CN106900552B - Culture medium kit and method for promoting rapid in-vitro propagation of fish - Google Patents

Culture medium kit and method for promoting rapid in-vitro propagation of fish Download PDF

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CN106900552B
CN106900552B CN201710133857.4A CN201710133857A CN106900552B CN 106900552 B CN106900552 B CN 106900552B CN 201710133857 A CN201710133857 A CN 201710133857A CN 106900552 B CN106900552 B CN 106900552B
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culture medium
culture
medium
rooting
agar
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CN106900552A (en
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胡秀
梁韩枝
吴永清
邓莎
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Guangzhou Ecological Research Co ltd
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Zhongkai University of Agriculture and Engineering
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    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/001Culture apparatus for tissue culture
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01GHORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G24/00Growth substrates; Culture media; Apparatus or methods therefor
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/008Methods for regeneration to complete plants

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Abstract

The invention discloses a culture medium kit for in vitro rapid propagation of a fish, which comprises: (1) medium for primary culture 1: adding MS culture medium as basic culture medium0.5-2 mg/L BA, 0.1mg/L NAA, 0-0.1 mg/L TDZ, 25-35 g/L sucrose and 6.5-7.5 g/L agar; (2) medium for subculture 2: taking MS culture medium as basic culture medium, adding 0.5mg/L BA, 0-0.1 NAA and 0-0.5 mg/L GA325-35 g/L sucrose and 6.5-7.5 g/L agar; (3) medium for strong seedlings 3: adding 25-35 g/L of sucrose and 6.5-7.5 g/L of agar by taking an MS culture medium as a basic culture medium; (4) medium for rooting 4: 1/2MS culture medium is used as basic culture medium, and 0.5-1.5 mg/L IBA, 25-35 g/L sucrose and 6.5-7.5 g/L agar are added. In addition, the invention also discloses a method for promoting the in vitro rapid propagation of the fish by adopting the culture medium kit. Under the optimized condition, the method is used for culturing the fish, so that the ideal breeding rate, rooting rate and transplanting survival rate can be obtained.

Description

Culture medium kit and method for promoting rapid in-vitro propagation of fish
Technical Field
The invention relates to a culture medium kit for plant tissue culture and a method thereof, in particular to a culture medium kit for in vitro rapid propagation of a fish rosewood clustered bud approach and a method thereof.
Background
The crawfish (Crateva religiosa G. Forster) is a small deciduous tree of the genus crawfish (Crateva) of the family Capparidaceae, and has beautiful tree shape and beautiful flower appearance, and the section of the crawfish is like a group of butterflies and is suitable for appreciation. The wood can be used as musical instruments and fine work materials; the fruit contains alkaloid and can be used as adhesive, and the pericarp can be used as dye.
The method is characterized in that the fish wood is usually propagated by adopting seed propagation and root-burying propagation, but the germination rate of the seeds is very low under natural conditions, and the root-burying propagation speed is limited, so that the large-scale popularization and planting of the fish wood are limited (Tyagi, 2010). The isolated culture rapid propagation has the advantages of non-seasonal dependence, gene consistency and no pathogenicity of plants (Husain, 2007; Vengadesan, 2007). At present, no report on the in vitro culture and rapid propagation of the fish is available.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a culture medium kit for promoting the in vitro rapid propagation of the fish;
another object of the present invention is to provide a method for promoting the in vitro rapid propagation of fish.
In order to achieve the purpose, the invention adopts the technical scheme that: a culture kit for promoting in vitro rapid propagation of fish, the culture kit comprising:
(1) medium for primary culture 1: MS culture medium is taken as basic culture medium, 0.5-2 mg/L, NAA (naphthylacetic acid) of BA (6-benzyladenine) is added, 0.1mg/L, TDZ (thidiazuron) is added, 0-0.1 mg/L of cane sugar is added, 25-35 g/L of cane sugar is added, and 6.5-7.5 g/L of agar is added;
(2) medium for subculture 2: MS culture medium is taken as basic culture medium, 0.5mg/L, NAA (naphthylacetic acid) 0-0.1 mg/L, GA (gibberellin) 0-0.5 mg/L, 25-35 g/L of sucrose and 6.5-7.5 g/L of agar are also added;
(3) medium for strong seedlings 3: taking an MS culture medium as a basic culture medium, and adding 25-35 g/L of sucrose and 6.5-7.5 g/L of agar;
(4) medium for rooting 4: 1/2-1 MS culture medium is used as basic culture medium, and 0.5-1.5 mg/L IBA (indolebutyric acid), 25-35 g/L sucrose and 6.5-7.5 g/L agar are also added;
BA, 6-benzyladenine, is a widely used cytokinin added to plant growth media, and has effects of inhibiting decomposition of chlorophyll, nucleic acid and protein in plant leaf, 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.
NAA, namely 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.
TDZ, thidiazuron, is a plant growth regulator promoter, and can be used for promoting callus growth, side bud and adventitious bud generation and promoting embryoid formation in plant in vitro rapid propagation.
GA, gibberellins, is a widely existing class of plant hormones, whose chemical structure belongs to diterpenoid acids, derived from tetracyclic scaffolds. Can be used for promoting the stem node of the dwarf plantlet to elongate in the in-vitro rapid propagation of the plant and stimulating the adventitious embryo to normally develop into the plantlet. As a preferred embodiment of the kit for a medium of the present invention, in the medium for subculture 2, the gibberellin is GA3
IBA (indolebutyric acid), namely 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.
In the culture medium kit, the culture mediums are matched with each other, the hormone types and contents in the culture mediums are well matched with the specific growth requirements of the fish plants, and the propagation rate, rooting rate and transplanting survival rate of the fish are obviously improved.
As a preferred embodiment of the culture kit, the culture kit further comprises a matrix formed by mixing peat soil and sand in a volume ratio of 2: 1.
As a preferred embodiment of the kit of culture media according to the present invention, in the culture medium 1 for primary culture, BA is 0.5mg/L, NAA is 0.1mg/L, TDZ is 0.1mg/L, sucrose is 30g/L and agar is 7 g/L.
As a preferred embodiment of the kit of culture media according to the present invention, in the culture medium 2 for subculture, BA is 0.5mg/L, NAA is 0.1mg/L, and GA30mg/L, 30g/L sucrose and 7g/L agar.
As a preferred embodiment of the kit of culture media according to the present invention, in the medium 4 for rooting, 1/2MS medium was used as a basal medium, IBA was 1.0mg/L, sucrose was 30g/L, and agar was 7 g/L.
The method for preparing the culture kit of the present invention is not particularly limited, but preferably, the culture kit of the present invention is prepared by the following method:
(1) preparing an MS culture medium;
(2) preparation of medium for primary culture 1: adding BA, NAA, TDZ, sucrose and agar on the basis of the MS culture medium prepared in the step (1);
(3) preparation of medium for subculture 2: adding BA, TDZ and GA on the basis of the MS culture medium prepared in the step (1)3Sucrose and agar;
(4) preparing a culture medium for strengthening seedlings 3: adding sucrose and agar on the basis of the MS culture medium prepared in the step (1);
(5) preparation of medium for rooting 4: adding IBA, sucrose and agar on the basis of the MS culture medium prepared in the step (1);
(6) preparing a matrix: according to the proportion of peat soil: sand 2:1 (volume ratio) was mixed.
The invention also provides a method for promoting the in vitro rapid propagation of the fish by adopting the culture medium kit.
As a preferred embodiment of the method for promoting the in vitro rapid propagation of the fish wood of the present invention, the method comprises the following steps:
(1) selecting roots of a stock plant of the fish, burying the roots in sand in a greenhouse to grow semi-lignified seedlings, and taking stem sections with axillary buds of the seedlings as explants;
(2) under aseptic conditions, sterilizing explants with mercuric chloride, and inoculating the sterilized explants to the culture medium 1 for primary culture to obtain primary aseptic seedlings;
(3) shearing the non-vitrified primary aseptic seedlings obtained in the step (2) into stem sections with buds with 1 node, inoculating the stem sections with buds to the culture medium 2 for subculture, obtaining cluster buds and obtaining the optimal subculture times;
(4) inoculating the cluster buds obtained in the step (3) on the culture medium 3 for strong seedlings to perform strong seedling culture;
(5) inoculating the healthy and strong seedlings obtained in the step (4) on the culture medium 4 for rooting to carry out rooting culture;
(6) and (5) cleaning the culture medium of the plant roots obtained in the step (5), and transplanting the culture medium on the substrate 1.
The method for promoting the in vitro rapid propagation of the fish wood specifically comprises the following steps: taking a stem section of a semi-lignified seedling newly grown after the root of a parent plant of the fish is buried as an explant, inoculating the explant on a primary culture medium after surface disinfection treatment, and determining the optimal primary culture medium according to indexes such as bud germination rate, multiplication coefficient, growth state and the like; a plant with good growth vigor formed on an optimal primary culture medium is taken, lateral buds with similar sizes are cut by a blade and inoculated on a subculture medium, and the optimal subculture times and a subculture formula are determined by multiple subcultures with the same formula and by taking a multiplication coefficient and vitrification as references, so that the blindness during subculture treatment in the production process is avoided. During rooting culture, the vitrified plants cannot induce the formation of roots, and after strong seedling culture is carried out on the vitrified plants on an MS blank culture medium without any plant growth regulator, the rooting induction culture can be carried out, so that the ratio of the vitrified seedlings is obviously reduced, and the availability of the tissue culture plants is greatly improved. The test establishes a rosewood cluster bud in-vitro rapid propagation system for the first time, and obtains complete and considerable rosewood tissue culture seedlings after alternate treatment, rooting and transplanting of primary generation, secondary generation and strong seedlings.
In a more preferred embodiment of the method for promoting the in vitro rapid propagation of the yuzuki, in the step (1), the root of the yuzuki stock is a lateral root of a robust stock with a thickness of 0.5-2 cm.
As a more preferred embodiment of the method for promoting the in vitro rapid propagation of the fish, the culture conditions in the steps (2) to (5) are all: the temperature is 24-26 ℃, the illumination time is 10-14 h/d, and the illumination intensity is 2500-3500 lx.
As a more preferred embodiment of the method for promoting the in vitro rapid propagation of the fish wood, wherein, in the step (2), the sterilization treatment process comprises: adding alcohol with the volume percentage of 70-75% to soak for 30-60 s; cleaning with sterile water, and soaking for 10-15 min with mercuric chloride solution with mass percentage concentration of 0.08-0.12%; after washing with sterile water, the surface water was blotted dry with sterile filter paper.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
1. firstly, performing primary culture on a culture medium 1 which is added with BA, NAA and TDZ and used for primary culture, and screening a proper primary culture medium by using bud multiplication rate and vitrification as indexes; removing TDZ and adding GA based on primary culture medium3Carrying out multiple subculture cluster buds induction culture on the culture medium 2 for subculture with reduced NAA concentration, and screening a suitable subculture medium and subculture times by using bud multiplication rate and vitrification as indexes; before rooting, adopting a strong seedling culture medium 3 without any plant growth regulator for strengthening seedlings, carrying out rooting culture on the strong plants on a rooting culture medium 4 containing IBA, and finally carrying out domestication and transplantation. The method establishes tissue culture rapid propagation system of the way of the rosewood cluster buds for the first time, generates tissue culture seedlings with gene consistency and capable of keeping excellent characters of mother plants, and solves the problems of high vitrification rate, serious callus and difficult rooting in the process of culturing the rosewood tissue. By taking 30 days as a culture period, the culture medium can ensure that the reproduction rate of the fish wood reaches 3-12 times, the rooting rate is 92%, and the transplanting survival rate is 100%.
2. The culture medium 1 for primary culture adopts the cooperation of BA, NAA and TDZ with different concentrations to induce the axillary buds of the explants to germinate for the primary generation, the growth state and the propagation rate of aseptic seedlings cultured on different culture media are determined, the culture period is 30 days, the propagation rate is 3.88-6.35 times, and the height of the plants is about 4-6 cm.
3. Media 2 for subculture used different concentrations of BA and NAA and GA3Cutting stem segment with one axillary bud of the plant obtained from the initial generation for subculture and 3 times of subculture treatment. And determining the appropriate subculture medium and subculture times by using the bud multiplication rate and vitrification as indexes. Using 30 days as culture period, the multiplication times is 3-12 times, and the plant height is about 4E6cm。
4. In the strong seedling stage, the culture medium 3 for strong seedlings (25-35 g/L of sucrose and 6.5-7.5 g/L of agar are added on the basis of the MS culture medium) can weaken the vitrification degree, ensure dark green leaves and strong growth, and ensure that the petioles have bud points, thereby meeting the requirements of rooting and seedling formation.
5. In the rooting stage, the strong and strong plants obtained in the culture medium 3 for strong seedlings are inoculated in a rooting culture medium 4 (0.5-1.5 mg/L of IBA, 30g/L of cane sugar and 7g/L of agar are added on the basis of 1/2-1 MS culture medium) for rooting culture, the rooting rate reaches 92% after 30 days of culture, and the rooting coefficient reaches 6.3.
6. The explant is obtained by newly growing semi-lignified seedlings from the mother plant roots after sand burying in a greenhouse, so that the pouring of external rainwater is avoided, fewer bacteria are carried, the pollution rate is low, the growth is vigorous, and the bud germination rate is high and is almost 100%. After primary culture and a certain number of times of subculture, the robust aseptic seedlings with 9-12 times of propagation multiple can be obtained by taking 30 days as a counting period, and the rooting rate and the transplanting survival rate are respectively 92% and 100%.
Drawings
FIG. 1 is a diagram showing the effect of the primary culture of the fish;
FIG. 2 is a graph showing the effect of a large number of cluster buds appearing in the first subculture;
FIG. 3 is a graph showing vitrification of strong seedlings during cultivation;
FIG. 4 is a plant of a fish after strong seedling culture;
FIG. 5 is a rooted plant;
FIG. 6 shows the plants that were transplanted to survive.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the following embodiments.
In this context, the acronyms in English have the following meanings:
BA: 6-benzylaminopurine;
TDZ: thidiazuron;
NAA: naphthylacetic acid;
IBA: is indolebutyric acid;
GA3: is gibberellin;
the terms:
the term "ex vivo" refers to a state in which a part of an organism is excised and released from the organism for various research purposes.
The term "explant" refers to a section of cultured tissue that is transferred into a new medium during subculture. In the invention, the "explant" specifically refers to the annual semi-lignified seedling grown from the root of the parent plant of the Yumu after sand burying in a greenhouse.
In the present invention, the term "vitrification phenomenon" refers to a phenomenon in which a plant develops a translucent shape and a tissue structure develops a deformed shape, which occurs in plant tissue culture. The plant with vitrification phenomenon can not be transplanted out of the test tube to survive. Compared with normal tissues, the chlorophyll content, protein content, activity of some enzymes, ethylene synthesis capacity and the like in the vitrified tissues are greatly changed.
1/2-1 MS culture medium refers to a culture medium prepared by reducing the amount of macroelements in MS culture medium to 1/2-1. For example, 1/2MS medium refers to a medium prepared by reducing the amount of various trace elements in MS medium to 1/2.
In the present example, the composition of the MS medium is shown in Table 1 below:
TABLE 1
Figure GDA0002354353070000081
Unless otherwise stated, the culture medium and the matrix involved in the method for promoting the in vitro rapid propagation of the fish wood in the embodiment of the invention are prepared according to the following methods respectively:
(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 NaOH, and diluting to 1L.
(2) Preparation of medium for primary culture 1: adding 0.5-2 mg/L BA, 0.1mg/L NAA, 0-0.1 mg/L TDZ, 25-35 g/L sucrose and 6.5-7.5 g/L agar on the basis of the MS culture medium prepared in the step (1);
(3) preparation of medium for subculture 2: adding 0.5mg/L BA, 0-0.1 NAA and 0-0.5 mg/L GA on the basis of the MS culture medium prepared in the step (1)325-35 g/L sucrose and 6.5-7.5 g/L agar;
(4) preparing a culture medium for strengthening seedlings 3: adding 25-35 g/L of sucrose and 6.5-7.5 g/L of agar on the basis of the MS culture medium prepared in the step (1);
(5) preparation of medium for rooting 4: adding 0.5-1.5 mg/L IBA, 25-35 g/L sucrose and 6.5-7.5 g/L agar on the basis of 1/2-1 MS culture medium;
(6) and (3) preparing a matrix 1, and mixing the matrix according to the volume ratio of the peat soil to the sand of 2: 1.
Unless otherwise stated, the conditions for inducing the aseptic seedlings and subculture in the following examples are 24-26 ℃, 12h/d of illumination and 2500-3500 lx of illumination intensity.
Example 1
Experiment of influence of BA, NAA and TDZ contents in culture medium 1 for primary culture on primary culture of fish
Experiment sets 1-6, in experiment sets 1-6, culture medium 2 for subculture is: taking an MS culture medium as a basic culture medium, and adding a culture medium of 0.5mg/L of 6-benzyladenine, 0-0.1 mg/L of naphthylacetic acid, 0-0.5 mg/L of gibberellin, 25-35 g/L of sucrose and 6.5-7.5 g/L of agar; the culture medium 3 for strong seedlings is as follows: taking an MS culture medium as a basic culture medium, and adding a culture medium containing 25-35 g/L of sucrose and 6.5-7.5 g/L of agar; the culture medium 4 for rooting is as follows: 1/2-1 MS culture medium is used as a basic culture medium, and a culture medium with 0.5-1.5 mg/L of indolebutyric acid, 25-35 g/L of sucrose and 6.5-7.5 g/L of agar is also added; the matrix is as follows: the volume ratio is 2:1, a matrix formed by mixing peat soil and sand; in the culture medium 1 for primary culture, the content of sucrose is 25-35 g/L, and the content of agar is: 6.5 to 7.5 g/L.
In the culture medium kit used in the test groups 1-6, the culture medium 2 for subculture, the culture medium 3 for strong seedlings, the culture medium 4 for rooting and the matrix are all the same, and the culture medium 1 for primary culture also has the same contents of sucrose and agar. The only difference of the culture medium kit used in the experimental groups 1-6 is that the culture medium 1 used for primary culture has different BA, NAA and TDZ contents, which are respectively as follows:
Figure GDA0002354353070000091
Figure GDA0002354353070000101
the following methods were used in each of test groups 1 to 6 (note: the methods for in vitro propagation of a fish wood described in the following examples were used unless otherwise specified):
taking the annual semi-lignified seedling grown after burying underground roots of the fish in sand in 10 months in 2015 as an explant, and germinating axillary buds of the explant through in vitro primary generation, subculture, seedling strengthening, rooting and transplanting, wherein the method comprises the following specific steps:
(1) selecting explants: no. 28/10 of 2015, underground roots of the stock plant of the yuzuki (preferably lateral roots of the robust stock plant of the yuzuki with the thickness of 0.5-2 cm) are taken to be buried in sand in a greenhouse, and sufficient water is poured. Sprouts were visible after 7 days. Semi-lignified seedlings were obtained after 45 days.
(2) Carrying out explant sterile treatment and primary culture: and (3) cutting the stem branches of the semi-lignified seedlings by using scissors in 15 days 12 months 2015, removing the leaves of the stem branches, and cutting the stem branches into 1-2 cm stem sections with 1 axillary bud as explants. Soaking the workpiece on an ultra-clean workbench for 30-60 s by using 75% alcohol, cleaning the workpiece by using sterile water, soaking the workpiece for 10-15 min by using a mercuric chloride solution with the mass percentage concentration of 0.08-0.12%, cleaning the workpiece for 6-8 times by using the sterile water, and sucking out surface water by using sterile filter paper. Directly inoculating to the culture medium 1 for primary culture to induce axillary bud germination. After 30 days, the germination rate of axillary buds is close to 100%, the multiplication rate of buds is 3.8-6.35, and the height of plants is about 3-4cm (see figure 1).
(3) First subculture: the primary cultured non-vitrified plants were cut into stem segments with 1 node, about 2cm in height, and inoculated on the medium 2 for subculture for a total of 30 stem segments. After 30 days of culture, the multiplication rate of the buds is 9.95-12, and the vitrification rate is 54.9-74.23%.
(4) Secondary subculture: the first subcultured plants were cut into stem segments with 1 node, about 2cm in height, and inoculated on medium 2 for subculture for a total of 30 stem segments. After 30 days of culture, the multiplication rate of the buds is 6.80-9.75, and the vitrification rate is 68-77.25%.
(5) And (3) carrying out third subculture: the plants of the second subculture were cut into stem segments with 1 node, about 2cm in height, and inoculated on medium 2 for subculture for a total of 30 stem segments. After 30 days of culture, the multiplication rate of the buds is 2.51-3.02, and the vitrification rate is 77.93-86.15%.
(6) Strengthening seedlings: the vitrification degree of the vitrification plant is reduced to 20 percent by the upper culture, the stem section is elongated, the leaf color is dark green, the growth is strong, the leaf stalk is provided with bud points, and the requirements of rooting and seedling formation are met.
(7) Rooting: cutting a stem section which is about 3-4cm long and strong, inoculating the stem section on a culture medium 4 for rooting, wherein the rooting rate is 92 percent after 30 days, the rooting coefficient is 6.33, the color of the plant leaf is dark green, the root is thick and strong, the length reaches 1-3cm, and the transplanting requirement is met.
(8) Transplanting: after acclimation and hardening-up, the plants after rooting induction are taken out from the culture bottle, the culture medium attached to the roots of the plants is carefully washed by tap water, and then the plants are transplanted into the matrix 1. The statistical survival rate is 100% after 40 days of transplantation.
The culture medium 1 for primary culture of test groups 1 to 6 was used for the primary culture of a fish tree in the above-described manner, respectively, and the test results are shown in table 2 below.
Table 2: influence of BA, NAA and TDZ with different concentrations on fish primary culture
Figure GDA0002354353070000111
As can be seen from Table 2, the shoots of each explant treated after 30d of culture all germinated, and the germination rate was 100%, but the proliferation coefficients were significantly different. In the range of BA concentration of 0.5 to 2.0mg/L, the bud growth factor increased with the increase in BA concentration, but when the BA concentration was 1.0mg/L or more (for example, test group 5 and test group 6), the bud growth factor did not increase significantly any more. The highest bud multiplication coefficient formula is: BA 1.0mg/L + NAA 0.1mg/L + TDZ0.1mg/L (test group 4), which can reach 6.35, but the blade shrinks and partial vitrification occurs. Although the explant cultured on the medium of BA0.5mg/L + NAA 0.1mg/L + TDZ0.1mg/L (test group 2) had the lowest bud growth factor of only 4.11, the leaf was verdant and extended, and the growth was good, and it could be used for further subculture.
Example 2
3BA, NAA and GA in Medium 2 for subcultureSubculturing the fish wood according to the content toAnd the number of subcultures are paired Effect test on reproduction of clumpy buds of Yumu
Experiment sets up 1 ~ 4 of test group, among test group 1 ~ 4, culture medium 1 for the primary culture is: taking an MS culture medium as a basic culture medium, and adding a culture medium of 0.5-2 mg/L of 6-benzyladenine, 0.1mg/L of naphthylacetic acid, 0-0.1 mg/L of thidiazuron, 25-35 g/L of sucrose and 6.5-7.5 g/L of agar; the culture medium 3 for strong seedlings is as follows: taking an MS culture medium as a basic culture medium, and adding a culture medium containing 25-35 g/L of sucrose and 6.5-7.5 g/L of agar; the culture medium 4 for rooting is as follows: 1/2-1 MS culture medium is used as a basic culture medium, and a culture medium with 0.5-1.5 mg/L of indolebutyric acid, 25-35 g/L of sucrose and 6.5-7.5 g/L of agar is also added; the matrix is as follows: the volume ratio is 2:1, a matrix formed by mixing peat soil and sand; in the culture medium 2 for subculture, the content of sucrose is 25-35 g/L, and the content of agar is: 6.5 to 7.5 g/L.
In the culture medium kit used in the test groups 1-4, the culture medium 1 for primary culture, the culture medium 3 for strong seedlings, the culture medium 4 for rooting and the matrix are all the same, and the culture medium 2 for secondary culture has the same content of sucrose and agar. The only difference between the medium kits used in test groups 1-4 is the use ofBA, NAA and GA in primary culture Medium 13Different contents are respectively as follows
Group of BA content (mg/L) NAA content (mg/L) GA3Content (mg/L)
Test group 1 0.5 0 0
Test group 2 0.5 0.01 0
Test group 3 0.5 0.1 0
Test group 4 0.5 0.1 0.5
The test groups 1 to 4 all adopt the method described in example 1 to carry out in vitro propagation of the fish wood:
the culture medium 2 for subculture described in test groups 1 to 4 was used for the subculture test of the fish wood by the method described above, and the test results are shown in table 3 below.
TABLE 3
Figure GDA0002354353070000121
Figure GDA0002354353070000131
As can be seen from Table 3, all the sprouts were germinated when the primary sterile axillary buds were transferred to the subculture medium for 5 days, and the germination rate was 100%. A large number of clumpy buds were visible around 20 days of culture (FIG. 2). In the first subculture, the multiplication coefficient of the explant bud cultured in the culture medium containing the ratio of BA and NAA is significantly higher than that of a single culture medium containing BA (test group 1) and can reach 12.09, and the vitrification ratio of the BA and NAA at the ratio of 5 is significantly lower than that of other treatment groups and is at least 54.9%, while the difference of the multiplication coefficients of the bud treated by BA0.5mg/L + NAA 0.1mg/L (test group 3) and BA0.5mg/L + NAA 0.01mg/L (test group 2) is not significant, and the former is only slightly higher than that of the latter. And adding GA3(test group 4) had no significant effect on the bud growth coefficient and the vitrification ratio. Unlike the first subculture, the bud growth coefficient of the treatment of BA0.5mg/L + NAA 0.01mg/L (test group 2) was the highest at the second subculture, and was 9.75, but significantly lower than that of the first subculture, and the vitrification rates were not significantly different among the treatments and were all higher than that of the first subculture. When the stem and branch of the plant after the subculture to the third generation are yellow and weak, the bud multiplication coefficient is obviously reduced, and the vitrification rate is obviously higher than that of the previous subcultures. It can be known that the accumulation of plant hormones in the bud has an influence on the bud multiplication coefficient and the vitrification rate, the bud multiplication coefficient tends to increase and then decrease with the increase of the subculture frequency, and the vitrification rate obviously increases. Considering the factors of bud multiplication coefficient, vitrification rate and the like comprehensively, the explant is subcultured for 1-2 times in a culture medium containing 0.5mg/L of BA and 0.1mg/L of NAA.
Example 3
Culture medium for strong seedlings 3 pairs of fishesEffect test of vitrified seedlings of Wood
In vitro propagation of fish using the method described in example 1, wherein the medium 1 for primary culture is: taking an MS culture medium as a basic culture medium, and adding a culture medium of 0.5-2 mg/L of 6-benzyladenine, 0.1mg/L of naphthylacetic acid, 0-0.1 mg/L of thidiazuron, 25-35 g/L of sucrose and 6.5-7.5 g/L of agar; the medium 2 for subculture was: taking an MS culture medium as a basic culture medium, and adding a culture medium of 0.5mg/L of 6-benzyladenine, 0-0.1 mg/L of naphthylacetic acid, 0-0.5 mg/L of gibberellin, 25-35 g/L of sucrose and 6.5-7.5 g/L of agar; the culture medium 3 for strong seedlings is as follows: taking an MS culture medium as a basic culture medium, and adding a culture medium containing 25-35 g/L of sucrose and 6.5-7.5 g/L of agar; the culture medium 4 for rooting is as follows: 1/2-1 MS culture medium is used as a basic culture medium, and a culture medium with 0.5-1.5 mg/L of indolebutyric acid, 25-35 g/L of sucrose and 6.5-7.5 g/L of agar is also added; the matrix is as follows: the volume ratio is 2:1, peat soil and sand. The strong seedling results of the medium for strong seedling 3 are shown in table 4 below.
TABLE 4
Figure GDA0002354353070000141
As can be seen from Table 4, compared with the vitrification ratio of 100% in inoculation, after 30 days of strong seedling culture, the vitrification degree of the plant is significantly reduced, and the obtained plant has long stem segment, dark green leaf color and thick stem and strong leaf stalk with bud points, which meets the requirements of rooting and seedling formation (FIG. 4).
Example 4
Effect test of culture Medium for rooting 4 on rooting Induction of Yumu
Experiment sets up experimental group 1 ~ 6, among experimental group 1 ~ 6, culture medium 1 for the primary culture is: taking an MS culture medium as a basic culture medium, and adding a culture medium of 0.5-2 mg/L of 6-benzyladenine, 0.1mg/L of naphthylacetic acid, 0-0.1 mg/L of thidiazuron, 25-35 g/L of sucrose and 6.5-7.5 g/L of agar; the medium 2 for subculture was: taking an MS culture medium as a basic culture medium, and adding a culture medium of 0.5mg/L of 6-benzyladenine, 0-0.1 mg/L of naphthylacetic acid, 0-0.5 mg/L of gibberellin, 25-35 g/L of sucrose and 6.5-7.5 g/L of agar; the culture medium 3 for strong seedlings is as follows: taking an MS culture medium as a basic culture medium, and adding a culture medium containing 25-35 g/L of sucrose and 6.5-7.5 g/L of agar; the matrix is as follows: the weight ratio is 2:1, a matrix formed by mixing peat soil and sand; in the culture medium 4 for rooting, 25-35 g/L of sucrose and 6.5-7.5 g/L of agar are used.
In the culture medium kit used in the test groups 1-6, the culture medium 1 for primary culture, the culture medium 2 for secondary culture, the culture medium 3 for strong seedlings and the matrix are all the same, and the contents of sucrose and agar in the culture medium 4 for rooting are also the same. The only difference between the culture medium kit used in the test groups 1-6 is that the basic culture medium 4 for rooting has different macroelement components and IBA contents, which are respectively as follows
Figure GDA0002354353070000142
Figure GDA0002354353070000151
The test groups 1 to 6 all adopt the method described in the embodiment 1 to carry out the in vitro propagation of the fish wood:
the culture medium 4 for rooting described in test groups 1 to 6 was used for the rooting induction test of the fish wood by the method described above, and the test results are shown in table 5 below.
TABLE 5
Figure GDA0002354353070000152
The rooting culture is divided into the rooting culture of vitrified seedlings and semi-lignified seedlings. As can be seen from Table 5, no rooting phenomenon occurs in any rooting medium for the vitrified plantlets, and the leaves of the plants are peeled off due to yellow flowers after 10 days of cultivation, and the whole plants die due to yellowing after 30 days of cultivation, which indicates that the vitrified plantlets are not suitable for rooting cultivation. For semi-lignified seedlings, after 7 days of culture, the seedlings containing 1.5mg/L IBA concentration treatment group were rooted first (with root length of 0.1cm as standard), while the seedlings of other treatment groups were rooted successively only after 9-12 days of culture. During the process of starting rooting, each treatment group shows different degrees of leaf fall. And observing after culturing for 40 days, wherein when the IBA concentration is in the range of 0.5-1.5 mg/L, the rooting rate and the rooting coefficient are in a direct proportion relation with the IBA concentration, and the rooting rate and the rooting coefficient are higher when the IBA concentration is higher. The rooting rate and rooting line are highest in 1/2MS + IBA1.5 mg/L (test group 5), and can reach 92% and 6.33 respectively. It is noted that, at the same IBA concentration, the rooting condition is that 1/2MS culture medium has short and dense roots and thicker roots than MS culture medium, but the rooting coefficient is smaller. It is known that the rooting rate mainly depends on the IBA concentration, and the rooting coefficient is influenced by the combination of the IBA concentration and the macroelement concentration in the culture medium.
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 (2)

1. A method of promoting rapid in vitro propagation of cratava meligiosa g.forster, comprising the steps of:
(1) selecting roots of a stock plant of the fish, burying the roots in sand in a greenhouse to grow semi-lignified seedlings, and taking stem sections with axillary buds of the seedlings as explants;
(2) under the aseptic condition, sterilizing the explant by mercuric chloride, and inoculating the sterilized explant to a culture medium 1 for primary culture to obtain primary aseptic seedlings;
(3) shearing the non-vitrified primary aseptic seedlings obtained in the step (2) into stem sections with buds and 1 node, and inoculating the stem sections with buds to a culture medium 2 for subculture to obtain cluster buds;
(4) inoculating the cluster buds obtained in the step (3) on a culture medium 3 for strong seedlings to perform strong seedling culture;
(5) inoculating the healthy and strong seedlings obtained in the step (4) on a culture medium 4 for rooting, and carrying out rooting culture;
(6) cleaning the culture medium of the plant roots obtained in the step (5), and transplanting the culture medium on a substrate formed by mixing peat soil and sand in a volume ratio of 2: 1;
wherein in the culture medium 1 for primary culture, MS culture medium is taken as basic culture medium, 6-benzyladenine is 0.5mg/L, naphthylacetic acid is 0.1mg/L, thidiazuron is 0.1mg/L, sucrose is 30g/L and agar is 7 g/L;
in the culture medium 2 for subculture, an MS culture medium is taken as a basic culture medium, 6-benzyladenine is 0.5mg/L, naphthylacetic acid is 0.1mg/L, gibberellin is 0mg/L, sucrose is 30g/L and agar is 7 g/L;
in the culture medium 3 for strong seedlings, an MS culture medium is used as a basic culture medium, and 25-35 g/L of sucrose and 6.5-7.5 g/L of agar are also added;
in the culture medium 4 for rooting, 1/2MS culture medium is used as a basic culture medium, indolebutyric acid is 1.0mg/L, sucrose is 30g/L and agar is 7 g/L.
2. The method according to claim 1, wherein the culture conditions in steps (2) to (5) are all: the temperature is 24-26 ℃, the illumination time is 10-14 h/d, and the illumination intensity is 2500-3500 lx.
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