CN113080059A - Tissue culture propagation method of linalool type sassafras - Google Patents
Tissue culture propagation method of linalool type sassafras Download PDFInfo
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Abstract
The application discloses a tissue culture propagation method of linalool type sassafras, which specifically comprises the following steps: collecting an S1 explant, processing the S2 explant, inducing an S3 axillary bud, carrying out proliferation culture on S4, carrying out rooting culture on S5, and carrying out transplantation on S6. Wherein the basic culture medium MS in the steps 3, 4 and 5 adopts electroplating-grade pure water, the sucrose content is 3%, the carrageenan content is 0.75%, the proliferation culture in the step 4 adopts MS +6-BA0.2mg/L-0.5mg/L + NAA0.1mg/L and MS +6-BA0.8mg/L + NAA0.2mg/L culture medium for the step propagation culture, and the efficient and rapid tissue culture propagation method is researched through screening of the hormone type, concentration and proportion of the culture medium. The invention aims to adopt a specific culture medium and carry out sectional taking, breeding and culturing on the buds so as to realize the purpose of quick propagation of the buds.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a tissue culture propagation method of linalool type sassafras.
Background
As is known, the sassafras is an evergreen arbor, the tree pose of the sassafras is beautiful, the trunk of the sassafras is obvious, straight and smooth, the sassafras grows quickly, the average growth amount in breast-to-neck years is 50 percent greater than that of the sassafras, the volume of wood is 100 percent greater, and the sassafras is an excellent fast-growing tree species. The sassafras have luxuriant branches and leaves, are evergreen in all seasons, are resistant to pruning, can be shaped, have good material use, ornamental value, ecological value and economic value, and have continuously expanded cultivation and afforestation areas in recent years.
The existing excellent linalool type sassafras propagation method mainly comprises three methods of seeding, cuttage and tissue culture, wherein the seeding is influenced by seasons and the excellent characters of all the seeded seedlings inherited by mother plants cannot be guaranteed; cuttage is also affected by seasons and has great damage to excellent stock plants; the tissue culture can not be affected by seasons, only a small amount of buds need to be collected, and no harm is caused to excellent stock plants. However, the problems of browning of primary buds, withered yellow necrosis, low bud multiplication coefficient and the like easily occur in the tissue culture process of the sassafras at present.
Disclosure of Invention
In order to solve the above problems in the prior art, the present invention aims to provide a tissue culture propagation method of linalool type sassafras, which aims to adopt a specific culture medium and perform segmental culture and harvest on sprouts so as to achieve the purpose of rapid propagation of the sprouts.
In order to realize the purpose of the invention, the technical scheme adopted by the invention is as follows:
a tissue culture propagation method of linalool type sassafras comprises the following steps:
s1, explant collection: taking a tender branch which is germinated in the current year of a sassafras parent plant growing for many years as an explant;
s2, explant treatment: cutting the explant in the step 1 into stem sections with at least one axillary bud, wherein the length of the stem sections is 0.5cm-1.5cm, putting the stem sections into a refrigerator for refrigeration for 10h-15h by using a closed container, and then taking out the stem sections for cleaning and disinfection for later use;
s3, axillary bud induction: firstly, inoculating the explant obtained in the step 2 into an MS culture medium, inoculating axillary buds into an MS +6-BA2.0mg/L + NAA0.2mg/L culture medium after the axillary buds grow to 0.5cm-1cm, carrying out dark culture for 18-25 days, transferring into the MS +6-BA0.3mg/L + NAA0.1mg/L culture medium, and culturing for 15-20 days to obtain cluster small bud masses;
s4, proliferation culture: cutting the cluster-shaped small bud masses in the step 3 into 3-5 buds/masses, transferring the buds/masses into a multiplication culture medium of MS +6-BA0.2mg/L-0.5mg/L + NAA0.1mg/L for 30 days, continuing the generation in a period of 30 days, generating 5-8 sub-buds once, after the generation is continued for 3-5 generations, selecting a robust individual plant with the length higher than 2.5cm from the bud masses, dividing the stem section of the robust individual plant into sections with the length of 0.3cm, transferring the robust individual plant into a culture medium of MS +6-BA0.8mg/L + NAA0.2mg/L, performing the clustered propagation growth, and performing the propagation in a period of 50 days;
s5, rooting culture: picking up the thick single bud higher than 2.5cm in step 4, and inoculating 1/2MS +
In a rooting culture medium of IAA0.3mg/L, after 15-20 days of culture, the roots begin to sprout, and after 40-45 days, 95% of buds can complete rooting to form seedlings;
s6, transplanting: putting the plantlets obtained in the step 5 into a pergola to harden the plantlets for 10 days, taking the plantlets out of a culture vessel, and transplanting the plantlets into a soil matrix for growth;
the MS culture medium comprises the following components:
720mg/LNHANO3, 1800mg/LKNO3, 200mg/L Ca (NO3)2, 170mg/LKH2PO1, 22.3mg/L MnSO 4.4H 20, 8.6mg/L ZnS 04.7H 20, 6.2mg/L H3BO3, 0.83mg/L KI, 0.25mg/L NazMoO 4.2H 20, 0.025mg/L CuSO 5H20, 0.025mg/L CoCl2 and 27.8mg/L FeSO 4.7H 20, wherein the MH culture medium adopts electroplating grade pure water, the sucrose content is 3%, the carrageenan content is 0.75%, the pH is 5.8, and the culture conditions are light irradiation for 10H/d-12H/d under the environment of 24 ℃ -26 ℃ and the light irradiation intensity of 1500-.
Optionally, the step of sterilizing the explant in step 2 specifically comprises: taking out the explant from the refrigerator, cleaning with sterile water, and cleaning with sterile water on an oscillator for 30-50 min; soaking with alcohol for 0.3-0.8 min, and washing with sterile water for 2-3 times; then sterilizing with 0.1% -0.2% mercuric chloride for 6min-12min, finally washing with sterile water for 3-5 times, and then sucking water with filter paper.
Optionally, the refrigerating temperature of the refrigerator in the step 2 is 4-6 ℃.
Alternatively, the axillary buds in step 3 have a pale green color at the base after culturing in the medium for 18 to 25 days in the dark.
Alternatively, each stem segment of the robust individual plant in the step 4 can be divided into 4-7 segments, and the multiplication multiple can be expanded to 9.0-12.0 times.
Optionally, the shoots in step 5 have a rooting length of 0.4cm to 0.6 cm.
Optionally, the culture medium of the plantlets is washed away after the plantlets are taken out of the culture vessel in step 6.
Optionally, when the clumpy buds appear after 15-20 days in step 4, step 5 is performed after 25-35 days of observation.
Optionally, after obtaining the cluster-shaped bud mass in step 4, cutting off excessive lateral buds and adventitious buds.
Optionally, after hardening off the seedlings for 10 days in the step 6, leaves of the seedlings are green.
Optionally, the explant is inoculated into the MS culture medium in the step 3, and after 36-42 days of culture, the axillary bud is picked.
Optionally, the clay substrate in step 6 comprises 1/3 coconut coir and 2/3 peat soil, and the coconut coir and the peat soil are mixed.
The MS culture medium in the technical scheme of the invention adopts electroplating-grade pure water, and the water quality is stable for a long time without other impurities, so that the phenomena of browning, leaf drop, withering and yellow death and the like of the product in the induction process due to the change of the water quality of the culture medium after the product is put into the culture medium are avoided. Meanwhile, after the explant induces and buds, the buds are cultured in a culture medium to form a small bud mass, then the small bud mass is cut into 3-5 buds/mass, and then the buds are transferred into a proliferation culture medium for proliferation culture, firstly, the small bud mass is cultured in the proliferation culture medium of MS +6-BA0.2mg/L-0.5mg/L + NAA0.1mg/L for 30 days, the regeneration is carried out in a period of 30 days, 5-8 sub-buds are generated by the regeneration once, after the regeneration is carried out for 3-5 generations, a robust single plant higher than 2.5cm is selected from the bud mass, the stem section is divided into sections of 0.3cm section, then the stem section is transferred into the culture medium of MS +6-BA0.8mg/L + NAA0.2mg/L, the robust single plant is grown in a mass propagation manner, the propagation is carried out in a period of 50 days, wherein each stem section of the robust single plant can be divided into 4-7 sections, the proliferation multiple can be increased to 9.0-12 times, and finally, picking up the strong single buds higher than 2.5cm, inoculating the strong single buds into a rooting culture medium, culturing for 15-20 days, and then beginning to root and sprout to form a plantlet which can be planted. Therefore, the robust single plant buds are subjected to segmented harvest culture in the propagation culture process, the propagation efficiency of the buds is greatly improved, and the propagation cost is reduced.
Drawings
FIG. 1 shows tissue-cultured induced buds of Cinnamomum camphora of the present invention;
FIG. 2 shows the tissue-cultured proliferated buds of Cinnamomum camphora of the present invention;
FIG. 3 shows the tissue-cultured rooting buds of cinnamomum camphora of the present invention;
FIG. 4 is a diagram of the transplantation of tissue culture seedlings of cinnamomum camphora of the present invention.
Detailed Description
The present invention is further illustrated below with reference to specific examples.
Example 1
As shown in fig. 1, this embodiment provides a tissue culture propagation method of linalool type sassafras, which includes the following steps:
s1, explant collection: taking a tender branch which is germinated in the current year of a sassafras parent plant growing for many years as an explant;
s2, explant treatment: cutting the explant in the step 1 into stem sections with at least one axillary bud, wherein the length of the stem sections is 0.5cm-1.5cm, putting the stem sections into a refrigerator for cold storage for 12h by using a closed container, and then taking out the stem sections for cleaning and disinfection for later use;
s3, axillary bud induction: firstly, inoculating the explant obtained in the step 2 into an MS culture medium, after 36-42 days, axillary buds grow to 0.5-1 cm, then inoculating the axillary buds into the culture medium of MS +6-BA2.0mg/L + NAA0.2mg/L, carrying out dark culture for 20 days, transferring into the culture medium of MS +6-BA0.3mg/L + NAA0.1mg/L, and culturing for 15 days to obtain cluster small bud masses;
s4, proliferation culture: cutting the cluster-shaped small bud masses in the step 3 into 3-5 buds/masses, transferring the buds/masses into a multiplication culture medium of MS +6-BA0.2mg/L-0.5mg/L + NAA0.1mg/L for 30 days, continuing the generation in a period of 30 days, generating 5-8 child buds once for the continued generation, after the continued generation is carried out for 3-5 generations, selecting a robust individual plant with the length higher than 2.5cm from the bud masses, dividing the stem section into sections with the length of 0.3cm, transferring the robust individual plant into a culture medium of MS +6-BA0.8mg/L + NAA0.2mg/L, carrying out clump propagation growth, and carrying out propagation in a period of 50 days;
s5, rooting culture: picking up the strong single buds higher than 2.5cm in the step 4, inoculating the strong single buds into a rooting culture medium of 1/2MS + IAA0.3mg/L, culturing for 15-20 days, then the roots begin to sprout, and after 40-45 days, 95% of buds can finish rooting to form seedlings;
s6, transplanting: putting the plantlets obtained in the step 5 into a pergola to harden the plantlets for 10 days, taking the plantlets out of a culture vessel, and transplanting the plantlets into 1/3 coconut coir +2/3 peat soil matrix for growth;
the MS culture medium comprises the following components:
720mg/LNHANO3, 1800mg/LKNO3, 200mg/L Ca (NO3)2, 170mg/LKH2PO1, 22.3mg/L MnSO 4.4H 20, 8.6mg/L ZnS 04.7H 20, 6.2mg/L H3BO3, 0.83mg/L KI, 0.25mg/L NazMoO 4.2H 20, 0.025mg/L CuSO 5H20, 0.025mg/L CoCl2 and 27.8mg/L FeSO 4.7H 20, wherein the MH culture medium adopts electroplating grade pure water, the sucrose content is 3%, the carrageenan content is 0.75%, the pH is 5.8, and the culture conditions are light irradiation for 10H/d-12H/d under the environment of 24 ℃ -26 ℃ and the light irradiation intensity of 1500-.
In the embodiment, the tender branches are adopted as the induction objects, so that the induction objects are easy to obtain, the period is short, the rooting rate is high, and the survival is easy. Taking young branches of perennial excellent large-leaf cinnamomum camphora stock plants which germinate in the current year, and cutting the young branches into small sections with length of about 1cm and axillary buds. A rusty scalpel is not used, the time for exposing the wound in the air is reduced, otherwise, the cut of the stem is browned due to the oxidation of polyphenol oxidase and the like, and the seed production difficulty is increased. The harvested stem segments are put into a refrigerator for about 12 hours after being packaged by a closed container, and then are cleaned by sterile water, and then are cleaned by oscillating more than 30min on an oscillator by sterile water. Then soaking with 70% alcohol for about 0.5min, and washing with sterile water for 2-3 times. And sterilizing with 0.1% mercuric chloride for 6min-10min and 0.2% mercuric chloride for 8min-12min respectively according to the size of the material, washing with sterile water for 3-5 times, adsorbing dry water with filter paper, inoculating into MS basic culture medium, and allowing the sterilized axillary bud to grow to about 0.5cm-1cm after 40 days. Picking up axillary buds, inoculating the axillary buds to a culture medium of MS +6-BA2.0mg/L + NAA0.2mg/L, performing dark culture for about 20 days, forming light yellow and green callus with moderate density and good quality, inoculating the callus to the culture medium of MS +6-BA0.3mg/L + NAA0.1mg/L, culturing for about 15 days to generate cluster buds, and cutting the bud groups into 3-5 buds/group for subculture after 30 days.
Selecting a robust individual plant with the length higher than 2.5cm from a bud mass, dividing a stem section of the robust individual plant into sections with the length of 0.3cm, transferring the stem section into a culture medium of MS +6-BA0.8mg/L + NAA0.2mg/L, carrying out conglobation propagation growth, and carrying out propagation in a cycle of 50 days, wherein each stem section of the robust individual plant can be divided into 4-7 sections, and the propagation multiple can be increased to 9.0-12.0 times. That is, each bud mass has 3-5 buds, and then a robust single plant is selected from the bud mass for segmental culture, namely, the plant is divided into 0.3cm segments, each stem segment can be divided into 4-7 segments, and one group can be divided into 20-35 segments, of course, each segment has at least one axillary bud, 5-8 sub-buds are generated in the next generation, and the multiplication multiple of the buds can be expanded to 9.0-12.0 times after the 3-5 generations. Finally, picking up the strong single buds after propagation, inoculating the strong single buds into a rooting culture medium of 1/2MS + IAA0.3mg/L, wherein the roots begin to sprout after 15-20 days, and after 40-45 days, 95% of seedlings grow 2-3 roots with the length of 0.4-0.6cm, thus completing the propagation of the seedlings.
And (3) putting the generated seedlings into a pergola, hardening the seedlings for about 10 days to ensure that the seedlings are fully lignified and the leaf color is changed to green, taking the seedlings out of a culture vessel, washing off a culture medium, transplanting the seedlings into a matrix of 1/3 coconut chaff +2/3 peat soil, keeping the temperature and moisture, and enabling the seedlings to grow normally.
The culture medium formula directly influences the differentiation and growth of explants, the proportion of various growth regulators is particularly critical, axillary buds with the length of 0.5cm-1cm are inoculated into induced callus culture media with different hormone proportions by taking 1 proliferation unit, and the test results are shown in table 1.
TABLE 1 test results of different hormone ratios on calli
From table 1, it can be seen that: axillary buds are more sensitive to 6-BA than KT and 2ip, and KT and 2ip are used at concentrations as high as 6-BA, but the callus formation rate is much lower. The 6-BA is best in the No. 4 culture medium, and the quality of the formed axillary buds is high. According to experimental results, the addition of NAA is easier to induce the callus than IAA, but the concentration of NAA is strictly controlled, otherwise, the vitrification phenomenon of the callus is easy to cause. The slight vitrification phenomenon can be responded by measures such as increasing the hardness of the culture medium, properly performing low-temperature treatment, enhancing illumination, reducing the concentration of ammonium nitrogen, increasing the concentration of nitrate nitrogen and the like.
Example 2
As shown in fig. 2, this embodiment provides a tissue culture propagation method of linalool type sassafras, including the following steps:
s1, explant collection: taking a tender branch which is germinated in the current year of a sassafras parent plant growing for many years as an explant;
s2, explant treatment: cutting the explant in the step 1 into stem sections with at least one axillary bud, wherein the length of the stem sections is 0.5cm-1.5cm, putting the stem sections into a refrigerator for cold storage for 12h by using a closed container, and then taking out the stem sections for cleaning and disinfection for later use;
s3, axillary bud induction: firstly, inoculating the explant obtained in the step 2 into an MS culture medium, after 36-42 days, axillary buds grow to 0.5-1 cm, then inoculating the axillary buds into the culture medium of MS +6-BA2.0mg/L + NAA0.2mg/L, carrying out dark culture for 20 days, transferring into the culture medium of MS +6-BA0.3mg/L + NAA0.1mg/L, and culturing for 15 days to obtain cluster small bud masses;
s4, proliferation culture: cutting the cluster-shaped small bud masses in the step 3 into 3-5 buds/masses, transferring the buds/masses into a multiplication culture medium of MS +6-BA0.2mg/L-0.5mg/L + NAA0.1mg/L for 30 days, continuing the generation in a period of 30 days, generating 5-8 child buds once for the continued generation, after the continued generation is carried out for 3-5 generations, selecting a robust individual plant with the length higher than 2.5cm from the bud masses, dividing the stem section into sections with the length of 0.3cm, transferring the robust individual plant into a culture medium of MS +6-BA0.8mg/L + NAA0.2mg/L, carrying out clump propagation growth, and carrying out propagation in a period of 50 days;
s5, rooting culture: picking up the strong single buds higher than 2.5cm in the step 4, inoculating the strong single buds into a rooting culture medium of 1/2MS + IAA0.3mg/L, culturing for 15-20 days, then the roots begin to sprout, and after 40-45 days, 95% of buds can finish rooting to form seedlings;
s6, transplanting: putting the plantlets obtained in the step 5 into a pergola to harden the plantlets for 10 days, taking the plantlets out of a culture vessel, and transplanting the plantlets into 1/3 coconut coir +2/3 peat soil matrix for growth;
the MS culture medium comprises the following components:
720mg/LNHANO3, 1800mg/LKNO3, 200mg/L Ca (NO3)2, 170mg/LKH2PO1, 22.3mg/L MnSO 4.4H 20, 8.6mg/L ZnS 04.7H 20, 6.2mg/L H3BO3, 0.83mg/L KI, 0.25mg/L NazMoO 4.2H 20, 0.025mg/L CuSO 5H20, 0.025mg/L CoCl2 and 27.8mg/L FeSO 4.7H 20, wherein the MH culture medium adopts electroplating grade pure water, the sucrose content is 3%, the carrageenan content is 0.75%, the pH is 5.8, and the culture conditions are light irradiation for 10H/d-12H/d under the environment of 24 ℃ -26 ℃ and the light irradiation intensity of 1500-.
In the embodiment, the tender branches are adopted as the induction objects, so that the induction objects are easy to obtain, the period is short, the rooting rate is high, and the survival is easy. Taking young branches of perennial excellent large-leaf cinnamomum camphora stock plants which germinate in the current year, and cutting the young branches into small sections with length of about 1cm and axillary buds. A rusty scalpel is not used, the time for exposing the wound in the air is reduced, otherwise, the cut of the stem is browned due to the oxidation of polyphenol oxidase and the like, and the seed production difficulty is increased. The harvested stem segments are put into a refrigerator for about 12 hours after being packaged by a closed container, and then are cleaned by sterile water, and then are cleaned by oscillating on an oscillator for more than 30min by sterile water. Soaking with 70% ethanol for about 0.5min, and washing with sterile water for 2-3 times. Sterilizing with 0.1% mercuric chloride for 6-10min and 0.2% mercuric chloride for 8-12min, washing with sterile water for 3-5 times, sucking dry water with filter paper, inoculating to MS minimal medium, and allowing the sterilized axillary bud to grow to 0.5-1 cm after 40 days. Axillary buds are picked and inoculated into a culture medium of MS +6-BA2.0mg/L + NAA0.2mg/L, after dark culture is carried out for about 20 days, the base parts of the axillary buds form light yellow and green callus with moderate density and good quality, the callus is inoculated into the culture medium of MS +6-BA0.3mg/L + NAA0.1mg/L, and cluster buds appear after culture is carried out for about 15 days. After 30 days, the bud clusters are cut into 3-5 buds/cluster for subculture. Selecting a robust individual plant with the length higher than 2.5cm from a bud mass, dividing a stem section of the robust individual plant into sections with the length of 0.3cm, transferring the stem section into a culture medium of MS +6-BA0.8mg/L + NAA0.2mg/L, carrying out conglobation propagation growth, and carrying out propagation in a cycle of 50 days, wherein each stem section of the robust individual plant can be divided into 4-7 sections, and the propagation multiple can be increased to 9.0-12.0 times. Finally, picking up the strong single buds after propagation, inoculating the strong single buds into a rooting culture medium of 1/2MS + IAA0.3mg/L, wherein the roots begin to sprout after 15-20 days, and after 40-45 days, 95% of seedlings grow 2-3 roots with the length of 0.4-0.6cm, thus completing the propagation of the seedlings.
And (3) putting the generated seedlings into a pergola, hardening the seedlings for about 10 days to ensure that the seedlings are fully lignified and the leaf color is changed to green, taking the seedlings out of a culture vessel, washing off a culture medium, transplanting the seedlings into a matrix of 1/3 coconut chaff +2/3 peat soil, keeping the temperature and moisture, and enabling the seedlings to grow normally.
Experimental surface the induction of shoots by different combinations of hormones varied, as shown in table 2:
TABLE 2 test results of different hormone combinations versus bud induction
By combining the data analysis, the culture medium MS +6-BA1.0mg/L + IBA0.5mg/L has better effect of inducing and sprouting from the callus. The auxin aspect is slightly superior to the IAA from the data aspect. In conclusion, the culture medium No. d is the best, the number of buds is the most, the callus grows obviously and the quality is not deteriorated.
Example 3
As shown in fig. 3 and fig. 4, this embodiment provides a tissue culture propagation method of linalool type sassafras, which includes the following steps:
s1, explant collection: taking a tender branch which is germinated in the current year of a sassafras parent plant growing for many years as an explant;
s2, explant treatment: cutting the explant in the step 1 into stem sections with at least one axillary bud, wherein the length of the stem sections is 0.5cm-1.5cm, putting the stem sections into a refrigerator for cold storage for 12h by using a closed container, and then taking out the stem sections for cleaning and disinfection for later use;
s3, axillary bud induction: firstly, inoculating the explant obtained in the step 2 into an MS culture medium, after 36-42 days, axillary buds grow to 0.5-1 cm, then inoculating the axillary buds into the culture medium of MS +6-BA2.0mg/L + NAA0.2mg/L, carrying out dark culture for 20 days, transferring into the culture medium of MS +6-BA0.3mg/L + NAA0.1mg/L, and culturing for 15 days to obtain cluster small bud masses;
s4, proliferation culture: cutting the cluster-shaped small bud masses in the step 3 into 3-5 buds/masses, transferring the buds/masses into a multiplication culture medium of MS +6-BA0.2mg/L-0.5mg/L + NAA0.1mg/L for 30 days, continuing the generation in a period of 30 days, generating 5-8 child buds once for the continued generation, after the continued generation is carried out for 3-5 generations, selecting a robust individual plant with the length higher than 2.5cm from the bud masses, dividing the stem section into sections with the length of 0.3cm, transferring the robust individual plant into a culture medium of MS +6-BA0.8mg/L + NAA0.2mg/L, carrying out clump propagation growth, and carrying out propagation in a period of 50 days;
s5, rooting culture: picking up the strong single buds higher than 2.5cm in the step 4, inoculating the strong single buds into a rooting culture medium of 1/2MS + IAA0.3mg/L, culturing for 15-20 days, then the roots begin to sprout, and after 40-45 days, 95% of buds can finish rooting to form seedlings;
s6, transplanting: putting the plantlets obtained in the step 5 into a pergola to harden the plantlets for 10 days, taking the plantlets out of a culture vessel, and transplanting the plantlets into 1/3 coconut coir +2/3 peat soil matrix for growth;
the MS culture medium comprises the following components:
720mg/LNHANO3, 1800mg/LKNO3, 200mg/L Ca (NO3)2, 170mg/LKH2PO1, 22.3mg/L MnSO 4.4H 20, 8.6mg/L ZnS 04.7H 20, 6.2mg/L H3BO3, 0.83mg/L KI, 0.25mg/L NazMoO 4.2H 20, 0.025mg/L CuSO 5H20, 0.025mg/L CoCl2 and 27.8mg/L FeSO 4.7H 20, wherein the MH culture medium adopts electroplating grade pure water, the sucrose content is 3%, the carrageenan content is 0.75%, the pH is 5.8, and the culture conditions are light irradiation for 10H/d-12H/d under the environment of 24 ℃ -26 ℃ and the light irradiation intensity of 1500-.
In the embodiment, the tender branches are adopted as the induction objects, so that the induction objects are easy to obtain, the period is short, the rooting rate is high, and the survival is easy. Taking young branches of perennial excellent large-leaf cinnamomum camphora stock plants which germinate in the current year, and cutting the young branches into small sections with length of about 1cm and axillary buds. A rusty scalpel is not used, the time for exposing the wound in the air is reduced, otherwise, the cut of the stem is browned due to the oxidation of polyphenol oxidase and the like, and the seed production difficulty is increased. The harvested stem segments are put into a refrigerator for about 12 hours after being packaged by a closed container, and then are cleaned by sterile water, and then are cleaned by oscillating on an oscillator for more than 30min by sterile water. Soaking with 70% ethanol for about 0.5min, and washing with sterile water for 2-3 times. Sterilizing with 0.1% mercuric chloride for 6-10min and 0.2% mercuric chloride for 8-12min, washing with sterile water for 3-5 times, sucking dry water with filter paper, inoculating to MS minimal medium, and allowing the sterilized axillary bud to grow to 0.5-1 cm after 40 days. Axillary buds are picked and inoculated into a culture medium of MS +6-BA2.0mg/L + NAA0.2mg/L, after dark culture is carried out for about 20 days, the base parts of the axillary buds form light yellow and green callus with moderate density and good quality, the callus is inoculated into the culture medium of MS +6-BA0.3mg/L + NAA0.1mg/L, and cluster buds appear after culture is carried out for about 15 days. After 30 days, the bud clusters are cut into 3-5 buds/cluster for subculture. Selecting a robust individual plant with the length higher than 2.5cm from a bud mass, dividing a stem section of the robust individual plant into sections with the length of 0.3cm, transferring the stem section into a culture medium of MS +6-BA0.8mg/L + NAA0.2mg/L, carrying out conglobation propagation growth, and carrying out propagation in a cycle of 50 days, wherein each stem section of the robust individual plant can be divided into 4-7 sections, and the propagation multiple can be increased to 9.0-12.0 times. Finally, picking up the strong single buds after propagation, inoculating the strong single buds into a rooting culture medium of 1/2MS + IAA0.3mg/L, wherein the roots begin to sprout after 15-20 days, and after 40-45 days, 95% of seedlings grow 2-3 roots with the length of 0.4-0.6cm, thus completing the propagation of the seedlings.
And (3) putting the generated seedlings into a pergola, hardening the seedlings for about 10 days to ensure that the seedlings are fully lignified and the leaf color is changed to green, taking the seedlings out of a culture vessel, washing off a culture medium, transplanting the seedlings into a matrix of 1/3 coconut chaff +2/3 peat soil, keeping the temperature and moisture, and enabling the seedlings to grow normally.
Experiments show that after 3-5 generations are continued by using the selected multiplication culture medium, a robust single plant with the height of more than 2.5CM is selected from a bud cluster, the stem section of the robust single plant is divided into sections with the length of about 0.3 CM/section, the sections are clustered and propagated by using the 6-BA culture medium with higher concentration, and the period is 50 days. Because each individual stem segment can be divided into 4-7 segments, the proliferation rate can be doubled and the quality of the seedlings can be ensured by multi-generation observation. Thereby achieving the purpose of mass production and continuous and stable seedling emergence, and the experimental results are shown in the table 3:
TABLE 3 growth morphology of robust individual plants in stages throughout the growth medium
As can be seen from Table 3, treatment medium No. 13 was the best for the proliferation of the segments by clumping.
Therefore, the tissue culture medium of the cinnamomum camphora of the present invention is selected as follows: the callus induction is preferably performed by using a culture medium of MS +6-BA2.0mg/L + NAA0.2mg/L; the bud induction from the callus is preferably performed by MS +6-BA0.3mg/L + NAA0.1mg/L culture medium; the continuous generation culture preferably comprises MS +6-BA0.2-0.5mg/L + NAA0.1mg/L culture medium; the rooting culture is preferably carried out in a medium of 1/2MS + IAA0.3, and all the above media are pure electroplating water.
Because linalool and other alcohol substances are easy to cause browning and withering leaf death of materials in the process of inducing callus, and the materials can slowly turn black and die even if the callus is induced, the culture medium is changed into pure water, the browning condition is greatly improved under the condition that an antioxidant and an adsorbent for preventing browning are not added, the callus with good quality is successfully induced, and buds are induced from the callus. It is also possible to add Vc or activated carbon to the medium to improve browning.
The present invention is not limited to the above-described alternative embodiments, and various other forms of products can be obtained by anyone in light of the present invention. The above detailed description should not be taken as limiting the scope of the invention, which is defined in the claims, and which the description is intended to be interpreted accordingly.
Claims (12)
1. A tissue culture propagation method of linalool type sassafras is characterized by comprising the following steps:
s1, explant collection: taking a tender branch which is germinated in the current year of a sassafras parent plant growing for many years as an explant;
s2, explant treatment: cutting the explant in the step 1 into stem sections with at least one axillary bud, wherein the length of the stem sections is 0.5cm-1.5cm, putting the stem sections into a refrigerator for refrigeration for 10h-15h by using a closed container, and then taking out the stem sections for cleaning and disinfection for later use;
s3, axillary bud induction: firstly, inoculating the explant obtained in the step 2 into an MS culture medium, inoculating axillary buds into an MS +6-BA2.0mg/L + NAA0.2mg/L culture medium after the axillary buds grow to 0.5cm-1cm, carrying out dark culture for 18-25 days, transferring into the MS +6-BA0.3mg/L + NAA0.1mg/L culture medium, and culturing for 15-20 days to obtain cluster small bud masses;
s4, proliferation culture: cutting the cluster-shaped small bud masses in the step 3 into 3-5 buds/masses, transferring the buds/masses into a multiplication culture medium of MS +6-BA0.2mg/L-0.5mg/L + NAA0.1mg/L for 30 days, continuing the generation in a period of 30 days, generating 5-8 child buds once for the continued generation, after the continued generation is carried out for 3-5 generations, selecting a robust individual plant with the length higher than 2.5cm from the bud masses, dividing the stem section into sections with the length of 0.3cm, transferring the robust individual plant into a culture medium of MS +6-BA0.8mg/L + NAA0.2mg/L, carrying out clump propagation growth, and carrying out propagation in a period of 50 days;
s5, rooting culture: picking up the strong single buds higher than 2.5cm in the step 4, inoculating the strong single buds into a rooting culture medium of 1/2MS + IAA0.3mg/L, culturing for 15-20 days, then the roots begin to sprout, and after 40-45 days, 95% of buds can finish rooting to form seedlings;
s6, transplanting: putting the plantlets obtained in the step 5 into a pergola to harden the plantlets for 10 days, taking the plantlets out of a culture vessel, and transplanting the plantlets into a soil matrix for growth;
the MS culture medium comprises the following components:
720mg/LNHANO3, 1800mg/LKNO3, 200mg/L Ca (NO3)2, 170mg/LKH2PO1, 22.3mg/L MnSO 4.4H 20, 8.6mg/L ZnS 04.7H 20, 6.2mg/L H3BO3, 0.83mg/L KI, 0.25mg/L NazMoO 4.2H 20, 0.025mg/L CuSO 5H20, 0.025mg/L CoCl2 and 27.8mg/L FeSO 4.7H 20, wherein the MH culture medium adopts electroplating grade pure water, the sucrose content is 3%, the carrageenan content is 0.75%, the pH is 5.8, and the culture conditions are light irradiation for 10H/d-12H/d under the environment of 24 ℃ -26 ℃ and the light irradiation intensity of 1500-.
2. The tissue culture propagation method of linalool type sassafras according to claim 1, wherein the step of disinfecting the explant in the step 2 is specifically as follows: taking out the explant from the refrigerator, cleaning with sterile water, and cleaning with sterile water on an oscillator for 30-50 min; soaking with alcohol for 0.3-0.8 min, and washing with sterile water for 2-3 times; then sterilizing with 0.1% -0.2% mercuric chloride for 6min-12min, finally washing with sterile water for 3-5 times, and then sucking water with filter paper.
3. The tissue culture propagation method of the linalool type sassafras according to claim 1, wherein the refrigeration temperature of the refrigerator in the step 2 is 4-6 ℃.
4. The tissue culture propagation method of linalool type sassafras according to claim 1, wherein the base part is light green after the axillary buds are cultured in the culture medium in the dark for 18 to 25 days in step 3.
5. The tissue culture propagation method of linalool type sassafras according to claim 1, wherein each stem segment of the stout single plant in step 4 can be divided into 4-7 segments, and the proliferation multiple can be amplified to 9.0-12.0 times.
6. The tissue culture propagation method of linalool type sassafras according to claim 1, wherein the rooting length of the bud in step 5 is 0.4cm-0.6 cm.
7. The tissue culture propagation method of linalool type sassafras according to claim 1, wherein the culture medium of the plantlets is washed away after the plantlets are taken out from the culture vessel in step 6.
8. The tissue culture propagation method of linalool type sassafras according to claim 1, wherein step 5 is performed after observing for 25-35 days when cluster-shaped buds appear after 15-20 days in step 4.
9. The tissue culture propagation method of linalool type sassafras according to claim 1, wherein after the cluster-shaped bud masses in step 4 are obtained, the excess lateral buds and adventitious buds need to be cut off.
10. The tissue culture propagation method of linalool type sassafras according to claim 1, wherein leaves of the seedlings are green after hardening for 10 days in step 6.
11. The tissue culture propagation method of linalool type sassafras according to claim 1, wherein the axillary buds are picked after the explants are inoculated into the MS culture medium in step 3 and cultured for 36-42 days.
12. The tissue culture propagation method of linalool type sassafras according to claim 1, wherein the clay substrate in step 6 comprises 1/3 coconut coir and 2/3 peat soil, and the coconut coir and the peat soil are mixed.
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