CN114145229A

CN114145229A - Tissue culture method for colorful Heguocao taro

Info

Publication number: CN114145229A
Application number: CN202111138180.6A
Authority: CN
Inventors: 蒋雄; 詹启成; 黎东均; 张花; 周博
Original assignee: Foshan Sanshui Young Plants Co ltd
Current assignee: Foshan Sanshui Young Plants Co ltd
Priority date: 2021-09-27
Filing date: 2021-09-27
Publication date: 2022-03-08
Anticipated expiration: 2041-09-27
Also published as: CN114145229B

Abstract

The invention discloses a tissue culture method of colorful Heguocao taro, which comprises the following steps: obtaining explants, and sterilizing and disinfecting the explants; inoculating and culturing explants; starting induction culture; cutting the tissue mass obtained by the induced induction culture, and then carrying out proliferation propagation culture, wherein the proliferation propagation culture medium comprises the components of MS, BA3mg/L, IAA 0.5-1 mg/L, sucrose 30g/L, carrageenan 5.8g/L and pH 6.2; cutting the proliferation blocks obtained by proliferation and propagation, and then performing strong seedling and seedling culture, wherein the components of a strong seedling and seedling culture medium comprise MS, AC0.2g/L, 30g/L of sucrose, 5.8g/L of carrageenan, and the pH value is 6.2; cutting the seedling obtained by strong seedling and seedling culture, and then carrying out rooting induction culture to obtain the tissue culture seedling, wherein the rooting induction culture medium comprises MS, AC1g/L, sucrose 30g/L, carrageenan 5.8g/L and pH is 6.2. The advantages include: the proliferation and propagation culture medium, the strong seedling culture medium and the rooting induction culture medium have proper components, can proliferate and culture the block seedlings in large quantity, strengthen the seedlings efficiently, improve the rooting efficiency and reduce the variation rate.

Description

Tissue culture method for colorful Heguocao taro

Technical Field

The invention relates to the technical field of plant tissue culture, in particular to synanthotamus plant tissue culture.

Background

Heguocao is a perennial evergreen herb of Heguo of Araceae. The leaves are in two types, arrow-shaped or halberd-shaped; small ear-shaped leaves are usually grown on both sides of the base lobe. The colorful Heguo taro is also named as Baumai, is a Heguo taro of Heguo of Araceae, has the Latin chemical name of Syngonium 'Dazle Color', has the original variety of neon, and is a variant variety which is independently selected and bred by an applicant in the tissue culture process. The leaf had a white main hue with red spots on it as shown in FIG. 1. The colorful Heguo taro is more and more favored by consumers after being popularized and sold in the market. Tissue culture is a main production mode of colorful Heguocao, but the conventional tissue culture method of Heguo is not suitable for the culture of the variety, has poor rooting efficiency, is easy to have variation, influences the yield and cannot meet the increasing market demand.

Disclosure of Invention

The invention aims to provide a tissue culture method of colorful synanthotagmus, which aims to solve the problems of low rooting efficiency and easy variation of the colorful synanthromus.

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

a tissue culture method of colorful Heguocao taro comprises the following steps:

(1) obtaining explants, and sterilizing and disinfecting the explants;

(2) inoculating and culturing explants;

(3) starting induction culture;

(4) cutting the tissue mass obtained by the induced culture starting, and then carrying out proliferation propagation culture, wherein the proliferation propagation culture medium comprises the components of MS, BA3mg/L, IAA 0.5-1 mg/L, sucrose 30g/L, carrageenan 5.8g/L and pH 6.2;

(5) cutting the proliferation blocks obtained by proliferation and propagation, and then performing strong seedling and seedling culture, wherein the components of a strong seedling and seedling culture medium comprise MS, AC0.2g/L, sucrose 30g/L, carrageenan 5.8g/L and pH is 6.2;

(6) cutting the seedling obtained by strong seedling and seedling culture, and then carrying out rooting induction culture to obtain the tissue culture seedling, wherein the rooting induction culture medium comprises the components of MS, AC1g/L, sucrose 30g/L, carrageenan 5.8g/L and pH is 6.2.

Further, the explant obtaining process in the step (1) is as follows: airing the colorful Heguo taro stock plant to dry the matrix to be grey white on the surface layer; cutting off the upper stem segment without lateral buds of the plant by using a dissecting knife in sunny days to form a shortened stem, then truncating the upper petiole, keeping the length of the petiole of the shortened stem to be 1cm, and keeping the lobule of the stem top;

cutting residual leaves or leafstalks on the stems from the base parts of the leafstalks to avoid cutting side buds and stem sections, starting from stem sections at the base parts of the stem sections, treating until stem sections at the top ends of the stem sections, if residual old leaves or old barks or other residual dirt exists on the surfaces of the stem sections, carefully scraping the stems by using a scalpel, and avoiding scraping the skins of the stems in the scraping treatment; cutting off each stem segment from the 2 nd node from the top end to obtain an explant for later use.

Further, the explant sterilization process in the step (1) is as follows: cutting the treated stem of the explant into single segments, cutting the short internodes into segments of 2-3 segments or cutting the short stem of the explant without segments, treating the short stem of the explant with 75% alcohol for 30-60s, and then treating the short stem of the explant with mercuric chloride for 25-35 min.

Further, the process of explant inoculation culture in the step (2) comprises the following steps: cutting off the incisions at the two ends of the stem segment of the explant, wherein the incisions are 2mm thick, clamping the morphological upper part of the stem segment of the explant, inserting the stem segment of the explant into an inoculation culture medium, inserting the stem segment of the explant into the inoculation culture medium with the depth of 3mm, placing the inoculation culture medium in an environment at 22 ℃, performing light scattering culture, performing 3000-4500lx, performing 16h light/8 h dark culture for 30-40 days, and germinating axillary buds on nodes;

the components of the inoculation culture medium comprise: MS, 3.0mg/L of BA, 30g/L of cane sugar, 5.8g/L of carrageenan and 6.2 of pH.

Further, the process of initiating the induction culture in the step (3) comprises: cutting off buds obtained by inoculation culture, transferring the buds to a fresh start induction culture medium, placing the buds at 26 ℃ for 30-45 days under 3000LX, growing single buds into cluster buds, completing the first start induction culture, cutting, transferring the buds to the fresh start induction culture medium, and inoculating 1-2 clusters in each bottle; thus, each time the induction culture is started for 40-45 days, the fresh induction culture medium is cut and transferred once, and the number of the block masses connected in the bottle is gradually increased according to the transfer times, and the block masses can reach 10 block masses at most.

Further, in the step (3), the cutting requirements when the bud obtained by the inoculation culture is cut off are as follows: in the case of small buds, some maternal tissues are left, and the top is removed for proliferation; removing the top and splitting into two halves under the condition of thick buds;

the cutting requirement after the induction culture is started for the first time is completed: removing tops and roots, cutting the small buds into 2-3 buds/clusters or 1-2 buds/clusters, and directionally dividing; cutting the bud into two halves after topping under the condition of thick buds, wherein the length of the stem remained during topping is 0.8-1 cm; removing withered yellow leaves;

and (3) inoculating a fresh start induction culture medium every time, wherein the cutting requirement refers to the cutting requirement after the first start induction culture is finished.

Further, the start induction medium: MS, 3mg/L, IAA 1mg/L of BA, 30g/L of cane sugar, 5.8g/L of carrageenan and 6.2 of pH.

Further, the process of propagation culture in step (4) comprises: starting induction culture to obtain a large number of blocks, and then carrying out proliferation propagation culture, wherein the cutting requirement is as follows: removing obvious blackheads; removing the top; removing roots; removing withered and yellow bracts, loosening and blackening the calluses; dividing cluster buds into 2-3 buds/cluster, and directionally dividing; if the variant seedlings with full red leaves are found, timely removing the variant seedlings; inoculating the cut bud group into a proliferation and propagation culture medium, and culturing for 30d at 26 ℃ under 3000LX light.

Further, the process of strengthening and growing the seedlings in the step (5) comprises the following steps: after a large amount of proliferation blocks are obtained by proliferation and propagation, strong seedling and seedling culture are carried out; selecting the proliferation blocks which grow vigorously and have high uniformity, directionally dividing the proliferation blocks, wherein each proliferation block comprises 3 buds, and inoculating the proliferation blocks into a strong seedling culture medium; if the variant seedlings with full red leaves are found, timely removing the variant seedlings; and placing the inoculated strong seedling and seedling culture medium at 26 ℃ for 3000LX light culture for 26d.

Further, the process of rooting induction in step (6) comprises: cutting the seedlings obtained by culturing strong seedlings into seedlings, wherein the cutting requirements are as follows: the plant height is more than or equal to 2 cm; the leaf width is more than or equal to 1 cm; the number of leaves is more than or equal to 1.5; 1-2 sections; removing withered and yellow leaves; root retention or removal; if the variant seedlings with full red leaves are found, timely removing the variant seedlings; after cutting, the cut pieces are inoculated into a rooting induction culture medium and are placed at 26 ℃ for 3000LX light culture for 15d.

The advantages of the invention include: the proliferation and propagation culture medium, the strong seedling culture medium and the rooting induction culture medium have proper components, can proliferate and culture the block seedlings in large quantity, strengthen the seedlings efficiently, improve the rooting efficiency and reduce the variation rate.

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 a colorful Heyu taro picture;

FIG. 2 is a photograph of the mother after pretreatment;

FIG. 3 is a picture of explants at inoculation;

FIG. 4 is a photograph showing the proliferation and propagation culture during inoculation;

FIG. 5 shows a proliferation pellet obtained after completion of proliferation and propagation culture;

FIG. 6 shows a seedling obtained after culturing strong seedlings;

FIG. 7 is a photograph at the time of inoculation of rooting-inducing culture;

FIG. 8 shows a tissue culture seedling cultured in a rooting induction medium containing 1g/L of AC;

FIG. 9 is a photograph of a tissue culture seedling meeting shipment standards;

FIG. 10 is a picture of the experiment at the time of treatment 1 inoculation in the relevant experiment of proliferation and propagation culture;

FIG. 11 is a picture of the experiment at the time of treatment 2 inoculation in the experiment relating to propagation culture;

FIG. 12 is a graph showing the state of the culture period of

treatments

1 and 2 in the experiments relating to the proliferation and proliferation culture;

FIG. 13 is a photograph showing the completion of the culture of

treatments

1 and 2 in the experiments relating to the proliferation and propagation culture;

FIG. 14 is an experimental picture of treatment 1 after completion of the relevant experiment for strong seedling and seedling culture;

FIG. 15 is an experimental picture of treatment 2 after completion of the relevant experiment for strong seedling and seedling culture;

FIG. 16 is an experimental picture of treatment 3 after completion of the relevant experiment for strong seedling and seedling culture;

FIG. 17 is a picture of the experiment of treatment 1 after completion of the experiment relating to rooting induction culture.

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.

Firstly, a culture medium used in each stage of tissue culture of colorful Heguocao taro:

inoculating a culture medium: MS, 3.0mg/L of BA, 30g/L of cane sugar, 5.8g/L of carrageenan and 6.2 of pH;

starting an induction culture medium: MS, 3mg/L, IAA 1mg/L BA, 30g/L sucrose, 5.8g/L carrageenan, and pH 6.2;

proliferation and propagation culture medium: MS, 3mg/L, IAA 0.5.5-1 mg/L BA, 30g/L sucrose, 5.8g/L carrageenan, and pH 6.2;

strong seedling and seedling culture medium: MS, AC0.2g/L, sucrose 30g/L, carrageenan 5.8g/L, pH 6.2;

rooting induction culture medium: MS, AC1g/L, sucrose 30g/L, carrageenan 5.8g/L, pH 6.2.

II, materials

The female parent requires: the colorful Heyou taro stock plant has the advantages of robust growth, no plant diseases and insect pests, and pure genetic character with the plant height of about 15-25 cm;

the treatment method comprises the following steps: drying in the dry and ventilated place, not watering, and preventing rain, so that the matrix is dried to be grey-white on the surface layer; selecting a sunny day, cutting off the upper stem section without the lateral bud of the plant by using a dissecting knife to form a shortened stem, and then cutting off the upper petiole to ensure that the length of the petiole of the shortened stem is kept about 1cm, and the lobule at the top of the stem can be reserved firstly for later use; cutting the residual leaves or petioles on the stem from the base of the petiole to avoid cutting the lateral buds and the stem segments, starting from the stem node at the base of the stem segment and treating until the stem node at the top of the stem segment, as shown in figure 2, if the surface of the stem segment has residual old leaves or old bark or other residual dirt, carefully scraping the surface of the stem segment with a scalpel is needed, and the scraping treatment avoids scratching the surface of the stem. Cutting off each stem segment from the 2 nd node from the top end to obtain an explant for later use.

The disinfection method comprises the following steps:

cutting the treated explant into single segments, cutting the internodes into 2-3 segments or the shortened stems into short segments without cutting, treating with 75% alcohol for 30-60s, and treating with mercuric chloride for 25-35 min.

Third, culture

Inoculation: cutting off the two ends of the stem segment of the explant to form incisions with the thickness of about 2mm, clamping the morphological upper part of the stem segment of the explant, inserting the stem segment of the explant into an inoculation culture medium with the depth of about 3mm, as shown in figure 3, placing the inoculation culture medium in an environment with the temperature of 22 ℃, performing light scattering culture, 3000-4500lx, 16h light/8 h dark, and performing culture for 30-40 days, wherein axillary buds on the nodes germinate.

Initiating induction culture: cutting off buds obtained by inoculating and culturing, wherein the cutting requirements are as follows: if the bud is smaller, some maternal tissues can be left, and the apical proliferation is removed; when the bud is bigger and thicker, the top of the bud can be removed and the bud can be cut into two halves. After cutting, transferring the bud into a fresh start induction culture medium, placing the bud in a condition of 26 ℃ and 3000LX for culturing for 30-45 days, then growing a single bud into multiple buds, completing the first start induction culture, and then cutting: removing tops and roots; cutting the mass with smaller bud into 2-3 buds/mass, cutting into 1-2 buds/mass if the bud is bigger, and directionally cutting; cutting the bigger bud into two halves after topping, wherein the stem remained during topping is 0.8-1 cm, otherwise, the bud is not easy to grow; removing withered yellow leaves. After cutting, transferring the cells into a fresh induction starting culture medium, and inoculating 1-2 clusters in each bottle. Thus, each time the induction culture is started for 40-45 days, the fresh induction culture medium is cut and transferred once, the cutting requirement refers to the cutting requirement after the first induction culture is started, the number of the block masses connected in the bottle is gradually increased according to the transfer times, and the block masses can reach 10 block masses at most.

Proliferation and propagation culture: after induction culture is started to obtain a large number of blocks, proliferation and propagation culture can be carried out, and the cutting requirements are as follows: removing obvious blackheads; removing the top; removing roots; removing withered and yellow bracts, loosening and blackening the calluses; the cluster buds are divided into 2-3 buds/group, and divided directionally. If the mutant seedlings with red leaves are found, the mutant seedlings should be removed in time. As shown in FIG. 4, the cut clusters were inoculated into a proliferation medium and cultured at 26 ℃ under 3000LX light for 30 days. FIG. 5 shows the proliferated pellet obtained after the proliferation and propagation culture.

Strong seedling and seedling culture: after a large amount of proliferation blocks are obtained by proliferation and propagation, strong seedling and seedling culture are carried out. And selecting the proliferation masses with vigorous growth and high uniformity, directionally dividing the proliferation masses, wherein each mass comprises about 3 larger buds, and inoculating the proliferation masses into a strong seedling culture medium. If the mutant seedlings with red leaves are found, the mutant seedlings should be removed in time. The inoculated strong seedling and seedling culture medium is placed at 26 ℃ and cultured for 26d by 3000LX light, and the seedling is obtained after the strong seedling and seedling culture as shown in figure 6.

Rooting induction culture: after the strong seedling and seedling culture is finished, carrying out rooting induction, cutting the seedling obtained by the strong seedling and seedling culture, wherein the cutting requirement is as follows: the plant height is more than or equal to 2 cm; the leaf width is more than or equal to 1 cm; the number of leaves is more than or equal to 1.5; 1-2 sections; removing withered and yellow leaves; the roots may be retained or removed. Generally, if the root is longer, the root can be cut off, and the root can be conveniently clamped into a bag or a bottle. If the mutant seedlings with red leaves are found, the mutant seedlings should be removed in time. After completion of the cutting as shown in FIG. 7, the cut pieces were inoculated into a rooting induction medium and incubated at 26 ℃ for 15 days under 3000LX light.

The tissue culture seedling obtained from the rooting induction medium with 1g/L of AC content is shown in FIG. 8, and meets the shipment standard.

The shipping criteria are shown in fig. 9.

The experiment related to the proliferation and propagation culture is shown in the following table 1:

TABLE 1

In the table, the proliferation and propagation medium contained 30g/L sucrose and 5.8g/L carrageenan in addition to the components shown in the table, and the pH was 6.2.

As shown in fig. 10 and 11, the experimental pictures of treatment 1 and treatment 2 during inoculation are shown; as shown in FIG. 12, it was observed that the leaf opening was much larger in treatment 2 than in treatment 1 during the culture. After the propagation, the propagated seedlings shown in FIG. 13 were obtained.

The relevant tests for strong seedling and seedling culture are shown in table 2 below:

in the table, the strong seedling and seedling culture medium comprises 30g/L of sucrose and 5.8g/L of carrageenan, and the pH value is 6.2 besides the components recorded in the table.

As can be seen from the table, MS is more suitable for strong seedling and seedling culture as a basic culture medium. Proper active carbon is added into the culture medium, which is favorable for strengthening the seedling.

The relevant experiments for rooting induction culture are shown in table 3 below:

table 3:

in the table, the rooting induction medium contained 30g/L sucrose and 5.8g/L carrageenan in addition to the components described in the table, and the pH was 6.2.

The rooting effect of the treatment 1 is slightly better than that of the treatment 2, but the two are not very different.

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, there may be variations in the specific implementation manners and application ranges, and in summary, the content of the present description should not be construed as a limitation to the present invention.

Claims

1. A tissue culture method of colorful Heguocao taro is characterized in that:

the method comprises the following steps:

(1) obtaining explants, and sterilizing and disinfecting the explants;

(2) inoculating and culturing explants;

(3) starting induction culture;

(4) cutting the tissue mass obtained by the induced induction culture, and then carrying out proliferation propagation culture, wherein the proliferation propagation culture medium comprises the components of MS, BA3mg/L, IAA 0.5.5-1 mg/L, sucrose 30g/L, carrageenan 5.8g/L and pH 6.2;

2. The tissue culture method of colorful Heyu taro as claimed in claim 1, wherein the tissue culture method comprises the following steps:

the explant acquisition process in the step (1) comprises the following steps: airing the colorful Heguo taro stock plant to dry the matrix to be grey white on the surface layer; cutting off the upper stem segment without lateral buds of the plant by using a dissecting knife in sunny days to form a shortened stem, then truncating the upper petiole, keeping the length of the petiole of the shortened stem to be 1cm, and keeping the lobule of the stem top;

3. The tissue culture method of colorful Heyu taro as claimed in claim 1, wherein the tissue culture method comprises the following steps:

the explant sterilization process in the step (1) comprises the following steps: cutting the treated stem of the explant into single segments, cutting the short internodes into segments of 2-3 segments or cutting the short stem of the explant without segments, treating the short stem of the explant with 75% alcohol for 30-60s, and then treating the short stem of the explant with mercuric chloride for 25-35 min.

4. The tissue culture method of colorful Heyu taro as claimed in claim 1, wherein the tissue culture method comprises the following steps:

the process of explant inoculation culture in the step (2) comprises the following steps: cutting off the incisions at the two ends of the stem segment of the explant, wherein the incisions are 2mm thick, clamping the morphological upper part of the stem segment of the explant, inserting the stem segment of the explant into an inoculation culture medium, inserting the stem segment of the explant into the inoculation culture medium with the depth of 3mm, placing the inoculation culture medium in an environment at 22 ℃, performing light scattering culture, performing 3000-4500lx, performing 16h light/8 h dark culture for 30-40 days, and germinating axillary buds on nodes;

5. The tissue culture method of colorful Heyu taro as claimed in claim 1, wherein the tissue culture method comprises the following steps:

the process of initiating induction culture in the step (3) comprises the following steps: cutting off buds obtained by inoculation culture, transferring the buds to a fresh start induction culture medium, placing the buds at 26 ℃ for 30-45 days under 3000LX, growing single buds into cluster buds, completing the first start induction culture, cutting, transferring the buds to the fresh start induction culture medium, and inoculating 1-2 clusters in each bottle; thus, each time the induction culture is started for 40-45 days, the fresh induction culture medium is cut and transferred once, and the number of the block masses connected in the bottle is gradually increased according to the transfer times, and the block masses can reach 10 block masses at most.

6. The tissue culture method of colorful Heyu taro as claimed in claim 5, wherein the tissue culture method comprises the following steps:

in the step (3), the cutting requirements when the buds obtained by the inoculation culture are cut off are as follows: in the case of small buds, some maternal tissues are left, and the top is removed for proliferation; removing the top and splitting into two halves under the condition of thick buds;

7. The tissue culture method of colorful Heyu taro as claimed in any one of claims 1, 5 and 6, wherein:

the components of the start induction culture medium comprise: MS, 3mg/L, IAA 1mg/L of BA, 30g/L of cane sugar, 5.8g/L of carrageenan and 6.2 of pH.

8. The tissue culture method of colorful Heyu taro as claimed in claim 1, wherein the tissue culture method comprises the following steps:

the process of proliferation and propagation culture in the step (4) comprises the following steps: starting induction culture to obtain a large number of blocks, and then carrying out proliferation propagation culture, wherein the cutting requirement is as follows: removing obvious blackheads; removing the top; removing roots; removing withered and yellow bracts, loosening and blackening the calluses; dividing cluster buds into 2-3 buds/cluster, and directionally dividing; if the variant seedlings with full red leaves are found, timely removing the variant seedlings; inoculating the cut bud group into a proliferation and propagation culture medium, and culturing for 30d at 26 ℃ under 3000LX light.

9. The tissue culture method of colorful Heyu taro as claimed in claim 1, wherein the tissue culture method comprises the following steps:

the process of strengthening and forming the seedlings in the step (5) comprises the following steps: after a large amount of proliferation blocks are obtained by proliferation and propagation, strong seedling and seedling culture are carried out; selecting the proliferation blocks which grow vigorously and have high uniformity, directionally dividing the proliferation blocks, wherein each proliferation block comprises 3 buds, and inoculating the proliferation blocks into a strong seedling culture medium; if the variant seedlings with full red leaves are found, timely removing the variant seedlings; and placing the inoculated strong seedling and seedling culture medium at 26 ℃ for 3000LX light culture for 26d.

10. The tissue culture method of colorful Heyu taro as claimed in claim 1, wherein the tissue culture method comprises the following steps:

the process of rooting induction in the step (6) comprises the following steps: cutting the seedlings obtained by culturing strong seedlings into seedlings, wherein the cutting requirements are as follows: the plant height is more than or equal to 2 cm; the leaf width is more than or equal to 1 cm; the number of leaves is more than or equal to 1.5; 1-2 sections; removing withered and yellow leaves; root retention or removal; if the variant seedlings with full red leaves are found, timely removing the variant seedlings; after cutting, the cut pieces are inoculated into a rooting induction culture medium and are placed at 26 ℃ for 3000LX light culture for 15d.