CN114158478B

CN114158478B - Tissue culture method for sea grapes

Info

Publication number: CN114158478B
Application number: CN202111100834.6A
Authority: CN
Inventors: 蒋雄; 陆小康; 詹启成; 黎东均; 王宏申
Original assignee: Guangzhou Baide Gardening Co ltd
Current assignee: Guangzhou Baide Gardening Co ltd
Priority date: 2021-09-18
Filing date: 2021-09-18
Publication date: 2022-08-23
Anticipated expiration: 2041-09-18
Also published as: CN114158478A

Abstract

The invention discloses a tissue culture method of a sea grape, which comprises the following steps: sterilizing and inoculating the explant; starting induction culture; performing proliferation culture, namely culturing in a proliferation culture medium 1, and then transferring to a proliferation culture medium 2 with the BA concentration lower than that of the proliferation culture medium 1 for culture; then culturing strong seedlings into seedlings, delivering the rooted seedlings which reach the delivery standard, continuing culturing the strong seedlings with the plant height which does not reach the standard, transferring the seedlings which reach the delivery standard but do not reach the delivery standard or the seedlings which reach the plant height but do not root to a rooting culture medium for rooting culture, and having the advantages of: the inoculation culture medium and the starting induction culture medium adopt proper hormone types and dosage, so that explants can be induced to grow buds when being terminal buds and stem segments with nodes; different hormone dosage is adopted in different stages of the proliferation culture medium, so that the proliferation rate of the proliferation seedlings is improved; after the propagation seedlings are cultured by strong seedling and seedling, different culture media are flexibly adopted for culture under the conditions of different plant heights and different rooting states, so that the tissue culture success rate and the delivery efficiency can be improved.

Description

Tissue culture method for sea grapes

Technical Field

The invention relates to the technical field of plant tissue culture, in particular to a method for culturing sea grape tissue.

Background

Vitis davidii (Coccoloba uvifera), Vitis davidii of Polygonaceae, evergreen shrub, and small arbor. The single leaf grows with each other, the male and female plants are different, the leaves are all marginal, the leaves are nearly round, and the leaf width is generally slightly larger than the leaf length. The new leaves are usually reddish brown, turn green after maturation, and have purplish red color on the surface of the aged leaves and clear veins. The female parent for greenhouse maintenance is basically green leaves.

Sea grapes are often used as street trees in coastal cities or as coastal windbreaker species, which can also be used as garden ornamentals. The sea grape fruit can be used for making jam or directly eaten. After the fruit ripens, the flower cover is purplish red, and the flesh is berry-shaped, has the diameter of about 2 cm and is clustered like grapes.

The way of stem branch culture bud growth is difficult to realize mass propagation in a short time. The effect of obtaining cluster buds by tissue culture is not good, the growth rate of new buds is low, the elongation growth is slow, and most calluses at cuts of explants are expanded and loose; and the requirement on explant materials is single, most of the explant materials need to adopt stem segments, and the success rate of inducing the growth of buds by adopting terminal buds as the explants is extremely low. A new tissue culture process is required to be researched and developed, so that the reproduction rate and the rooting rate are improved, and the production requirement is met.

Disclosure of Invention

The invention aims to provide a tissue culture method of a vitis amurensis so as to solve the problems of low reproduction rate and low rooting rate of the tissue culture method of the vitis amurensis in the prior art.

In order to achieve the purpose, the invention adopts the following technical scheme:

a tissue culture method of Vitis heyneana comprises the following steps:

(1) obtaining explants and sterilizing;

(2) inoculating an explant;

(3) after germination, performing initiation induction culture to obtain tissue culture seedlings, wherein the components of an initiation induction culture medium comprise MS, BA, NAA, sucrose and carrageenan, and the pH value is 6.2;

(4) starting induction to obtain a tissue culture seedling with stable proliferation rate and stable seedling bud number of a block, and entering a proliferation and propagation stage, firstly, adopting a proliferation and propagation culture medium 1 for culture, wherein the proliferation and propagation culture medium 1 contains MS, BA, NAA, sucrose and carrageenan, and the pH value is 6.2; along with the increase of the generation number, the conditions of small leaves, deformity, excessive foam callus, short internodes of the proliferated seedlings and few buds, or the conditions of seedling vitrification and foam callus increase/deformity are generated, the culture medium 2 with the BA concentration lower than that of the proliferated culture medium 1 is needed to be transferred for culture, and the proliferated culture medium 2 contains MS, BA, NAA, sucrose and carrageenan, and the pH is 6.2;

(5) carrying out strong seedling and seedling culture on the proliferated seedlings obtained after proliferation and propagation, directly bagging and delivering rooted seedlings which reach the delivery standard after the strong seedling and seedling culture, continuously carrying out strong seedling and seedling culture when the plant height does not reach the standard, and transferring the seedlings which reach the delivery standard but do not reach the standard or the seedlings which reach the standard but do not root to a rooting induction culture medium for rooting culture;

(6) rooting induction culture, wherein the rooting induction culture medium contains 1/2MS, NAA, sucrose and carrageenan, and the pH is 6.2.

Further, the step (1) of obtaining the explant comprises the following steps: pretreating, cleaning up dust accumulated on stems and leaves of the female parent, draining the stems and leaves of the female parent, sterilizing by sunlight ultraviolet rays, selecting branches of the same year with the green skin, cutting cleaner, healthy and strong branches, and cutting off redundant leaves; performing fine treatment, performing girdling on a leaf supporting sheath on the branch, taking care not to damage the stem, cutting the branch into single nodes, and reserving 1cm internode parts above and below each node to obtain an explant: apical bud and segmented stem; the whole process is kept clean, the explant after the fine treatment is standard, the stem section is required to be clean, the stem is not cut unless the stem is damaged by worms and rotten, and the nodes cut off the leaf supporting sheath are not stained with dust;

the explant sterilization and disinfection process comprises the following steps: wiping the surface of the explant by 75% alcohol, pouring reserved mercuric chloride into an explant material bottle, submerging the explant by the mercuric chloride liquid level, and screwing a bottle cap; in the mercuric chloride disinfection process, forcibly shaking the explant bottle every 2-3 minutes to ensure that the explant and the inner wall of the bottle are thoroughly disinfected by the mercuric chloride, and after rapidly shaking for 7-8 times each time, putting the explant bottle in the right direction and in the reverse direction in turn; if the mercuric chloride solution is discolored or turbid, when the mercuric chloride disinfection time reaches half of the target time, pouring the waste mercuric chloride into a bottle containing the waste mercuric chloride, then adding new mercuric chloride till the liquid level of the submerged explant, tightly covering the explant bottle cap, and continuing disinfection by the method; if the mercuric chloride solution does not discolor or become turbid, the mercuric chloride solution does not need to be replaced midway;

cleaning with sterile water: after mercuric chloride disinfection is finished, taking a sterile water bottle, opening a cover, pouring sterile water into the explant material bottle, holding the lower part of the explant material bottle by a left hand, slightly shaking to clean the explant and the inner wall of the bottle, shaking for 7-8 times, pouring out the cleaned sterile water into an empty wide-mouth bottle for storage, then opening a second bottle of sterile water cover, and continuously cleaning, wherein the disinfection is finished after 5-6 times of cleaning;

and (3) disinfection time: sterilizing with 0.1% mercuric chloride for 8-12 min.

Further, the inoculation process of step (2) comprises: cutting off the wound of the explant caused by mercuric chloride disinfection, cutting the terminal bud and the stem segment with the node into 1cm, clamping the upper part of the explant, inserting the lower part of the explant into an inoculation culture medium, wherein the depth of the insertion culture medium is at least 3mm, and the explant is preferably not laid down, and one explant is inoculated in each bottle; placing the culture medium at 26-30 ℃, performing astigmatism culture for 45-60 days, and then germinating explants;

the inoculation medium comprises MS, concentration of 0.5mg/LBA, concentration of 0.01mg/LNAA, concentration of 30g/L sucrose and concentration of 5.5g/L carrageenan, and pH is 6.2.

Further, the step (3) of initiating the induction culture operation comprises: transferring the explant to a starting induction culture medium after the bud grown from the explant is larger; the cutting requirements during switching are as follows: removing part of leaves of the buds with larger leaves, and removing black heads, withered yellow leaves and callus; the bud with long internodes can be transversely cut into a single section, and the bud with short internodes can be transversely cut into a plurality of sections with the length of 1-1.5 cm; after the transfer, placing the culture medium at 26 ℃ for 3000LX light culture for 45-60 days;

the start induction medium comprises the following components: MS, BA with the concentration of 0.5mg/L, BA with the concentration of 0.01mg/LNAA, sucrose with the concentration of 30g/L and carrageenan with the concentration of 5.5g/L, and the pH value is 6.2.

Further, the operation process of the step (4) is as follows: the proliferation rate of the tissue culture seedling obtained by starting induction is stable, the number of the buds of the clump seedling is stable, the tissue culture seedling enters a proliferation and propagation period, when the tissue culture seedling is transferred, black heads, withered and yellow leaves and callus tissues of the tissue culture seedling are removed, the buds with long internodes can be transversely cut into single sections, the buds with short internodes can be transversely cut into a plurality of sections, preferably 1-1.5 cm, the sections are transferred to a proliferation and propagation culture medium 1 for culture, and the culture medium is placed at 26 ℃ for 3000LX light culture; along with the increase of the algebra, the conditions of small and deformed leaves, excessive foam callus, short internodes of the proliferated seedlings and few buds, or the conditions of vitrification of the seedlings and increase/deformation of the foam callus are required to be transferred to a proliferation and propagation culture medium 2 with the BA concentration lower than that of the proliferation and propagation culture medium 1 for continuous culture, and the proliferation and propagation culture period is 45-60 d;

the proliferation and propagation medium 1 comprises the following components: MS, BA with the concentration of 0.3mg/L, 0.01mg/LNAA, sucrose with the concentration of 30g/L and carrageenan with the concentration of 5.5g/L, and the pH value is 6.2;

the proliferation and propagation medium 2 comprises the following components: MS, BA with the concentration of 0.1mg/L, BA with the concentration of 0.01mg/LNAA, sucrose with the concentration of 30g/L and carrageenan with the concentration of 5.5g/L, and the pH value is 6.2.

Further, the operation process of the step (5) comprises: carrying out strong seedling and seedling culture on the proliferated and expanded seedling, removing black heads, withered yellow leaves and callus during transfer, carrying out transverse cutting on the bud with long internodes into a single section, carrying out transverse cutting on the bud with short internodes into a plurality of sections with the length of 1-1.5 cm, transferring the bud into a strong seedling and seedling culture medium, placing the culture medium at 26 ℃, carrying out 3000LX light culture, and carrying out 45-60 d culture period; after the strong seedling and the mature seedling are cultured, the rooting seedling which reaches the shipment standard can be directly bagged and shipped, the seedling with the plant height which does not reach the shipment standard can be continuously cultured for the strong seedling and the mature seedling, and the seedling which reaches the shipment standard but does not reach the shipment or the seedling with the plant height which reaches the shipment standard but does not root is transferred to a rooting induction culture medium to be subjected to next rooting culture;

the medium for strengthening and forming the seedlings comprises the following components: 1/4MS, 0.2g/LAC concentration, 30g/L sucrose concentration and 5.5g/L carrageenan concentration, and pH is 6.2.

Further, the operation process of the step (6) comprises the following steps: cutting the tissue culture seedlings obtained in the strong seedling and seedling stage into single plants according to requirements, and directionally dividing; removing blackheads; removing the callus; removing abnormal leaves, transferring to rooting induction culture medium, culturing at 26 deg.C under 2000LX light for 20-25d, after rooting induction culture, delivering the long root, and transferring the non-long root to fresh rooting induction culture medium for rooting; the longest storage period of the rooted seedlings is not more than 2 months, and if the rooted seedlings cannot be delivered after more than 2 months due to the above reasons, the rooted seedlings are transferred to a rooting induction culture medium again according to the requirements to take roots and then can be delivered;

the rooting medium comprises the following components: 1/2MS, 0.7-1.5 mg/LNAA concentration, 30g/L sucrose concentration, 5.5g/L carrageenan concentration, and pH 6.2.

The advantages of the invention include:

the inoculation culture medium and the starting induction culture medium adopt proper hormone types and dosage, so that explants can be well induced to grow buds when being terminal buds and stem segments with nodes;

different hormone dosage is adopted in different stages of the multiplication medium, so that the multiplication rate of the tissue culture seedling is improved;

after the tissue culture seedlings are cultured by strengthening and seedling growing, different culture media are flexibly adopted for culture under the conditions of different plant heights and different rooting states, so that the tissue culture success rate and the delivery efficiency can be improved.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention:

FIG. 1 is an experimental diagram of explants;

FIG. 2 is a diagram of an explant experiment with successful inoculation culture;

FIGS. 3 to 4 are experimental graphs of tissue culture seedlings obtained by initiating induction culture;

FIG. 5 is an experimental diagram of the cut proliferated shoots obtained from the proliferation medium 1;

FIG. 6 is an experimental diagram showing the case where the proliferated plantlets obtained in the proliferation and proliferation medium 1 are cut and inoculated into the proliferation and proliferation medium 2;

FIG. 7 is a diagram showing the experimental results of the proliferated plantlets during the culture of the proliferation medium 2;

FIG. 8 is an experimental diagram of the culture and inoculation of strong seedlings and their seedlings;

FIG. 9 is a diagram showing experimental results of tissue culture seedlings obtained by culturing strong seedlings into seedlings;

FIG. 10 is a schematic diagram showing the shipment standard of tissue culture seedlings;

FIG. 11 shows a tissue culture seedling cultured in the rooting induction medium of the number 1 in tables 3 and 4;

FIG. 12 shows a tissue culture seedling cultured in the rooting induction medium of the number 2 in tables 3 and 4;

FIG. 13 shows a tissue culture seedling cultured in the rooting induction medium of the number 3 in tables 3 and 4;

FIG. 14 shows a tissue culture seedling cultured in rooting induction medium No. 4 of tables 3 and 4;

FIG. 15 shows a tissue culture seedling cultured in the rooting induction medium of the number 5 in tables 3 and 4;

FIG. 16 shows a tissue culture seedling cultured in rooting induction medium No. 6 of tables 3 and 4;

FIG. 17 shows a tissue culture seedling cultured in the rooting induction medium of the number 7 in tables 3 and 4;

FIG. 18 shows tissue-cultured seedlings cultured on rooting induction medium No. 8 in tables 3 and 4.

Detailed Description

The present invention will be described in detail with reference to the drawings and specific embodiments, which are illustrative of the present invention and are not to be construed as limiting the present invention.

Example one

1. Examples culture media used in the different culture stages:

STG1

the inoculation medium comprises the following components: MS, 0.5mg/LBA concentration, 0.01mg/LNAA concentration, 30g/L sucrose concentration and 5.5g/L carrageenan concentration, and pH is 6.2;

STG2

the induction initiating medium comprises the following components: MS, BA with the concentration of 0.5mg/L, NAA with the concentration of 0.01mg/L, sucrose with the concentration of 30g/L and carrageenan with the concentration of 5.5g/L, and the pH value is 6.2;

STG3

the proliferation and propagation medium 1 comprises the following components: MS, BA with the concentration of 0.3mg/L, NAA with the concentration of 0.01mg/L, sucrose with the concentration of 30g/L and carrageenan with the concentration of 5.5g/L, and the pH value is 6.2;

the proliferation and propagation medium 2 comprises the following components: MS, BA with the concentration of 0.1mg/L, NAA with the concentration of 0.01mg/L, sucrose with the concentration of 30g/L and carrageenan with the concentration of 5.5g/L, and the pH value is 6.2;

STG4

the medium for strengthening and forming the seedlings comprises the following components: 1/4MS, 0.2g/LAC concentration, 30g/L concentration of sucrose and 5.5g/L concentration of carrageenan, pH 6.2;

STG5

the rooting induction culture medium comprises the following components: 1/2MS, 0.7-1.5 mg/LNAA concentration, 30g/L sucrose concentration, 5.5g/L carrageenan concentration, and pH 6.2.

2. Experimental Material

The female parent requires: strong, disease and insect pest-free and pure genetic character mother plants grow;

the treatment method comprises the following steps: pretreating, cleaning dust accumulated on stems and leaves of the female parent, draining the stems and leaves of the female parent, preferably sterilizing by sunlight and ultraviolet rays, selecting branches of the current year with green epidermis as much as possible, cutting cleaner, healthy and strong branches, and cutting off redundant leaves; performing fine treatment, performing girdling on a leaf supporting sheath on the branch, taking care not to damage the stem, cutting the branch into single nodes, and reserving 1cm internode parts above and below each node to obtain an explant: apical bud and node stem. The whole process is kept clean, the explant after the fine treatment is standard, the stem section is required to be clean, the stem cannot be cut (unless the stem is damaged by worms or rotten, the stem must be cut), and the nodes cut off the leaf supporting sheaths are not stained with dust.

And (3) explant sterilization and disinfection: wiping the surface of the explant by 75% alcohol, pouring prepared mercuric chloride into the explant material bottle, submerging the explant by the mercuric chloride liquid level, and screwing the bottle cap. In the mercuric chloride disinfection process, forcibly shaking the explant bottle every 2-3 minutes to ensure that the explant and the inner wall of the bottle are thoroughly disinfected by the mercuric chloride, and after rapidly shaking for 7-8 times each time, putting the explant bottle in the right direction and putting the explant bottle upside down in turn. If the mercuric chloride solution is discolored or turbid, when the mercuric chloride disinfection time reaches half of the target time, pouring the waste mercuric chloride into a bottle containing the waste mercuric chloride, then adding new mercuric chloride till the liquid level of the submerged explant, tightly covering the explant bottle cap, and continuing disinfection by the method. If the mercuric chloride solution does not discolor or become turbid, the mercuric chloride solution does not need to be replaced midway.

Cleaning with sterile water: and after mercuric chloride disinfection is finished, taking a sterile water bottle, opening a cover, pouring sterile water into the explant material bottle, holding the lower part of the explant material bottle by a left hand, slightly shaking to clean the explant and the inner wall of the bottle, shaking for 7-8 times, pouring out the cleaned sterile water into an empty wide-mouth bottle for storage, then opening a second bottle of sterile water cover, continuously cleaning, and finishing disinfection after 5-6 times of cleaning.

And (3) disinfection time: the explant is terminal bud, 0.1% mercury bichloride is disinfected for 8-12min, and the success rate is 33% -67%; the explant is a stem section with nodes, 0.1 percent of mercury bichloride is disinfected for 8-12min, and the success rate is 10-20 percent.

The sterilized marine grape explants are shown in FIG. 1.

3. Tissue culture process

Inoculation: cutting off the wound of the explant caused by mercuric chloride disinfection, cutting the terminal bud and the segmented stem into 1cm, clamping the upper part of the explant form, inserting the lower part of the explant form into an inoculation culture medium, wherein the insertion depth of the inoculation culture medium is at least 3mm (the explant is not prone), and inoculating one explant per bottle. Placing the culture medium at 26-30 ℃, performing light scattering culture for 45-60 days, then germinating the explant, and successfully inoculating the cultured explant as shown in figure 2;

initiating induction: after the explant grows bigger buds, transferring to a starting induction culture medium. The cutting requirements during switching are as follows: removing part of leaves from the bud with larger leaves, and removing black heads, withered yellow leaves and callus. The bud with long internodes can be transversely cut into a single section, and the bud with short internodes can be transversely cut into a plurality of sections, preferably 1-1.5 cm. After the transfer, the culture medium is placed at the temperature of 26 ℃ and is cultured by 3000LX light for 45-60 days; the priming induced shoots were obtained as shown in FIGS. 3-4.

Propagation and propagation: after hormone accumulation, the proliferation rate of the tissue culture seedlings obtained by starting induction tends to be stable, and the mass seedling bud number is stable, so that the tissue culture seedlings can enter a proliferation period; during transfer, removing black heads, withered yellow leaves and callus from the tissue culture seedlings, wherein the buds with long internodes can be transversely cut into single sections, the buds with short internodes can be transversely cut into a plurality of sections, preferably 1-1.5 cm, then transferring the buds into a multiplication and propagation culture medium 1, and placing the culture medium at 26 ℃ for 3000LX light culture; after the generation number is increased, the conditions of small and abnormal leaves, excessive foam callus, short internodes of the proliferated seedlings, few buds and the like occur, or the conditions of seedling vitrification, foam callus increase or abnormal formation are caused by certain factors, the proliferated seedlings are timely transferred to a proliferation and proliferation culture medium 2 with low BA concentration for continuous culture, and as shown in figure 5, the proliferated seedlings are cut after being cultured by a proliferation and proliferation culture medium 1; FIG. 6 shows the state in which the cut proliferated seedlings cultured in the proliferation and proliferation medium 1 are inoculated into the proliferation and proliferation medium 2; as shown in FIG. 7, the propagation culture medium 2 is cultured to the 32 nd state, the propagation seedling in this state can be transferred to the strong seedling culture medium for culture, and of course, the culture time can be prolonged to 45 th-60 th days and then transferred to the strong seedling culture medium, and the whole propagation culture period is 45 d-60 d.

And (3) in the seedling strengthening and growing stage: and (3) carrying out strong seedling and seedling culture on the proliferated and expanded seedlings, removing blackheads, withered yellow leaves and callus during transfer, enabling the internode long buds to be cut into single sections in a transverse mode, enabling the internode short buds to be cut into a plurality of sections in a transverse mode, preferably 1-1.5 cm, then transferring the buds into a strong seedling and seedling culture medium, and placing the culture medium at 26 ℃ for 3000LX light culture with the culture period of 45-60 d as shown in figure 8. After the culture, the results are shown in FIG. 9.

The minimum shipment standard of the rooting seedlings is as follows: the plant height of each plant is 3-4.5 cm, the leaf length is 2-3.5 cm, the leaf width is 1.5-2.5 cm, the number of leaves is 3-6, the number of roots is 1-5, and the root length is 1-6 cm, as shown in figure 10, the plant meets the minimum shipment standard. After strong seedling and seedling culture, the rooting seedlings reaching the shipment standard can be directly bagged and shipped, the seedlings with unqualified plant height can continue strong seedling and seedling culture, and the seedlings which reach the shipment standard but not shipped or the seedlings with qualified plant height but not rooting can be transferred to a rooting induction culture medium for rooting culture.

And (3) rooting induction period:

cutting the tissue culture seedlings obtained in the seedling strengthening and growing period into single plants according to the requirements of the varieties of the tissue culture seedling product quality standard list in the product quality standard annex, wherein the indexes comprise plant height, leaf number and the like, and directionally cutting; removing black heads; removing the callus; removing abnormal leaves, and transferring to rooting induction culture medium; culturing at 26 deg.C under 2000LX light, and rooting induction for 20-25 d. After the rooting induction culture, the long roots can be delivered, and the non-long roots are continuously transferred to a fresh rooting induction culture medium for rooting. The longest storage period of the rooted seedlings is not more than 2 months, and if the rooted seedlings cannot be delivered for more than 2 months, the rooted seedlings are transferred to a rooting induction culture medium again according to the requirements to carry out rooting culture so as to be delivered. The obtained rooted shoots all meet shipment standards as shown in FIGS. 16-18.

Example two

During the development process, the inventors tried different ways of initiating induction culture, as shown in table 1:

table 1:

in the table, 30g/L sucrose and 5.8g/L carrageenan were added to the medium except for the minimal medium and the hormone, and the pH was 6.2.

As can be seen from Table 1, the influence of different hormone dosages on the growth states of different types of explants is different, and the culture medium formula in the first embodiment of the invention can achieve a good induction starting effect on both terminal buds and node stem sections of the explants, so that the growth state of the explants can be optimal.

In the development process, the inventors also tried different propagation culture modes, as shown in table 2:

table 2:

Therefore, when the BA in the multiplication and expansion culture medium 1 is 0.3mg/L and the BA in the multiplication and expansion culture medium 2 is 0.1mg/L, the multiplication rate of the seedlings is the highest. The seedlings cultured by the proliferation and propagation culture mode of the invention grow well, and the number of the foam callus is less.

In the development process, the inventor also tries different rooting culture modes, as shown in tables 3 and 4:

table 3:

TABLE 4

The tissue culture plantlets cultured in the rooting induction medium of number 1 in tables 3 and 4 are shown in FIG. 11; the tissue culture seedling cultured by the rooting induction medium with the serial number 2 is shown in figure 12; the tissue culture seedling cultured by the rooting induction medium with the serial number 3 is shown in figure 13; the tissue culture seedling cultured by the rooting induction medium with the serial number 4 is shown in figure 14; the tissue culture seedling cultured by the rooting induction medium with the serial number 5 is shown in figure 15; the tissue culture seedling cultured by the rooting induction medium with the serial number 6 is shown in figure 16; the tissue culture seedling cultured by the rooting induction medium with the serial number 7 is shown in figure 17; the tissue culture plantlets cultured in the rooting induction medium No. 8 are shown in FIG. 18, and the rooted plantlets in FIGS. 16 to 18 meet the shipment standards and have high rooting rates.

In summary, tables 3 to 4 and FIGS. 11 to 18 show that:

1. comparing the treatments, the foamed callus is large and the root length is short, which is not beneficial to rooting;

the rooting condition of the NAA hormones is superior to the rooting condition of the IBA hormones;

when the IBA concentration is more than 1.0mg/L, foam callus can appear; when the NAA concentration is more than 1.0mg/L, foam callus is also generated.

4. The foam callus is similar to fine sand, and the foam callus is scattered after being twisted and is grey white.

5. Taking NAA0.7mg/L-NAA1.5mg/L as the most suitable rooting culture medium, the rooting rate can reach 75% -86.1%.

The way of culturing buds and growing buds by the stem-moving branches of the sea grapes is difficult, and the effect of obtaining cluster buds is not as good as that of other varieties. Only a single section or multiple sections of branches can be used, the branches are grown into branches by single section or multiple sections, and then the branches are cut into multiple single sections or multiple sections to realize proliferation. If too much foam callus exists in the proliferation process, the BA concentration needs to be reduced, the rooting rate of the variety is low, but the tissue culture method can ensure that the rooting rate reaches 75-86.1 percent, the rooting seedlings reaching the standard can be delivered, and the non-rooted seedlings can be continuously transferred to a rooting culture medium for rooting, so that the tissue culture success rate is improved.

The technical solutions provided by the embodiments of the present invention are described in detail above, and the principles and embodiments of the present invention are explained herein by using specific examples, and the descriptions of the embodiments are only used to help understanding the principles of the embodiments of the present invention; meanwhile, for a person skilled in the art, according to the embodiments of the present invention, the specific implementation manners and the application ranges may be changed, and in conclusion, the content of the present specification should not be construed as limiting the invention.

Claims

1. Sea grapeCoccoloba uviferaA tissue culture method, characterized by:

the method comprises the following steps:

(1) obtaining terminal buds and stem sections with nodes of explants, and sterilizing and disinfecting;

(2) inoculating an explant;

the components of the inoculation culture medium comprise MS, BA with the concentration of 0.5mg/L, NAA with the concentration of 0.01mg/L, sucrose with the concentration of 30g/L and carrageenan with the concentration of 5.5g/L, and the pH value is 6.2;

(3) after germination, starting induction culture to obtain tissue culture seedlings;

(4) starting induction to obtain tissue culture seedlings with stable proliferation rate and stable seedling bud number of the block, entering a proliferation and propagation stage, and firstly adopting a proliferation and propagation culture medium 1 for culture; along with the increase of the generation number, the conditions of small and deformed leaves, excessive foam callus, short internodes of the proliferated seedlings and few buds, or the conditions of seedling vitrification and foam callus increase/deformation are generated, and the proliferated seedlings need to be transferred to a proliferated culture medium 2 with the BA concentration lower than that of the proliferated culture medium 1 for culture;

the propagation and multiplication medium 1 comprises the following components: MS, BA with the concentration of 0.3mg/L, NAA with the concentration of 0.01mg/L, sucrose with the concentration of 30g/L and carrageenan with the concentration of 5.5g/L, and the pH value is 6.2;

(5) carrying out strong seedling and seedling culture on the proliferated seedlings obtained after proliferation and propagation, directly bagging the rooting seedlings which reach the shipment standard after the strong seedling and seedling culture, carrying out the strong seedling and seedling culture on the rooting seedlings which reach the shipment standard, continuing the strong seedling and seedling culture on the rooting seedlings which reach the shipment standard but do not reach the shipment standard or the rooting seedlings which reach the plant height but do not root to a rooting induction culture medium for rooting culture;

the medium for strengthening and forming the seedlings comprises the following components: 1/4MS, 0.2g/L AC, 30g/L sucrose and 5.5g/L carrageenan, and has pH of 6.2;

(6) rooting induction culture, wherein the rooting induction culture medium comprises the following components: 1/2MS, NAA with concentration of 0.7-1.5 mg/L, sucrose with concentration of 30g/L and carrageenan with concentration of 5.5g/L, and pH is 6.2.

2. A sea grape according to claim 1Coccoloba uviferaA tissue culture method, characterized by:

the process for obtaining the explant in the step (1) comprises the following steps: pretreating, cleaning dust accumulated on stems and leaves of the female parent, draining the stems and leaves of the female parent, sterilizing by sunlight ultraviolet rays, selecting current-year branches with green epidermis, cutting cleaner, healthy and strong branches, and cutting off redundant leaves; performing fine treatment, namely performing girdling on a leaf supporting sheath on the branch, taking care not to damage the stem, cutting the branch into single nodes, and reserving 1cm internode parts above and below each node; the whole process is kept clean, the explant after the fine treatment is standard, the stem section is required to be clean, the stem is not cut unless the stem is damaged by worms and rotten, and the nodes cut off the leaf supporting sheath are not stained with dust;

the explant sterilization and disinfection process comprises the following steps: wiping the surface of the explant by 75% alcohol, pouring reserved mercuric chloride into an explant material bottle, submerging the explant by the mercuric chloride liquid level, and screwing a bottle cap; in the mercuric chloride disinfection process, forcibly shaking the explant bottle every 2-3 minutes to ensure that the explant and the inner wall of the bottle are thoroughly disinfected by the mercuric chloride, and after rapidly shaking for 7-8 times each time, alternately placing the explant bottle forwards and backwards; if the mercuric chloride solution is discolored or turbid, when the mercuric chloride disinfection time reaches half of the target time, pouring the waste mercuric chloride into a bottle containing the waste mercuric chloride, then adding new mercuric chloride till the liquid level of the submerged explant, tightly covering the explant bottle cap, and continuing disinfection by the method; if the mercuric chloride solution does not discolor or become turbid, the mercuric chloride solution does not need to be replaced midway;

cleaning with sterile water: after mercuric chloride disinfection is finished, taking a sterile water bottle, opening a cover, pouring sterile water into the explant material bottle, holding the lower part of the explant material bottle by a left hand, slightly shaking to clean the explant and the inner wall of the bottle, shaking for 7-8 times, pouring out the cleaned sterile water into an empty wide-mouth bottle for storage, then opening a sterile water cover of a second bottle, and continuously cleaning, wherein the sterilization is finished after 5-6 times of cleaning;

3. A sea grape according to claim 1Coccoloba uviferaA tissue culture method, characterized by:

the inoculation process of the step (2) comprises the following steps: cutting off the wound of the explant caused by mercuric chloride disinfection, cutting the terminal bud and the stem segment with the node into 1cm, clamping the upper part of the explant, inserting the lower part of the explant into an inoculation culture medium, wherein the depth of the insertion culture medium is at least 3mm, so that the explant does not fall down, and one explant is inoculated in each bottle; placing the culture medium at 26-30 ℃, performing light scattering culture for 45-60 days, and then germinating the explant.

4. Sea grape according to claim 1Coccoloba uviferaA tissue culture method characterized by:

the step (3) of starting the induction culture operation comprises the following steps: transferring to a starting induction culture medium after the bud grown from the explant is larger; the cutting requirements during switching are as follows: removing part of leaves from the buds with larger leaves, and removing black heads, withered yellow leaves and callus; the bud with long internodes is transversely cut into single sections, and the bud with short internodes is transversely cut into a plurality of sections with the length of 1-1.5 cm; after the transfer, the culture medium was incubated at 26 ℃ under 3000lx light for 45 days to 60 days.

5. Sea grape according to claim 1Coccoloba uviferaA tissue culture method, characterized by:

the operation process of the step (4) is as follows: starting induction to obtain a tissue culture seedling with stable proliferation rate and stable bud number of a block seedling, entering a proliferation and propagation period, removing black heads, withered yellow leaves and callus from the tissue culture seedling in the condition during transfer, transversely cutting buds with long internodes into single sections, transversely cutting buds with short internodes into a plurality of sections with the length of 1-1.5 cm, transferring the buds into a proliferation and propagation culture medium 1 for culture, placing the culture medium at 26 ℃, and carrying out 3000lx light culture; and (3) along with the increase of the generation number, the conditions of small leaves, deformity, excessive foam callus, short internodes of the proliferated seedlings and few buds, or the conditions of seedling vitrification and foam callus increase/deformity are generated, the seedlings need to be transferred to a proliferation and propagation culture medium 2 with the BA concentration lower than that of the proliferation and propagation culture medium 1 for continuous culture, and the proliferation and propagation culture period is 45-60 d.

6. Sea grape according to claim 1Coccoloba uviferaA tissue culture method, characterized by:

the operation process of the step (5) comprises the following steps: carrying out strong seedling and seedling culture on the proliferated and expanded seedlings, removing black heads, withered yellow leaves and callus during transfer, transversely cutting the buds with long internodes into single joints, transversely cutting the buds with short internodes into a plurality of sections with the length of 1-1.5 cm, transferring the buds into a strong seedling and seedling culture medium, and culturing the medium at 26 ℃ under 3000lx light for 45-60 days; and directly bagging the rooting seedlings which reach the shipment standard after the strong seedling and the seedling growth culture, continuously culturing the strong seedlings and the seedlings with the plant height which does not reach the shipment standard, transferring the seedlings which reach the shipment standard but do not reach the shipment standard or the seedlings with the plant height which reaches the shipment standard but do not root to a rooting induction culture medium, and carrying out the next step of rooting culture.

7. A sea grape according to claim 1Coccoloba uviferaA tissue culture method characterized by:

the operation process of the step (6) comprises the following steps: cutting the tissue culture seedlings obtained in the strong seedling and seedling stage into single plants according to requirements, and directionally dividing; removing blackheads; removing the callus; removing abnormal leaves, transferring to rooting induction culture medium, culturing at 26 deg.C under 2000lx light for 20-25d, discharging long root, and transferring to fresh rooting induction culture medium for rooting; the longest storage period of the rooted seedlings is not more than 2 months, and if the rooted seedlings cannot be delivered after more than 2 months, the rooted seedlings need to be transferred to a rooting induction culture medium again according to the requirements for delivering the products.