CN112715367A - Method for carrying out tissue culture subculture propagation on cornus walteri by utilizing lanthanum nitrate - Google Patents
Method for carrying out tissue culture subculture propagation on cornus walteri by utilizing lanthanum nitrate Download PDFInfo
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Abstract
The invention belongs to the technical field of forest tissue culture and breeding, and provides a method for tissue culture and subculture propagation of cornus walteri by utilizing lanthanum nitrate in order to solve the problems of low propagation rate, poor propagation quality and the like of the conventional cornus walteri tissue culture system. Adopting MS as a basic culture medium, taking young stem segments of cornus walteri as explants, inducing axillary buds, and transferring the axillary buds to a medium added with 0.2 mg.L‑1Subculture proliferation is carried out in a proliferation culture medium of lanthanum nitrate to induce and generate a large amount of axillary buds and adventitious buds, and the proliferation rate and the proliferation coefficient respectively reach 91.5 percent and 18.1 percent. The method can obtain a large amount of secondary propagation materials in a short time, remarkably improves the tissue culture secondary propagation effect of the cornus walteri, and shortens the propagation period of the secondary buds, thereby reducing the production cost of the tissue culture industrialized seedling of the cornus walteri.
Description
Technical Field
The invention belongs to the technical field of forest tissue culture and breeding, and particularly relates to a method for carrying out tissue culture subculture propagation on cornus walteri by using lanthanum nitrate.
Background
Cornus walteri (A)Cornus walteriWanger), also named as blackwood 26891 ], seed, chartrema and the like, belong to cornus of dogwood family, and are intensively distributed in Shanxi, Shandong, Shaanxi, Henan and the like. The oil content of the fruits of cornus walteri is 31.8-41.3%, the oil content of the fruit peels is 24.9-25.7%, and the cornus walteri can be used for producing novel woody edible oil, health products and biodiesel for development and is an important biomass energy tree species. The tree has drought resistance, barren resistance, strong adaptability, strong root system and low requirement on soil, and is a good tree species for greening barren mountains and maintaining water and soil; the tree shape is beautiful, the branches and leaves are dense, and the white and black fruits can be used as landscaping tree species.
The method has the advantages that the walter dogwood is developed vigorously, the cultivation area is enlarged, the base forest of raw materials is cultured, and a large number of high-quality strong seedlings are required to be provided. In recent years, cornus walteri has been listed as deeply developed rural plants in Beijing, cornus walteri has been listed as a key developed tree species in Shandong province, and cornus walteri has been developed and utilized as a valuable rural tree species in Shanxi province. At present, the propagation mode of cornus walteri is mainly seed propagation, but the seed germination rate is low [1-2], the seedlings emerge unevenly, the phenomenon of seedling emergence every other year exists, the propagation time is long, variation is easy to generate, and the excellent characteristics of the variety are not easy to maintain; although cuttage propagation can be adopted, the survival rate is low, rooting is very difficult, and the method is limited by seasons and resources; the survival rate of grafting is low, and the method is not suitable for large-scale propagation. Therefore, there is a great need to search an effective method for breeding cornus walteri. The method has the advantages of simple plant tissue culture operation and high propagation speed, can greatly shorten the period of woody plants, is favorable for the popularization and large-scale production of the plants, and is an inevitable choice for rapidly propagating cornus walteri on a large scale.
At present, the research on the tissue culture of the cornus walteri is less at home and abroad. Only Zhang Dan and other [3] are used for researching influence factors of primary culture of walter Cornus walteri; the students of Bowman sweet and the like [4] establish a tissue culture system of a fine clone of Cornus walteri. However, the existing research has the defects of low proliferation rate, poor proliferation quality and the like, and the requirement of large-scale production cannot be met quickly and efficiently.
Disclosure of Invention
The invention provides a method for carrying out tissue culture subculture propagation on cornus walteri by utilizing lanthanum nitrate, aiming at solving the problems of low propagation rate, poor propagation quality and the like of the conventional cornus walteri tissue culture system.
The invention is realized by the following technical scheme: a method for tissue culture subculture proliferation of Cornus walteri by using lanthanum nitrate comprises adopting MS as basic culture medium, using young stem of Cornus walteri as explant, inducing axillary bud, and transferring axillary bud to the medium containing 0.2 mg.L-1Subculture proliferation culture is carried out in a proliferation culture medium of lanthanum nitrate, and axillary buds and adventitious buds are induced to generate.
The method comprises the following specific steps:
(1) selecting an explant: collecting current-year branches for water culture in 1-3 months to obtain young stem segments of 4-5 cm; or selecting robust young stem section without diseases and insect pests as explant material in 4-5 months;
(2) and (3) explant sterilization: soaking the obtained young stem with 75% alcohol solution for 30s, washing with sterile water for 3 times, draining, and adding 0.1% HgCI2Sterilizing in solution for 4-5 min, washing with sterile water for 5 times, and cutting into 1.5-18cm of stem segments, each with at least one axillary bud for use;
(3) primary culture: inoculating the cut stem segments on a primary culture medium for primary culture, wherein the primary culture medium is as follows: MS + BA 0.5 mg.L-1-1.0 mg·L-1 + NAA 0.1 mg·L-1 + IBA 0.1 mg·L-1+ sucrose 20 g.L-1+ agar 6.5 g.L-1(ii) a The pH value of the culture medium is 5.8-5.9, the culture temperature is 25 +/-2 ℃, the humidity is 60 +/-2%, the illumination intensity is 2500 + 3000lx, the illumination time is 12-14h/d, the culture time is 20-30 days, and when the axillary buds germinate to 3cm, the axillary buds are cut off for later use;
(4) subculture multiplication culture: transferring the axillary buds cut off in the step (3) into a subculture multiplication medium for culturing, wherein the subculture multiplication medium comprises: MS + BA 1.0 mg.L-1+ NAA 0.2 mg·L-1+La(NO3)3·6H2O 0.2 mg·L-1+ sucrose 20 g.L-1+ agar 6.5 g.L-1(ii) a The pH value of the culture medium is 5.8-5.9, the culture temperature is 25 +/-2 ℃, the humidity is 60 +/-2%, the illumination intensity is 2500 + 3000lx, the illumination time is 12-14h/d, and the culture time is 20-30 days, cluster buds are formed around the base part of the stem segment, axillary buds are germinated, the stem segment is cut into segments with stem nodes, and the test-tube plantlet is propagated in an expanding way.
In the invention, BA is plant growth hormone 6-benzylamino adenine, has the main functions of promoting the formation of buds, inducing callus to generate, promoting cell division, promoting differentiation of non-differentiated tissues, promoting accumulation of substances in organisms, promoting lateral bud generation and preventing aging, and is cytokinin in plant tissues and cell culture.
IBA is a plant growth hormone indolebutyric acid, is mainly used for rooting of cuttings, can induce the formation of root protomer, promote cell differentiation and division, is beneficial to the generation of new roots and the differentiation of vascular bundle systems, and promotes the formation of adventitious roots of the cuttings.
NAA is plant growth hormone naphthylacetic acid, can be used for plant tissue culture and rapid propagation when the plant is propagated by using a cutting method, can promote cell division and expansion, induce to form adventitious roots, increase fruit setting, prevent fruit drop, change the ratio of female flowers and male flowers and the like, can enter the plant through the tender epidermis of leaves and branches, and can be guided to the whole plant along with nutrient flow.
The method can obtain a large amount of secondary propagation materials in a short time, remarkably improves the tissue culture secondary propagation effect of the cornus walteri, and shortens the propagation period of the secondary buds, thereby reducing the production cost of the tissue culture industrialized seedling of the cornus walteri.
According to the method, lanthanum nitrate with a specific concentration is used for carrying out tissue culture subculture proliferation on the cornus walteri, MS is used as a basic culture medium, and the optimal culture medium components and the optimal proportion are obtained by screening a primary culture medium and a proliferation culture medium. Adopting young and tender stems of cornus walteri to perform start culture, adding BA, NAA and IBA into an MS culture medium to perform regulation and control, and inducing axillary buds to germinate and grow; adding BA, NAA, and La (NO) with appropriate concentration into MS culture medium3)3Regulating the subculture multiplication of axillary buds and establishing a high-efficiency and stable subculture multiplication method. The subculture multiplication method has the advantages of short culture time, high multiplication efficiency and highest multiplication coefficient of 18.1, can provide a large amount of high-quality cornus walteri test-tube plantlets with consistent hereditary characters in a short period, and meets the requirements of large-scale industrialized seedling culture and industrialized development.
Drawings
FIG. 1 is a drawing of axillary bud induction of Cornus walteri stems;
FIG. 2 is a drawing of a multiplication culture of Cornus walteri test-tube plantlets;
fig. 3 is a picture of cornus walteri test-tube plantlet.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments; all other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
1. Test materials: and transferring axillary buds induced by the cornus walteri stems to a multiplication culture medium for subculture multiplication culture. The method adopts a single-factor completely random design, and the process is repeated for 3 times for each 20 bottles.
2. The experimental method comprises the following steps:
A. adding TDZ alone or in combination with NAA, IBA and 2,4-D for culture: transferring the induced axillary buds to an MS basic culture medium, adding TDZ independently or combining with NAA, IBA and 2,4-D respectively, counting the proliferation rate and the proliferation coefficient of the test-tube plantlet after 4 weeks, and researching the influence of adding TDZ independently or combining with NAA, IBA and 2,4-D on the proliferation of the cornus walteri test-tube plantlet.
B. Adding BA alone or in combination with NAA and 2,4-D for culture: transferring the induced axillary buds to an MS minimal medium, adding BA alone or combining with NAA and 2,4-D respectively, counting the proliferation rate and proliferation coefficient of the test-tube plantlet after 4 weeks, and researching the influence of adding BA alone or combining with NAA and 2,4-D on the proliferation of the cornus walteri test-tube plantlet.
C. And (3) performing combined culture of lanthanum nitrate, BA, NAA and IBA: transferring the induced axillary buds to an MS basic culture medium, adding a combination of BA, NAA, IBA and lanthanum nitrate, counting the proliferation rate and the proliferation coefficient of the test-tube plantlet after 4 weeks, and researching the influence of the combination of BA, NAA, IBA and lanthanum nitrate on the proliferation of the cornus walteri test-tube plantlet.
D. And (3) performing combined culture of lanthanum nitrate, BA and NAA with different concentrations: transferring the induced axillary buds to MS minimal medium, adding 1.0 mg.L-1 BA、0.2 mg·L-1 NAA, lanthanum nitrate (0.05, 0.1, 0.2, 0.5, 1.0 mg. L) was added to each of the solutions at different concentrations-1) And after 4 weeks, counting the proliferation rate and proliferation coefficient of the test-tube plantlets, and researching the influence of the combination of lanthanum nitrate, BA and NAA with different concentrations on the proliferation of the cornus walteri test-tube plantlets.
E. And (3) proliferation cycle selection: taking the selected optimal culture medium as a basic culture medium, respectively transferring the test-tube plantlets cultured for 20d, 30d and 40d to a new culture medium for propagation culture, counting the propagation rate and the propagation coefficient of the test-tube plantlets after 4 weeks, and researching the influence of the propagation period on the propagation of the cornus walteri test-tube plantlets.
F. The culture conditions are as follows: the culture temperature is 25 +/-2 ℃, the illumination intensity is 2500 lx-3000 lx, and the illumination time is 14-16 h/d; sucrose (20 g. L) was added to all media-16.5 g.L of agar-1。
3. Data statistics and analysis: the data obtained from the experiment were analyzed for variance using SPSS 17.0 and the mean was tested for significance of differences using the Duncan multiple comparison method. The specific indexes are calculated as follows:
4. results and analysis
A. Influence of TDZ alone or in combination with NAA, IBA and 2,4-D on the proliferation of Cornus walteri test-tube plantlets: the test researches the influence of TDZ added alone or combined with NAA, IBA and 2,4-D on the proliferation of the cornus walteri test-tube plantlet. The results are shown in Table 1, and show that only a small amount of yellowish or whitish, soft callus is differentiated from the base of the test-tube plantlet, and the test-tube plantlet is not proliferated (Table 1). Therefore, TDZ alone or in combination with NAA, IBA and 2,4-D is not suitable for propagation culture of cornus walteri test-tube plantlets.
TABLE 1 Effect of TDZ or in combination with NAA, IBA, 2,4-D on test-tube plantlet proliferation
B. The influence of the single addition of BA or the combination of BA and NAA and 2,4-D on the proliferation of the cornus walteri test-tube plantlet is as follows: the experiment researches the influence of adding BA alone or respectively combining with NAA and 2,4-D on the proliferation of the cornus walteri test-tube plantlet. The research results are shown in table 2, and the results show that only a small amount of light green, compact or light white and loose callus is differentiated from the base of the test-tube plantlet; when the concentration of BA is too high (not less than 3.0 mg. L)-1) In the meantime, stem segment was vitrified, and the base was enlarged, but neither of them achieved test-tube plantlet proliferation. Therefore, the addition of BA alone or in combination with NAA and 2,4-D in the culture medium is not suitable for propagation culture of cornus walteri test-tube plantlets.
TABLE 2 Effect of BA or combination with NAA, 2,4-D on test-tube plantlet proliferation
C. Influence of lanthanum nitrate in combination with BA, NAA and IBA on the proliferation of Cornus walteri test-tube plantlets: this experiment investigated nitric acidInfluence of lanthanum in combination with BA, NAA and IBA on the proliferation of Cornus walteri test-tube plantlet. Analysis of variance shows that the lanthanum nitrate and BA, NAA and IBA with different concentrations are combined to the multiplication rate of cornus walteri test-tube plantlets (P<0.001), growth factor: (P= 0.002) effect was significant. The multiple comparison results are shown in Table 3.
TABLE 3 multiple comparison of lanthanum nitrate in combination with BA, NAA, IBA for test-tube plantlet proliferation
In all the combinations, the base parts of the test-tube plantlets are differentiated into a small amount of light green and compact callus tissues and are differentiated into a large amount of axillary buds, so that the test-tube plantlets grow well and belong to a cluster bud generation type. The results are shown in Table 3, and show that when 0.2 mg.L-1Lanthanum nitrate and 1.0 mg. L-1 When BA is combined, the proliferation rate is 47.4 percent, and the proliferation coefficient is 3.7; when 0.2 mg.L-1Lanthanum nitrate and 1.0 mg. L-1 BA、0.2 mg·L-1 When NAA is combined, the proliferation rate is the highest and is 91.5%, and the proliferation coefficient is 18.1; when 0.2 mg.L-1Lanthanum nitrate and 0.5 mg. L-1 BA、0.05 mg·L-1 When NAA is combined, the proliferation rate is 64.2 percent, and the proliferation coefficient is 4.2; when 0.2 mg.L-1Lanthanum nitrate and 0.5 mg. L-1 BA、0.05 mg·L-1 NAA、0.1 mg·L-1 The IBA combination showed a growth rate of 70.4% and a growth coefficient of 5.4 (Table 3).
As can be seen, when 0.2 mg.L was added to the medium-1Lanthanum nitrate and 1.0 mg. L-1 BA、0.2 mg·L-1 NAA combination is the best multiplication culture medium of cornus walteri test-tube plantlets, and belongs to the cluster bud generation type.
D. Influence of combination of lanthanum nitrate with different concentrations, BA and NAA on proliferation of walter dogwood test-tube plantlet: in order to further screen the optimal concentration of lanthanum nitrate, the test researches the influence of the combination of lanthanum nitrate with different concentrations, BA and NAA on the proliferation of cornus walteri test-tube plantlets. The result of the anova shows that the combination of lanthanum nitrate with different concentrations, BA and NAA can increase the multiplication rate of the cornus walteri test-tube plantlets (P<0.001), growth factor: (P= 0.015) effect was significant. The multiple comparison results are shown in Table 4.
TABLE 4 multiple comparison of lanthanum nitrate with BA, NAA combinations at different concentrations for test-tube plantlet proliferation
Research results show that the proliferation rate and the proliferation coefficient both show a trend of increasing first and then decreasing with the increase of the concentration of lanthanum nitrate. When 0.05 mg.L-1Lanthanum nitrate and 1.0 mg. L-1 BA、0.2 mg·L-1 When NAA is combined, the proliferation rate is 40.2 percent, and the proliferation coefficient is 7.7; as the concentration of lanthanum nitrate is increased, the proliferation rate and the proliferation coefficient are gradually increased. When the concentration of lanthanum nitrate is increased to 0.2 mg.L-1When the cell growth rate is higher than that of the cell growth rate, the proliferation rate and the proliferation coefficient are both the highest and are respectively 91.5 percent and 18.1 percent; when the concentration of lanthanum nitrate continues to increase, the proliferation rate and the proliferation coefficient begin to decrease; when the concentration of lanthanum nitrate is 1.0 mg.L-1When the growth rate was high, the growth rate and the growth coefficient were low, and 72.6% and 8.2, respectively.
As can be seen, when 0.2 mg.L was added to the medium-1Lanthanum nitrate and 1.0 mg. L-1 BA、0.2 mg·L-1 NAA composition is the best propagation culture medium for Cornus walteri test-tube plantlets.
E. Influence of proliferation cycle on proliferation of cornus walteri test-tube plantlets: the test uses MS +1.0 mg.L-1 BA+0.2 mg·L-1 NAA+0.2 mg·L-1 Lanthanum nitrate is used as a basic culture medium to study the influence of different proliferation cycles on the proliferation of test-tube plantlets. The results of the anova are shown in table 5; the results show that the proliferation cycle is related to the proliferation rate of the test-tube plantlet: (P= 0.074) without significant effect on the proliferation factor: (P= 0.025) was significant.
TABLE 5 multiple comparison of proliferation cycles to proliferation of test-tube plantlets
Researches show that when the proliferation cycle is 20d and 30d, the test-tube plantlet starts to enter an obvious growth stage after being transferred for 4d-5d, grows to the highest peak after 20d and can be sustained to about 30d, the proliferation rates are high and are respectively 91.5% and 89.9%, and the proliferation coefficients are respectively 18.1 and 17.7; the test-tube plantlets transferred by 40 days of culture are obviously inferior to the test-tube plantlets in growth, the proliferation rate is slightly lower and is 79.8%, and the proliferation coefficient is 10.4. Therefore, 20 d-30 d is the optimal multiplication period of the cornus walteri test-tube plantlets.
The minimal medium can only keep the axillary bud induction or adventitious bud induction, but the key factors determining the test-tube plantlet proliferation are the type, concentration and proportion of plant growth regulators, particularly the concentration ratio of cytokinin and auxin. Generally, when the concentration ratio of the two is high, the bud differentiation is facilitated, when the concentration ratio is moderate, the bud growth is facilitated, and when the concentration ratio is low, the rooting is facilitated. Therefore, only by adding a certain proportion of plant growth regulator into the culture medium, the effective proliferation of the test-tube plantlet can be ensured, thereby obtaining more tissue culture plantlets in a shorter time.
The differentiation and proliferation of different plant materials varies widely with different cytokinin and auxin combinations, but most species are most effective with BA. Wangqiqing et al [5]In tissue culture of cornus stolonifera, the adventitious bud is found to be 1/2MS +0.5 mg.L-1 BA+0.25 mg·L-1 IBA medium can be effectively proliferated. Zhao Qing Hua [ 6)]It was found in the proliferation culture of the Reunion test-tube plantlet that the proliferation of the shoots was expressed as M (CaCl in MS medium)2·2H2The concentration of O is 1200 mg.L-1) Adding 0.5 mg.L into the minimal medium-1 BA and 0.05 mg. L-1 IBA, the cluster bud grows best, the multiplication rate reaches 73.5%, and the multiplication coefficient is 2.32. However, in the research, TDZ is added into the culture medium alone or combined with NAA, IBA and 2,4-D respectively, and only a small amount of yellowish or pale and soft callus is differentiated from the base of the test-tube plantlet, so that the test-tube plantlet proliferation is not realized; the BA is added independently or combined with NAA and 2,4-D respectively, only a small amount of light green, compact or light white loose callus is differentiated from the base of the test-tube plantlet, and the test-tube plantlet proliferation is not realized.
The proper amount of rare earth elements can increase the content of endogenous hormones in plants, thereby promoting the differentiation and growth of tissue culture seedlings, and the high concentration of rare earth elements can destroy the action on cell membranes, influence the permeability of the cell membranes and ensure that cytoplasm is in cytoplasmK of+The loss of the nutrient ions hinders the normal nutrient metabolism of the plants. This study showed that when 0.2 mg.L was added to the medium-1Lanthanum nitrate and 1.0 mg. L-1 BA、0.2 mg·L-1 When NAA is combined, a small amount of light green and compact callus is differentiated at the base of the test-tube plantlet, a large amount of axillary buds are differentiated, the growth vigor is good, the culture medium belongs to a cluster bud generation type, and is the best propagation culture medium for the test-tube plantlet of cornus walteri, so that lanthanum nitrate is beneficial to propagation of the test-tube plantlet of the cornus walteri.
The proliferation cycle is also important for the proliferation of test-tube plantlets. The test researches the influence of different proliferation cycles on the proliferation of the cornus walteri test-tube plantlets, and research results show that when the proliferation cycles are 20d and 30d, the test-tube plantlets start to enter an obvious growth stage after being transferred for 4d-5d, grow to the highest peak after 20d, and can be continued to about 30d, the proliferation rates are high and are respectively 88.5% and 88.1%; the growth of the test-tube plantlets transferred by 40 days is obviously inferior to that of the test-tube plantlets transferred by the 40 days, the proliferation rate is slightly low, and the transferred test-tube plantlets are in a stasis state and are not easy to recover to grow. The observation shows that the test-tube seedling leaves after 60 days of culture become yellow and withered.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Reference documents:
[1] kangyongxing, Yuyujie, Zhao Baoxin, Chenmianfan, Kangjin, Cornus walteri seed germination characteristics and dormancy releasing technology research [ J ] seed, 2011,30(09):22-28+33.
[2] Kangyongxing, Yuyujie, Zhao Baoxin, Chenmianfan, Kangjin, Cornus walteri seed germination characteristics and seedling growth law research [ J ]. academy of northwest forest, 2012,27(03):62-67+112.
[3] Zhangdan, Kangyongxian, Xueliyan, Yexinhua, Cornus walteri Primary culture influencing factor research [ J ]. northern horticulture, 2012(07): 123-.
[4] The tissue culture system of the excellent clone of the Rous walteri, namely the sweet, the Sushushizhu, the Highe, the Chenyifan, the cornus walteri, is established in the university of science and technology of the south China, 2017,37(06) and 70-74+95.
[5] Establishment of tissue culture and regeneration system of Queen autumn celebration, Liuhua, Zhujinqi, Wangchuting, Zhangxia Queenus, (English) [ J ] molecular cell biology report, 2008(03): 227-.
[6] Zhao Qinghua, tissue culture and rapid propagation of Reyno [ J ] Phytology report 2008(02): 220.
Claims (5)
1. A method for carrying out tissue culture subculture propagation on cornus walteri by utilizing lanthanum nitrate is characterized by comprising the following steps: MS is used as a basic culture medium, Cornus walteri water culture stem segments are used as explants, axillary bud induction is carried out, and then the axillary buds are transferred to be added with 0.2 mg.L-1Subculture proliferation culture is carried out in a proliferation culture medium of lanthanum nitrate, and axillary buds and adventitious buds are induced to generate.
2. The method for tissue culture and subculture propagation of cornus walteri by using lanthanum nitrate as claimed in claim 1, wherein the method comprises the following steps: the method comprises the following specific steps:
(1) selecting an explant: collecting current-year branches for water culture in 1-3 months to obtain young stem segments of 4-5 cm; or selecting robust young stem section without diseases and insect pests as explant material in 4-5 months;
(2) and (3) explant sterilization: soaking the obtained young stem with 75% alcohol solution for 30s, washing with sterile water for 3 times, draining, and adding 0.1% HgCI2Sterilizing in solution for 4-5 min, washing with sterile water for 5 times, cutting into 1.5-1.8cm stem segments with at least one axillary bud;
(3) primary culture: inoculating the cut stem segments on a primary culture medium for primary culture at the culture temperature of 25 +/-2 ℃, the humidity of 60 +/-2%, the illumination intensity of 2500 & lt- & gt 3000lx, the illumination time of 12-14h/d and the culture time of 20-30 days, and cutting off the axillary buds for later use when the axillary buds germinate to 3 cm;
(4) subculture multiplication culture: transferring the axillary buds cut off in the step (3) into a subculture multiplication mediumCulturing, wherein the subculture multiplication culture medium comprises: MS + BA 1.0 mg.L-1+ NAA 0.2 mg·L-1+La(NO3)3·6H2O 0.2 mg·L-1+ sucrose 20 g.L-1+ agar 6.5 g.L-1(ii) a The pH value of the culture medium is 5.8-5.9, the culture temperature is 25 +/-2 ℃, the humidity is 60 +/-2%, the illumination intensity is 2500 + 3000lx, the illumination time is 12-14h/d, and the culture time is 20-30 days, cluster buds are formed around the base part of the stem segment, axillary buds are germinated, the stem segment is cut into segments with stem nodes, and the test-tube plantlet is propagated in an expanding way.
3. The method for tissue culture and subculture propagation of cornus walteri by using lanthanum nitrate as claimed in claim 2, wherein the method comprises the following steps: the primary culture medium is as follows: MS + BA 0.5 mg.L-1-1.0 mg·L-1 + NAA 0.1 mg·L-1 + IBA 0.1 mg·L-1+ sucrose 20 g.L-1+ agar 6.5 g.L-1(ii) a The pH value of the culture medium is 5.8-5.9.
4. The method for tissue culture and subculture propagation of cornus walteri by using lanthanum nitrate as claimed in claim 2, wherein the method comprises the following steps: in the step (4), the subculture multiplication culture time is 20d, the multiplication coefficient is 18.1, and the multiplication rate is 91.5%.
5. The method for tissue culture and subculture propagation of cornus walteri by using lanthanum nitrate as claimed in claim 2, wherein the method comprises the following steps: and (4) performing proliferation culture for 30d, wherein the proliferation coefficient is 17.7, and the proliferation rate is 89.9%.
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| CN112715368A (en) * | 2021-02-04 | 2021-04-30 | 山西省林业和草原科学研究院 | Cornus walteri stem induced plant regeneration culture medium and tissue culture and rapid propagation method thereof |
| CN113234735A (en) * | 2021-06-07 | 2021-08-10 | 山西省林业和草原科学研究院 | Poplar PtNF-YC1 gene and application thereof in promoting premature flowering of plants |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112715368A (en) * | 2021-02-04 | 2021-04-30 | 山西省林业和草原科学研究院 | Cornus walteri stem induced plant regeneration culture medium and tissue culture and rapid propagation method thereof |
| CN112715368B (en) * | 2021-02-04 | 2022-06-28 | 山西省林业和草原科学研究院 | Cornus walteri stem induced plant regeneration culture medium and tissue culture and rapid propagation method thereof |
| CN113234735A (en) * | 2021-06-07 | 2021-08-10 | 山西省林业和草原科学研究院 | Poplar PtNF-YC1 gene and application thereof in promoting premature flowering of plants |
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