those of the comparative example 3, and the composition of the culture medium in the example 3 can obviously improve the rooting rate and the rooting amount of the proliferated seedlings. FIG. 2 shows that the transplanted seedlings can grow into excellent chicory nodularis on the basis of rooting.
Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and all of the embodiments are included in the scope of the present invention.
Claims (10)
1. A tissue culture method of chicory leaves is characterized by comprising the following steps:
1) Carrying out induction culture on sterile leaf blades of the chicory in an induction culture medium to generate adventitious buds; the induction medium comprises: MS culture medium, 6-BA 0.5-1 mg/L, NAA 0.1-0.2 mg/L, GA 3 1.0mg/L；
2) Transferring the adventitious bud into a proliferation culture medium for proliferation culture to obtain a proliferation seedling; the multiplication medium comprises: MS culture medium, 6-BA0.5mg/L, NAA 0.02-0.06 mg/L and adenine 5.0mg/L;
3) Transferring the proliferated seedling into a rooting culture medium for rooting culture to obtain a seedling, wherein the rooting culture medium comprises a 1/2MS culture medium, 0.8-1 mg/L IBA and 3g/L active carbon;
4) And (3) hardening the seedlings, transplanting the seedlings to a substrate for culture, and obtaining the chicory seedlings with the nodulation.
2. The tissue culture method of the chicory leaf discs of claim 1, wherein the size of the sterile chicory leaf discs is 0.2-0.3 mm.
3. The method for tissue culture of leaves of chicory according to claim 1, wherein the aseptic processing of leaves of chicory comprises the following steps: the leaves are disinfected by 84 disinfectant and alcohol in sequence and washed by distilled water for later use.
4. The tissue culture method of the noded chicory leaves as claimed in claim 1, characterized in that the MS culture medium further comprises sucrose and agar, the concentration of the sucrose is 25-35 g/L, and the concentration of the agar is 5-8 g/L.
5. The method for tissue culture of leaf of chicory of claim 1 or 4, wherein the induction medium comprises MS medium, 6-BA0.5mg/L, NAA 0.1.1 mg/L, GA 3 1.0mg/L。
6. The tissue culture method of the chicory leaf discs of claim 1, wherein the inducing culture conditions are as follows: the illumination intensity is 1500-2000 Lx, and the temperature is 24-26 ℃; the induction culture time is 30d.
7. The method for tissue culture of leaf of chicory according to claim 1 or 4, wherein the multiplication medium comprises MS medium, 6-BA0.5mg/L, NAA 0.04.04 mg/L, adenine 5.0mg/L.
8. The tissue culture method of the chicory leaf discs of claim 1, wherein the proliferation culture conditions are as follows: the illumination intensity is 2000 to 3500Lx, and the illumination time is 12h/d; the temperature is 24-26 ℃, and the humidity is 80%; the proliferation culture time is 20-23 d.
9. The method for tissue culture of leaf of chicory according to claim 1 or 4, wherein the rooting medium comprises 1/2MS medium, IBA0.8mg/L, activated charcoal 3g/L.
10. The tissue culture method of the leaves of chicory, which is characterized in that the rooting culture conditions are as follows: the illumination intensity is 2000-3500 Lx, and the illumination time is 12h/d; the temperature is 24-26 ℃, and the humidity is 80%; the rooting culture time is 10-25 days.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211650540.5A CN115885852A (en) | 2022-12-21 | 2022-12-21 | Tissue culture method for leaf of chicory |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211650540.5A CN115885852A (en) | 2022-12-21 | 2022-12-21 | Tissue culture method for leaf of chicory |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115885852A true CN115885852A (en) | 2023-04-04 |
Family
ID=86478853
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211650540.5A Pending CN115885852A (en) | 2022-12-21 | 2022-12-21 | Tissue culture method for leaf of chicory |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115885852A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117121813A (en) * | 2023-06-30 | 2023-11-28 | 江苏省中国科学院植物研究所 | Efficient chicory regeneration method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1820584A (en) * | 2006-03-13 | 2006-08-23 | 西北大学 | High efficiency quick breeding method for cichorium intybus test tube seedling |
| UA24324U (en) * | 2007-02-21 | 2007-06-25 | Uman State Agrarian University | Method for obtaining reproductive shoots of common chicory in an in vitro culture |
| CN104604689A (en) * | 2015-02-09 | 2015-05-13 | 鲁东大学 | Method for rapidly obtaining chicory explants and improving callus rate of chicory explants |
-
2022
- 2022-12-21 CN CN202211650540.5A patent/CN115885852A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1820584A (en) * | 2006-03-13 | 2006-08-23 | 西北大学 | High efficiency quick breeding method for cichorium intybus test tube seedling |
| UA24324U (en) * | 2007-02-21 | 2007-06-25 | Uman State Agrarian University | Method for obtaining reproductive shoots of common chicory in an in vitro culture |
| CN104604689A (en) * | 2015-02-09 | 2015-05-13 | 鲁东大学 | Method for rapidly obtaining chicory explants and improving callus rate of chicory explants |
Non-Patent Citations (3)
| Title |
|---|
| 张玉等: "植物生长调节剂对菊苣再生的影响", 《草学》, no. 3, pages 10 - 2 * |
| 程林梅等: "菊苣再生体系建立及转AFL2基因的研究", 《草地学报》, vol. 12, no. 3, pages 200 * |
| 程林梅等: "菊苣组织培养与植株再生的研究", 《草业学报》, vol. 11, no. 4, pages 105 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117121813A (en) * | 2023-06-30 | 2023-11-28 | 江苏省中国科学院植物研究所 | Efficient chicory regeneration method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101507415B (en) | In-vitro culture method of antlerpilose grass | |
| KR20150026554A (en) | Development of a micropropagation technique in Prunus avium clones | |
| CN113142059A (en) | Method for synchronously culturing tissue culture buds of galangal flowers and proliferating and rooting | |
| CN115885852A (en) | Tissue culture method for leaf of chicory | |
| CN113080063B (en) | Rapid rooting method for tissue culture of coarse chaff tree | |
| CN110384044B (en) | Cultivation method of virus-free seed stems of taros | |
| CN116267620A (en) | Method for constructing callus induction and adventitious bud regeneration system by utilizing atractylis lancea leaf stalks | |
| CN115968786A (en) | Culture medium and culture method for tea tree tissue culture | |
| CN106613973B (en) | Utilize the method for tissue-cultured seedling leaf regeneration adventitious bud fast breeding Chinese azalea | |
| CN112616675B (en) | Tissue culture and rapid propagation method for Zingiber dance | |
| CN111280058B (en) | Rapid breeding method of detoxified seedlings of stem tips of salvia miltiorrhiza bunge | |
| CN115024221A (en) | Method for rapidly propagating tissue culture seedlings of large-leaf morinda officinalis and application of method | |
| CN114793903A (en) | Breeding method of virus-free strawberry seedlings | |
| CN108112479A (en) | A kind of stem section of papaya sprout Bud Differentiation vacantly plants leaf promoting root growth method | |
| CN113907005A (en) | Simple, convenient, rapid and efficient direct regeneration tissue culture method for mung beans | |
| CN113826549A (en) | Ornamental dendrobium crossbreeding method | |
| CN113875585A (en) | Method for in-vitro rapid propagation and seedling raising of roxburgh rose | |
| CN109362571B (en) | Method for tissue culture and rapid propagation of seedlings through ophiopogon japonicus seeds | |
| CN112931226A (en) | Tissue culture and rapid propagation method for alnus orientalis | |
| Zhai et al. | Shoot multiplication and plant regeneration in Caragana fruticosa (Pall.) Besser | |
| Gabryszewska | Propagation In vitro of hellebores (Helleborus L.) review | |
| CN111328715A (en) | Tissue culture and rapid propagation method for parietal buds of monilia petiolata | |
| CN111316900A (en) | Method for improving transplanting survival rate of virus-free seedlings of purple-meat sweet potatoes | |
| CN111448985A (en) | Tissue culture method of rosa tenuifolia | |
| CN111149703A (en) | Simple, convenient, efficient and high-quality papaya tissue culture seedling rooting method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |