CN112841032B - Infinite bud multiplication medium for mallotus maritima and method for in-vitro rapid propagation of mallotus maritima - Google Patents
Infinite bud multiplication medium for mallotus maritima and method for in-vitro rapid propagation of mallotus maritima Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Abstract
The invention discloses an adventitious bud multiplication medium for mallotus maritima and a method for in-vitro rapid propagation of mallotus maritima. The invention takes the stem segment with the lateral bud of the maritime tung (Guettarda speciosa L.) as the explant, discusses the influence of plant growth regulators with different concentrations and types on the proliferation of the adventitious buds of the maritime tung, discusses the influence of different matrixes on the induction of the adventitious buds of the maritime tung to the rooting and the induction of tissue culture seedlings of the maritime tung from different sources, discusses the influence of the tissue culture seedlings of the maritime tung from different sources on the survival rate of the domesticated and transplanted seedlings of the maritime tung, establishes a set of complete plant tissue culture method for synchronizing the propagation and rooting induction of the maritime tung bud, effectively shortens the breeding period of the tissue culture seedlings of the maritime tung, provides technical support for the large-scale industrial production of the maritime tung, provides a basis for the germplasm preservation of the maritime tung and fills the blank of the maritime tung in the plant tissue culture technology.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to an adventitious bud multiplication medium for mallotus maritima and an in-vitro rapid propagation method for mallotus maritima.
Background
Tung coast (Guettaria speciosa L.) is evergreen small arbor of Tung coast (Guettaria) of Rubiaceae (Rubiaceae), also known as Henderia plant; coastal tung trees are widely distributed in tropical coastal areas, particularly in eastern and western parts of Malaysia. In china, it is mainly distributed in tropical regions such as taiwan, south of the sea and south islands of the sea. The coast tung tree is a evergreen small arbor, the height of which is 3-5 m, and rarely reaches 8 m. The weather conditions of the islands in south China sea are basically characterized by high temperature and high humidity, much rainfall, clear dry and wet seasons, and frequent influence of disastrous weather such as tropical cyclone, storm, drought and the like. Coastal tung is one of the tree species of coastal tides, and is a typical coastal plant. Coastal tung trees are commonly found in coastal sandy bushes, reef gaps, and gravel beaches. The vernicia maritima has strong properties, good salt resistance and salt fog resistance, high temperature resistance, drought resistance, calcium preference, fertility, light preference, yin tolerance and impoverishment resistance, and is suitable for being used as a vegetation recovery tool species in tropical coral island reefs or similar environments. In addition, the research shows that the coast tung tree is a common medicinal plant in Africa and India, and the medicinal materials are originally the bark, branches and leaves of the coast tung tree and are mainly used for treating ulcer, wound and abscess. The wood of the coast tung is an excellent furniture material, and the wood has dark stripes.
The invention content is as follows:
the invention aims to provide a culture medium for propagation of adventitious buds of mallotus maritima and a method for in-vitro rapid propagation of mallotus maritima.
In order to realize the purpose, the invention adopts the following technical scheme:
the propagation culture medium for the adventitious buds of the coastal tung tree contains 0.2-2.0 mg of 6-BA, 0.01-0.1 mg of NAA, 25-35 g of cane sugar and a proper amount of agar per liter, and the balance is an MS culture medium.
Preferably, the propagation medium for adventitious buds of the coast tung tree contains 1.0mg of 6-BA, 0.1mg of NAA, 30g of cane sugar and 7g of agar per liter, and the rest is MS medium with pH of 6.0.
Another object of the present invention is to provide a method for the rapid propagation of erythrina maritima ex vivo, comprising the steps of:
a. obtaining sterile buds: taking a stem section of a healthy branch of the coast tung tree, cutting off redundant leaves, taking the stem section with side buds as an explant, and inoculating the sterilized explant to an aseptic bud induction culture medium for culture to obtain an aseptic bud;
b. adventitious bud induction: b, inoculating the sterile bud in the step a on an adventitious bud induction culture medium to induce adventitious buds to obtain adventitious buds germinated from the coast tung tree;
c. adventitious bud proliferation: cutting the adventitious bud in the step b into a single bud, inoculating the single bud on the propagation medium of the adventitious bud of the coast tung tree as described in claim 1 or 2, and performing propagation culture to obtain the adventitious bud of the coast tung tree;
d. rooting culture and transplanting: and c, cutting the adventitious buds in the step c into single buds, inoculating the single buds on a rooting culture medium for rooting culture to obtain a rooting tissue culture seedling of the mallotus maritima, hardening the rooting tissue culture seedling, and transplanting the hardening tissue culture seedling into a culture medium to obtain the mallotus maritima seedling.
Preferably, the sterile buds obtained in the step a are cultured under the conditions of (25 +/-1) ° c, the illumination time is 12h/d, and the illumination intensity is 2000 lx; the sterile bud induction culture medium contains per liter: sucrose 30g and agar 7g, the rest is MS culture medium, pH 6.0.
Preferably, the culture condition for inducing the adventitious bud is (25 +/-1) DEG C, the illumination time is 12h/d, and the illumination intensity is 2000 lx; the adventitious bud induction culture medium contains per liter: 1.0mg of 6-BA, 0.01mg of NAA, 30g of cane sugar and 7g of agar, and the rest is MS culture medium with pH of 6.0.
Preferably, the culture conditions of the proliferation culture are (25 +/-1) ° C, the illumination time is 12h/d, and the illumination intensity is 2000 lx.
Preferably, the culture condition of the rooting culture is (25 +/-1) DEG C, the illumination time is 12h/d, and the illumination intensity is 2000 lx; the rooting culture medium comprises a liquid culture medium and vermiculite, wherein each liter of the liquid culture medium contains: IBA 0.5mg, NAA 0.1mg and sucrose 30g, the rest is MS culture medium, pH 6.0.
Preferably, the disinfection treatment specifically comprises: putting the explant into mercuric chloride solution with the mass fraction of 0.1%, soaking and sterilizing for 8min, rinsing for 4-5 times by using sterile water, airing water on the surface of the explant on sterile filter paper, and cutting off two ends.
Preferably, the culture medium comprises vermiculite and river sand, and the volume ratio of the vermiculite to the river sand is 1: 1.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
1. in order to discuss the influence of plant growth regulators with different concentrations and types on the proliferation of adventitious buds of the coast tung tree, an MS culture medium is used as a basic culture medium, KT and 6-BA are independently added or 6-BA and NAA are simultaneously added, and after proliferation culture is carried out for 60 days, the optimal formula of the adventitious bud proliferation culture medium is determined by taking the proliferation multiple of buds of the coast tung tree as an index.
2. In order to discuss the influence of different matrixes on the induction of adventitious bud rooting of the coast tung tree, agar and vermiculite are selected as matrixes to carry out rooting culture, and the result shows that: the rooting ratio in the vermiculite-based medium was 100%, and in the agar-based medium was 90%. The most suitable substrate is determined to be vermiculite.
3. In order to discuss the influence of the tissue culture seedlings of the mallotus japonicus from different sources on the survival rate of the acclimatized seedlings after transplantation, a culture medium taking vermiculite as a matrix and a culture medium taking agar as a matrix are selected to culture the obtained tissue culture seedlings of the mallotus japonicus for transplantation, the transplantation survival rate of the tissue culture seedlings of the mallotus japonicus obtained by the culture medium taking vermiculite as a matrix is 81.8%, and the transplantation survival rate of the tissue culture seedlings of the mallotus japonicus obtained by the culture medium taking agar as a matrix is 63.6%. Finally, the highest transplanting survival rate of the tissue culture seedlings of the coast tung tree obtained by culturing the culture medium with vermiculite as a matrix is determined.
The invention takes the stem segment with the lateral bud of the maritime tung (Guettarda speciosa L.) as the explant, discusses the influence of plant growth regulators with different concentrations and types on the proliferation of the adventitious buds of the maritime tung, discusses the influence of different matrixes on the induction of the adventitious buds of the maritime tung to the rooting and the induction of tissue culture seedlings of the maritime tung from different sources, discusses the influence of the tissue culture seedlings of the maritime tung from different sources on the survival rate of the domesticated and transplanted seedlings of the maritime tung, establishes a set of complete plant tissue culture method for synchronizing the propagation and rooting induction of the maritime tung bud, effectively shortens the breeding period of the tissue culture seedlings of the maritime tung, provides technical support for the large-scale industrial production of the maritime tung, provides a basis for the germplasm preservation of the maritime tung and fills the blank of the maritime tung in the plant tissue culture technology.
Drawings
FIG. 1 is a graph showing the effect of adventitious bud induction in coast tung tree.
FIG. 2 is a graph showing the effect of the proliferation of adventitious buds of coastal tung tree.
FIG. 3 is a graph showing the effect of Tung coast on the induction of rooting in a vermiculite-based medium.
Fig. 4 is a graph of the survival effect of the transplantation of the coast tung tree.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the following description is given with reference to specific examples, but the present invention is not limited thereto.
In this context, the acronyms in English have the following meanings:
IBA: indolebutyric acid is a plant endogenous auxin and is used for promoting cell division and cell growth in the in-vitro rapid propagation process of plants, inducing the formation of adventitious roots, increasing fruit setting in agricultural production, preventing fruit drop, changing the ratio of female flowers to male flowers and the like.
NAA: naphthylacetic acid is a broad-spectrum plant growth regulator, is used for inducing cell division and root differentiation in the process of in vitro culture of plants, and can influence the phenomena of elongation, tropism, apical dominance, leaf shedding and the like of stems and nodes.
KIN or KT: kinetin, an artificially synthesized cytokinin. Can promote cell differentiation, division and growth, promote cell division, and differentiate adventitious bud on callus or organ.
6-BA: 6-benzylaminopurine is a widely used cytokinin added to a plant growth culture medium, and has the effects of inhibiting the decomposition of chlorophyll, nucleic acid and protein in plant leaves, keeping green and preventing aging; the amino acid, the auxin, the inorganic salt and the like are transferred to a treatment part, and the like, and are widely used in various stages from germination to harvest of agricultural, fruit tree and horticultural crops; in vitro rapid propagation of plants, cell division and differentiation of adventitious buds from callus or organs can be promoted, and simultaneously, axillary buds can be released under the inhibition of apical dominance.
The term "ex vivo" refers to a state in which a part of an organism is extirpated and advantageous outside the organism for various research purposes.
The term "explant" refers to a section of tissue that is transferred into a new medium for culture during subculture. In the present invention, the "explant" specifically refers to a stem segment with lateral buds of the coast tung tree.
In the present example, the composition of the MS medium is shown in Table 1 below:
TABLE 1 composition of MS Medium
Unless otherwise specified, the culture medium and the substrate involved in the method for the ex vivo rapid propagation of jatropha curcas in the examples of the present invention were prepared as follows:
(1) preparing 1L of MS culture medium: accurately weighing each compound described in Table 1, dissolving in appropriate amount of distilled water, stirring with glass rod to promote dissolution, adjusting pH to 6.0 with KOH, and diluting to 1L.
(2) Preparing a sterile bud induction culture medium: adding 30g/L of sucrose and 7.0g/L of agar to the MS culture medium prepared in the step (1), and adjusting the pH to 6.0; sterilizing at 121 deg.C for 20 min.
(3) Preparing an adventitious bud induction culture medium: adding 6-BA1.0mg/L, NAA 0.01.01 mg/L, 30g/L of cane sugar and 7.0g/L of agar to the MS culture medium prepared in the step (1), and adjusting the pH value to 6.0; sterilizing at 121 deg.C for 20 min.
(3) Preparing an adventitious bud proliferation culture medium: on the basis of the MS culture medium prepared in the step (1), 0.2-2.0 mg/L, KT 0.2.2-2.0 mg/L of 6-BA is added independently or 0.2-2.0 mg/L of 6-BA and 0.01-0.1 mg/L of NAA, 30g/L of sucrose and 7.0g/L of agar are added in combination, and the pH is adjusted to 6.0; sterilizing at 121 deg.C for 20 min.
(4) Preparing a rooting culture medium: when agar is taken as a substrate: adding IBA 0.5mg/L, NAA 0.1.1 mg/L, sucrose 30g/L and agar 7.0g/L on the basis of the MS culture medium prepared in the step (1), and adjusting the pH to 6.0; sterilizing at 121 deg.C for 20 min. When vermiculite is used as a substrate: firstly, preparing a liquid culture medium, adding IBA 0.5mg/L, NAA 0.1.1 mg/L and sucrose 30g/L on the basis of the MS culture medium prepared in the step (1), adjusting the pH value to 6.0, and then uniformly mixing the liquid culture medium and vermiculite according to the addition ratio of 30mL to 10 g; sterilizing at 121 deg.C for 20 min.
(5) Preparing culture medium, and mixing the culture medium according to the volume ratio of vermiculite to river sand of 1: 1.
Example 1
(1) Obtaining sterile buds:
taking a stem section of a healthy branch of a coastal tung (Guettarda speciosa L.), cutting off redundant leaves, and taking a stem section which is 2-3 cm long and has 1 lateral bud as an explant; putting the explant into 0.1% mercuric chloride solution by mass percent under aseptic condition, soaking and sterilizing for 8min, rinsing for 4-5 times by using sterile water, airing water on the surface of the explant on sterile filter paper, cutting off two ends, inoculating the explant on a sterile bud induction culture medium for culturing, wherein the culture condition is (25 +/-1) ° C, the illumination time is 12h/d, the illumination intensity is 2000lx, and culturing for 60d to obtain a sterile bud; sterile sprout induction medium contains per liter: sucrose 30g and agar 7g, the rest is MS culture medium, pH 6.0.
(2) Adventitious bud induction:
inoculating the sterile bud in the step (1) on an adventitious bud induction culture medium to induce adventitious buds, wherein the culture condition is (25 +/-1) DEG C, the illumination time is 12h/d, the illumination intensity is 2000lx, and culturing for 60d to obtain the adventitious buds germinated from the coast tung tree; the adventitious bud induction medium contains per liter: 1.0mg of 6-BA, 0.01mg of NAA, 30g of cane sugar and 7g of agar, and the balance is MS culture medium with pH of 6.0.
(3) Adventitious bud proliferation:
in order to investigate the influence of different plant growth regulators on the proliferation of adventitious buds of the coast tung tree, 9 groups (test groups 1-9) are set in the test, and the formulas of the adventitious bud proliferation culture media of the test groups 1-9 are as follows: the MS culture medium is used as a basic culture medium, and 0.2-2.0 mg/L, KT 0.2.2-2.0 mg/L of 6-BA is added independently or 0.2-2.0 mg/L of 6-BA and 0.01-0.1 mg/L of NAA, 30g/L of sucrose and 7.0g/L of agar are added in combination, and the pH value is 6.0 (the specific ratio is shown in Table 2). The specific test steps are as follows, the adventitious bud in the step (2) is taken, cut into single buds under the aseptic condition, and respectively inoculated to the adventitious bud proliferation culture medium in the table 2 for proliferation culture, 3 single buds are inoculated to each bottle, each bottle is treated by 10 bottles, the culture condition is (25 +/-1) ° C, the illumination time is 12h/d, the illumination intensity is 2000lx, the adventitious bud of the coast tung tree is obtained after culture for 30d and 60d, and the proliferation multiple of the bud body of the coast tung tree is counted. The bud growth factor is the total number of buds after inoculation culture/the total number of buds at the time of inoculation.
TABLE 2 ingredients of adventitious bud growth medium and bud monthly growth factor for test groups 1-9
The plant growth regulator has great influence on the multiplication factor of the coast tung tree buds, and the multiplication factor of the buds is lower when KT or 6-BA is added independently. Although the increase is observed with the increase of the culture time, the increase is small. When 6-BA and NAA are added into the culture medium at the same time, the proliferation multiple of the bud body is obviously improved, the bud body is generally bred in 2.53 months in the culture medium added with KT and NAA, the bud body can be bred in 3.87 months at most and in 4.56 months in the culture medium added with 6-BA alone, and the bud body can be bred in 5.47 months at most and in 6.12 months at most when 6-BA and NAA are mixed for use. The optimal adventitious bud propagation medium thus obtained is: contains 1.0mg of 6-BA, 0.1mg of NAA, 30g of sucrose and 7g of agar per liter, and the rest is MS culture medium with pH of 6.0.
(4) Rooting culture:
to investigate the effect of different substrates on adventitious bud rooting induction in the coast tung tree, 2 groups (test groups 10 and 11) were set up for the experiment. The rooting medium of test group 10 comprises a liquid medium and vermiculite as a substrate, the liquid medium containing per liter: IBA 0.5mg, NAA 0.1mg and sucrose 30g, the rest is MS culture medium, pH 6.0, wherein the addition ratio of liquid culture medium and vermiculite is 30mL:10 g; the rooting medium of test group 11 contained per liter: IBA 0.5mg, NAA 0.1mg, sucrose 30g and agar 7.0g, the rest is MS culture medium, pH 6.0. The specific test steps are as follows, the adventitious buds of the mallotus maritima obtained after the test group 3 of the step (3) is cultured for 60 days are cut into single buds under the aseptic condition, the single buds are respectively inoculated to a rooting culture medium for rooting culture, 3 buds are inoculated to each bottle, 10 bottles are processed, the culture condition is (25 +/-1) ° C, the illumination time is 12h/d, the illumination intensity is 2000lx, the tissue culture seedlings rooted by the mallotus maritima are obtained after the culture for 30 days, and the rooting number and the rooting rate are counted.
The test results show that: the rooting rate in the culture medium using vermiculite as a substrate was 100%, and the number of roots was 10.3. On the other hand, the rooting rate in the agar-based medium was 90%, and the number of roots was 6.7. Obviously, the adinandra nitida grows better in the culture medium taking vermiculite as a matrix, and the rooting number and the rooting rate are higher than those of the culture medium taking agar as a matrix. The optimal rooting culture medium is obtained by using vermiculite as a matrix.
(5) Transplanting:
in order to investigate the influence of the tissue culture seedlings of the mallotus japonicus from different sources on the survival rate of the mallotus japonicus after hardening and transplanting, 2 groups (test groups 12 and 13) are set in the test, the culture substrates of the test groups 12 and 13 are substrates formed by mixing vermiculite and river sand according to the volume ratio of 1:1, the tissue culture seedlings transplanted by the test group 12 are the tissue culture seedlings of the mallotus japonicus obtained after the test group 10 is cultured for 30d in the step (4), and the tissue culture seedlings transplanted by the test group 13 are the tissue culture seedlings of the mallotus japonicus obtained after the test group 11 is cultured for 30d in the step (4). The specific test steps are that the tissue culture bottle cap of the tissue culture seedling of the mallotus maritima is unscrewed, the tissue culture bottle cap is put under natural light to be hardened for 7d, then the tissue culture seedling is transplanted into a culture medium, watering and shading are paid attention to, and thereby the mallotus maritima seedling is obtained. And after 30 days of transplanting, observing and recording the growth condition and counting the transplanting survival rate of the tissue culture seedlings of the coastal tung tree, wherein the transplanting survival rate of the test group 12 is 81.8 percent, and the transplanting survival rate of the test group 13 is 63.6 percent. The results show that the tissue culture seedling obtained by rooting culture in the culture medium taking vermiculite as a matrix has higher transplanting survival rate.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (8)
1. A method for the in vitro rapid propagation of the mallotus maritima, which is characterized by comprising the following steps:
a. obtaining sterile buds: taking a stem section of a healthy branch of the coast tung tree, cutting off redundant leaves, taking the stem section with side buds as an explant, and inoculating the sterilized explant to an aseptic bud induction culture medium for culture to obtain an aseptic bud;
b. adventitious bud induction: b, inoculating the sterile bud in the step a on an adventitious bud induction culture medium to induce adventitious buds to obtain adventitious buds germinated from the coast tung tree;
c. adventitious bud proliferation: b, cutting the adventitious bud in the step b into single buds, inoculating the single buds to an adventitious bud multiplication culture medium of the coast tung tree for multiplication culture to obtain the adventitious bud of the coast tung tree;
d. rooting culture and transplanting: c, cutting the adventitious buds in the step c into single buds, inoculating the single buds on a rooting culture medium for rooting culture to obtain rooting tissue culture seedlings of the mallotus maritima, hardening the rooting tissue culture seedlings, and transplanting the hardening tissue culture seedlings into a culture medium to obtain mallotus maritima seedlings;
the sterile bud induction culture medium contains per liter: 30g of sucrose and 7g of agar, and the balance of MS culture medium, pH 6.0;
the adventitious bud induction culture medium contains per liter: 1.0mg of 6-BA, 0.01mg of NAA, 30g of cane sugar and 7g of agar, and the balance is MS culture medium with pH of 6.0;
the rooting culture medium comprises a liquid culture medium and vermiculite, wherein each liter of the liquid culture medium contains: IBA 0.5mg, NAA 0.1mg and sucrose 30g, the rest is MS culture medium, pH 6.0;
the propagation medium for the adventitious buds of the coastal tung tree is composed of the following components per liter: 0.2-2.0 mg of 6-BA, 0.01-0.1 mg of NAA, 25-35 g of cane sugar, a proper amount of agar and the balance of MS culture medium.
2. The method for the ex vivo rapid propagation of mallotus maritima according to claim 1, wherein the culture medium for propagation of adventitious buds of mallotus maritima per liter is composed of the following components: 1.0mg of 6-BA, 0.1mg of NAA, 30g of cane sugar and 7g of agar, and the rest is MS culture medium with pH of 6.0.
3. The method for the ex vivo rapid propagation of coast tung tree according to claim 1, wherein the aseptic buds obtained in step a are cultured at (25 ± 1) ° c for 12h/d at an intensity of 2000 lx.
4. The method for the ex vivo rapid propagation of coast tung oil according to claim 1 wherein the adventitious bud is induced under the culture conditions of (25 ± 1) ° c, the illumination time is 12h/d and the illumination intensity is 2000 lx.
5. The method for the ex vivo rapid propagation of coast tung oil according to claim 1, characterized in that the culture conditions of the proliferation culture are (25 ± 1) ° c, the illumination time is 12h/d, and the illumination intensity is 2000 lx.
6. The method for the ex vivo rapid propagation of coast tung tree according to claim 1, characterized in that the culture conditions of the rooting culture are (25 ± 1) ° c, the illumination time is 12h/d, and the illumination intensity is 2000 lx.
7. The method for the ex vivo rapid propagation of coastal waters as claimed in any one of claims 1 to 6, characterized in that said disinfection treatment is in particular: putting the explant into mercuric chloride solution with the mass fraction of 0.1%, soaking and sterilizing for 8min, rinsing for 4-5 times by using sterile water, airing water on the surface of the explant on sterile filter paper, and cutting off two ends.
8. The method for the ex vivo rapid propagation of coast tung oil according to any one of claims 1 to 6 wherein the culture substrate is vermiculite and river sand, the volume ratio of vermiculite to river sand being 1: 1.
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| 单头切花菊‘白扇’组培快繁技术;周婷等;《热带作物学报》;20190425;第40卷(第04期);第715-723页，尤其是第1.2.1节 * |
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