CN111280056A - Subculture breeding method of stingless pepper tissue culture seedlings - Google Patents

Subculture breeding method of stingless pepper tissue culture seedlings Download PDF

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Publication number
CN111280056A
CN111280056A CN202010136322.4A CN202010136322A CN111280056A CN 111280056 A CN111280056 A CN 111280056A CN 202010136322 A CN202010136322 A CN 202010136322A CN 111280056 A CN111280056 A CN 111280056A
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culture
subculture
stem
tissue culture
zanthoxylum
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陈泽雄
唐宁
娄娟
许锋
刘霞
刘华敏
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Yangtze University
Chongqing University of Arts and Sciences
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Yangtze University
Chongqing University of Arts and Sciences
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Priority to CN202110603511.2A priority Critical patent/CN113331055A/en
Priority to CN202010136322.4A priority patent/CN111280056A/en
Publication of CN111280056A publication Critical patent/CN111280056A/en
Priority to PCT/CN2020/134255 priority patent/WO2021174933A1/en
Priority to NL2027681A priority patent/NL2027681B1/en
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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
    • A01H4/008Methods for regeneration to complete plants
    • 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
    • A01H4/002Culture media for tissue culture

Abstract

The invention discloses a subculture breeding method of stingless pepper tissue culture seedlings, and belongs to the technical field of vegetative propagation of cash crops. The method specifically comprises the following steps: primary culture, cutting and subculture proliferation, wherein the subculture proliferation is finished until the amount of PPM in the culture medium is reduced to 0, and the generation number of the subculture proliferation is not less than 4. The method for subculturing the tissue culture seedlings of the stingless Chinese prickly ash establishes a technical system for subculturing the tissue culture seedlings of the stingless Chinese prickly ash by optimizing culture conditions, properly adjusting the composition of culture medium components according to the times of subculturing, comprehensively researching factors such as a special cutting process and the like, so that the stem section of the initial culture of the stingless Chinese prickly ash can quickly and stably realize subculture proliferation, thereby realizing large-scale breeding in a short time and having great practical value in the breeding production of the stingless Chinese prickly ash.

Description

Subculture breeding method of stingless pepper tissue culture seedlings
Technical Field
The invention belongs to the technical field of vegetative propagation of commercial crops, and particularly relates to a subculture propagation method of stingless pepper tissue culture seedlings.
Background
Zanthoxylum bungeanum is a spice plant of Zanthoxylum Linn of Rutaceae, has a long cultivation history in China, is a spice plant and a traditional medicinal plant, and has high economic value and water and soil conservation benefit in cultivation of Zanthoxylum bungeanum. The zanthoxylum bungeanum is distributed in various provinces of yellow river, Yangtze river and Zhujiang river basin, Yunnan, Guizhou, Tibet and the like in China, and can be classified into northern zanthoxylum bungeanum, southern zanthoxylum bungeanum and zanthoxylum bungeanum produced in Shandong according to commodity types.
About 200 kilohm of pepper is planted in China at present2The annual total yield is about 600 million yuan, and the pepper becomes a pillar type economic tree species supporting economic sources of farmers in more than 5000 million mountainous areas in China. However, the trunks, branches and leaves of the pepper have thorns, so that the harvesting is difficult and the cost is extremely high. The harvesting cost of the zanthoxylum bungeanum in 2011 accounts for 1/2-2/3 of sales income, and 200 ten thousand hm of the zanthoxylum bungeanum nationwide2The annual harvesting cost of the Chinese prickly ash reaches 300 plus 400 million yuan. Therefore, the difficulty in harvesting the zanthoxylum bungeanum is a key problem to be solved urgently in zanthoxylum bungeanum production, and if a stingless zanthoxylum bungeanum variety can be used for replacing the stingless zanthoxylum bungeanum, the harvesting efficiency can be greatly improved, and the harvesting cost can be saved.
Since the 50 s of the 20 th century, Chinese prickly ash species improvement is carried out successively, and thornless prickly ash species discovery and cultivation research is carried out continuously. In the beginning of the 20 th century in the 80 th era, China introduced Japanese stingless prickly ash which is planted in Hebei, Henan and the like, in 1994, Japan also introduced Sichuan when implementing assistance projects in Sichuan, and in 2010, introduced in Shaanxi county from Hebei; the Japanese stingless pepper is different from Chinese pepper in nature, has insufficient spicy flavor, low yield, deep sunken parts of fruit surface glands and weak cold resistance, has strong spicy flavor and high yield of green pepper in south China and red pepper in north China, and bulges the fruit surface glands like bubbles, so the Japanese stingless pepper is not popularized and cultivated in Chinese pepper production.
At present, the breeding method of the stingless pepper tree is mainly grafting breeding, but the grafting period is longer, and the influence of external environmental conditions and plant diseases and insect pests is great, so that the seedling quality of the grafted stingless pepper tree is poor, the survival rate is low, and the quality and the yield of the pepper tree are influenced.
Disclosure of Invention
In view of the above, the invention aims to provide a subculture breeding method of a zanthoxylum bungeanum tissue culture seedling. So as to solve the problems of low survival rate and low yield of the stingless pepper by adopting grafting propagation,
in order to achieve the purpose, the invention provides the following technical scheme:
a subculture breeding method of a stingless pepper tissue culture seedling comprises the following steps:
s1, primary culture: placing the sprouting stem section of the zanthoxylum lunulatum in a culture medium, culturing for 5-9 days in a dark environment, then culturing for 6-8 days under the condition that the illumination intensity is 900-1100 lux, then increasing the illumination intensity to 1900-2100 lux, and culturing for 12-16 days until the sprouting stem section of the zanthoxylum lunulatum grows to 3-4cm in height and 3-4 internodes, and the number of axillary buds is not less than 2;
s2, cutting: placing the bud stem section of the prickless prickly ash after primary culture in an aseptic inoculation tray on a super clean workbench, cutting off the tissue of the base part of the bud stem section of the prickless prickly ash, namely one end immersed in a culture medium, by using a sterilized tool to expose fresh tissue, and then cutting into 1-3 leaf stem sections;
s3, second generation culture: placing the cut stem segments in S2 in a culture medium, culturing for 5-9 days in a dark environment, timely placing the stem segments in a condition that the illumination intensity is 900-1100 lux, culturing for 6-8 days, then increasing the illumination intensity to 1900-2100 lux, culturing for 12-16 days until the stem segments grow to 3-4cm in height and 3-4 internodes, and the number of axillary buds is not less than 2;
the components of the culture medium in the primary culture process and the secondary culture process are as follows: MS basal medium, Zeatin (ZT) 2.0 mg.L-10.1 mg. L of naphthylacetic acid (NAA)-1The amount of carrageenan is 6.0 g.L-1Sucrose (30 g. L)-1And plant tissue culture antibacterial agent (PPM) 0.1%;
s4, repeating the steps S2 and S3 to carry out subculture proliferation, and ending when the amount of PPM in the culture medium is 0, wherein the generation number of subculture proliferation is not less than 4;
the components of the culture medium for the subculture are as follows: MS basal medium, ZT 1.0-2.0 mg.L-1NAA of 0.1 mg. L-1The amount of carrageenan is 6.0 g.L-1Sucrose (30 g. L)-1And PPM is 0-0.05%;
and when the generation number of the subculture is increased by 1-2 times, the PPM dosage is reduced by 0.01%.
Wherein the sprouting stem section of the zanthoxylum armatum is a 2-3 cm stem section cut from a zanthoxylum armatum tree seedling, and the sprouting stem section grows after being cultured in a culture medium for 30-40 days.
The bud stem section of the stingless prickly ash is cultured in the culture process, one purpose of culturing under dark conditions is to avoid browning at the cut of the newly cut stem section, the culturing under dark conditions is favorable for the growth of the bud of the stem section, the other purpose is to prevent the stem section of the newly cut from growing too fast and aging, the third purpose is that the body of the stingless prickly ash is formed by grafting from the stingless prickly ash, and the stem section can be prevented from growing axillary buds with stings in the culturing under dark conditions. After a period of dark condition culture, the illumination culture intensity is gradually increased, so that the stem section is gradually adapted to the illumination condition, and after the stem section is adapted to the external condition, the stem section is placed under the stronger illumination condition, so that the stem section is rapidly grown and aged.
Preferably, in S4, ZT content in the medium components of the third generation culture and the fourth generation culture is 2.0 mg.L-1
Preferably, in S4, after the subculture multiplication number exceeds 4, the ZT dosage in the medium composition needs to be adjusted according to the length between the stem segments of the proliferated axillary buds and the number of the axillary buds, and when the axillary buds are dense and clustered, the ZT dosage is 1.0-1.25 mg.L-1When the axillary bud stem segment internodes grow and the axillary bud germinates less, the ZT dosage is 1.25-1.5 mg.L-1
Preferably, in S4, the amount of PPM decreases by 0.01% each time as the number of subcultures increases during the subculture.
After 3-4 generations of subculture, the stem of the proliferated axillary bud gradually adapts to the in vitro environment, meanwhile, the in vivo endophytic fungi are inhibited, the in vivo hormone level is gradually accumulated, the composition of the subculture medium needs to be properly adjusted according to the situation, on the basis of the original MS basic medium, the cell division number and the ratio of auxin are gradually reduced along with the continuous increase of the subculture multiplication frequency, and meanwhile, the dosage of PPM is gradually reduced until the dosage of PPM reaches 0.
Preferably, in the S2 step, in the cutting process of the stingless pepper seedlings, the terminal buds are cut into stem sections which are 1-2cm long and have 2-4 leaves.
Preferably, in the S2, in the cutting process of the prickless pepper seedlings, all the other leaves are required to be reserved except for the old leaves which are close to the water stain shape of the culture medium and the yellow flowers.
Wherein, the cutting process is one of the key factors for efficient subculture multiplication of the stingless pepper. The same material, with different cutting methods, has a great difference in subsequent proliferation. Therefore, the cutting process for the subculture multiplication of the stingless pepper mainly comprises the following four points: 1) placing the material to be cut in an aseptic inoculation tray on a clean bench, and cutting old tissues at the base of the stem segment (one end inserted into a culture medium) by using a sterilized tool to expose fresh tissues; 2) cutting is carried out in a manner that axillary buds proliferate microscopically. Namely, cutting 3-4cm of stem section material into stem sections with 1-3 leaves, which is determined according to the germination condition of axillary buds of the stem sections. If the internodes of the stem section are longer and the axillary buds germinate better, the stem section can be cut into 1 leaf and 1 bud; if the internodes of the stem sections are compact and the axillary buds are not germinated enough, the stem sections can be selected to be cut into 2-3 pieces; 3) cutting the apical bud of the stem segment into 1-2cm long stem segments with 2-4 leaves; 4) all the other leaves need to be completely reserved except for old leaves which are close to the water stain shape of the culture medium and yellow flowers which need to be cut.
In the cutting process, if the base part of the sprouting stem section of the stingless prickly ash and old leaves close to the water stain shape and yellow flowers of the culture medium are not cut off, the base part and the old leaves can absorb nutrition in the subculture multiplication process of the stem section, so that the new stem section is slowly multiplied, and meanwhile, part of the stem section with weak growth can be withered.
Preferably, the illumination culture time is 10h and the temperature is 23-27 ℃ during the whole subculture multiplication culture period.
The invention has the beneficial effects that:
1) according to the subculture breeding method of the stingless pepper tissue culture seedling, dark culture is performed firstly, then the illumination intensity is gradually increased, so that the hormone in the tissue culture seedling gradually tends to be stable, and the generation number of subculture proliferation can be greatly reduced by adopting a special cutting process, so that the aim of rapidly breeding the stingless pepper tree seedling is fulfilled;
2) according to the subculture breeding method of the tissue culture seedling of the stingless prickly ash, the culture condition is optimized, the composition of the components of the culture medium is properly adjusted according to the times of subculture, and the comprehensive research on factors such as a special cutting process is carried out, so that a technical system of subculture of the tissue culture seedling of the stingless prickly ash is established, the subculture proliferation of the stem section of the primary culture of the stingless prickly ash is quickly and stably realized, the large-scale breeding is realized in a short time, and the subculture ash breeding method has great practical value in the breeding production of the stingless prickly ash;
3) the subculture breeding method of the stingless pepper tissue culture seedling is not influenced by external plant diseases and insect pests, is simple and quick, can save the cost of manpower and material resources caused by grafting of the stingless pepper, can effectively ensure the quality and the quantity of the seedling, and can greatly improve the yield of the pepper in the subsequent planting and pepper fruiting processes.
Drawings
FIG. 1 is a diagram of a prickless pepper seedling after primary culture in example 1;
fig. 2 is a diagram of the cut stem segment of the present example 1.
Detailed Description
The present invention is further described with reference to specific examples to enable those skilled in the art to better understand the present invention and to practice the same, but the examples are not intended to limit the present invention.
Example 1
A subculture breeding method of a stingless pepper tissue culture seedling comprises the following steps:
s1, primary culture: placing the sprouting stem section of the zanthoxylum armatum without prick in a culture medium, culturing for 8-9 days in a dark environment, culturing for 7-8 days under the condition of 1000lux of illumination intensity, then increasing the illumination intensity to 2000lux, culturing for 13-14 days until the sprouting stem section of the zanthoxylum armatum without prick grows to 3-4cm in height and 3-4 internodes, and the number of axillary buds sprouting is not less than 2, wherein the cultured sprouting stem section seedlings of the zanthoxylum armatum without prick are shown in figure 1;
s2, cutting: placing the bud stem section of the prickless prickly ash after primary culture in an aseptic inoculation tray on a super clean workbench, cutting off the tissue of the base part of the bud stem section of the prickless prickly ash, namely one end immersed in a culture medium, by using a sterilized tool to expose fresh tissue, then cutting the terminal bud into stem sections with 2-4 leaves and 1-3 leaf stem sections, wherein the length of the stem sections is 1-2cm, and the rest are shown in figure 2;
s3, second generation culture: placing the cut stem segments in S2 in a culture medium, culturing for 8-9 days in a dark environment, timely placing the stem segments under the condition that the illumination intensity is 1000lux, culturing for 7-8 days, then increasing the illumination intensity to 2000lux, culturing for 13-14 days until the stem segments grow to the height of 3-4cm, 3-4 internodes and the number of axillary buds is not less than 2;
wherein, the components of the culture medium in the primary culture process and the secondary culture process are as follows: MS basal medium, ZT dosage of 2.0 mg.L-1The dosage of NAA is 0.1 mg.L-1The dosage of the carrageenan is 6.0 g.L-1The amount of sucrose is 30 g.L-1And PPM amount is 0.1%;
s4, repeating the steps S2 and S3 to carry out subculture proliferation, wherein the components of a culture medium of the subculture proliferation are as follows: MS basal medium, ZT dosage in third generation and fourth generation culture medium is 2.0 mg.L-11.20 mg. L-1The dosage of NAA is 0.1 mg.L-1The dosage of the carrageenan is 6.0 g.L-1The amount of sucrose is 30 g.L-1And PPM is 0-0.05%, the amount of PPM in the third generation culture medium of subculture proliferation is 0.05%, the amount of PPM is reduced by 0.01% every 1 time, and finally the generation of subculture proliferation is seven generations;
during the whole proliferation culture period, the illumination culture time is 10h every day, and the temperature is 23-25 ℃.
By the breeding method, the final culture of the thornless pepper seedlings requires 30-40 days in the whole process, and the survival rate after transplanting reaches more than 95%.
Rooting culture:
(1) cutting and inoculating the bud seedlings obtained by subculture proliferation into a rooting culture medium, wherein the height of the bud seedlings is more than or equal to 2.0cm, the stem thickness is more than or equal to 2.0mm, and the depth of the bud seedlings inserted into the culture medium is 3.0-4.0 mm;
(2) the formula of the culture medium is as follows: 1/2MS + IBA0.5mg. L-1+ AC (activated carbon) 50 mg. L-1+ carrageenin 6.0 g.L-1+ sucrose 20 g.L-1
(3) The culture conditions are as follows: after inoculation, the mixture is placed in a dark environment for culture for 7 days, and then is transferred to a light environment for continuous culture for 7-14 days, wherein the light culture time is 10 h/d. The temperature in the whole rooting culture process is 23-25 ℃, and the illumination intensity is 2000 lux;
(4) and after culturing for 14-21 days, selecting rooting seedlings with the number of roots being more than or equal to 2, the root length being more than or equal to 2.0cm and normal leaves for transplanting.
Domestication and transplantation:
(1) domestication: before transplanting, the prickless prickly ash bottle seedlings are placed in a greenhouse with the shading rate of 65% -80%, the culture bottle caps are loosened, and the seedlings are hardened at normal temperature for about 10 days to thicken and thicken the leaves of the rooting seedlings.
(2) Transplanting: washing the rooting seedling culture medium with clear water, soaking in 2000 times of carbendazim solution for 1-2min, planting in a prepared substrate plug, transplanting the substrate according to the proportion of peat: and (3) pouring root fixing water after the perlite is planted, covering a film, preserving heat and moisture, culturing in a greenhouse, paying attention to ventilation in the morning and evening during transplanting culture, spraying with 0.5% compound fertilizer (N: P: K: 15:15:15) after 10 days, removing the film after every 7 days and 15 days, and taking out the nursery after 30-45 days.
In conclusion, the subculture breeding method of the tissue culture seedling of the zanthoxylum simulans establishes a technical system of subculture of the tissue culture seedling of the zanthoxylum simulans by comprehensively researching factors such as optimized culture conditions, proper adjustment of the composition of the culture medium according to the times of subculture, special cutting process and the like, so that the subculture proliferation of the stem section of the primary culture of the zanthoxylum simulans is quickly and stably realized, the large-scale breeding is realized in a short time, and the subculture breeding method of the tissue culture seedling of the zanthoxylum simulans has great practical value in the breeding production of the zanthoxylum simulans.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Claims (7)

1. A subculture breeding method of a stingless pepper tissue culture seedling is characterized by comprising the following steps:
s1, primary culture: placing the sprouting stem section of the zanthoxylum lunulatum in a culture medium, culturing for 5-9 days in a dark environment, then culturing for 6-8 days under the condition that the illumination intensity is 900-1100 lux, then increasing the illumination intensity to 1900-2100 lux, and culturing for 12-16 days until the sprouting stem section of the zanthoxylum lunulatum grows to 3-4cm in height and 3-4 internodes, and the number of axillary buds is not less than 2;
s2, cutting: placing the bud stem section of the prickless prickly ash after primary culture in an aseptic inoculation tray on a super clean workbench, cutting off the tissue of the base part of the bud stem section of the prickless prickly ash, namely one end immersed in a culture medium, by using a sterilized tool to expose fresh tissue, and then cutting into 1-3 leaf stem sections;
s3, second generation culture: placing the cut stem segments in S2 in a culture medium, culturing for 5-9 days in a dark environment, timely placing the stem segments in a condition that the illumination intensity is 900-1100 lux, culturing for 6-8 days, then increasing the illumination intensity to 1900-2100 lux, culturing for 12-16 days until the stem segments grow to 3-4cm in height and 3-4 internodes, and the number of axillary buds is not less than 2;
the components of the culture medium in the primary culture process and the secondary culture process are as follows: MS basal medium, Zeatin (ZT) 2.0 mg.L-10.1 mg. L of naphthylacetic acid (NAA)-1The amount of carrageenan is 6.0 g.L-1Sucrose (30 g. L)-1And plant groupThe antibacterial agent is 0.1 percent (PPM);
s4, repeating the steps S2 and S3 to carry out subculture proliferation, and ending when the amount of PPM in the culture medium is 0, wherein the generation number of subculture proliferation is not less than 4;
the components of the culture medium for the subculture are as follows: MS basal medium, ZT 1.0-2.0 mg.L-1NAA of 0.1 mg. L-1The amount of carrageenan is 6.0 g.L-1Sucrose (30 g. L)-1And PPM is 0-0.05%;
and when the generation number of the subculture is increased by 1-2 times, the PPM dosage is reduced by 0.01%.
2. The method for subculturing the tissue culture plantlets of zanthoxylum armatum DC according to claim 1, wherein the ZT dosage in the culture medium components of the third generation culture and the fourth generation culture in S4 is 2.0 mg.L-1
3. The method for the subculture propagation of the tissue culture plantlets of Zanthoxylum armatum in claim 1, wherein in S4, when the subculture multiplication generation exceeds 4 generations, the ZT dosage in the medium composition is adjusted according to the internode length of the axillary bud stem and the number of axillary buds, and when the axillary bud stem is dense and the axillary buds are clustered, the ZT dosage is 1.0-1.25 mg-L-1When the axillary bud stem segment internodes grow and the axillary bud germinates less, the ZT dosage is 1.25-1.5 mg.L-1
4. The method for subculture propagation of tissue culture plantlets of zanthoxylum armatum DC.H. Chen in claim 1, wherein in S4, the amount of PPM decreases by 0.01% each time with the increase of the number of subcultures during the subculture propagation.
5. The subculture breeding method of the zanthoxylum acanthopanax tissue culture seedling according to claim 1, wherein in S2, during the cutting process of the zanthoxylum acanthopanax tissue culture seedling, the terminal bud is cut into stem segments with 2-4 leaves and length of 1-2 cm.
6. The method for subculture propagation of zanthoxylum armatum tissue culture seedling as claimed in claim 1, wherein in S2, all the leaves except the old leaves close to the culture medium water stain and the yellow flowers need to be cut off during cutting of zanthoxylum armatum tissue culture seedling.
7. The subculture propagation method of the tissue culture seedling of zanthoxylum armatum DC as claimed in claim 1, wherein the light culture time per day is 10h and the temperature is 23-27 ℃ during the whole subculture propagation culture period.
CN202010136322.4A 2020-03-02 2020-03-02 Subculture breeding method of stingless pepper tissue culture seedlings Pending CN111280056A (en)

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CN202110603511.2A CN113331055A (en) 2020-03-02 2020-03-02 Cutting method of stingless pepper tissue culture seedlings
CN202010136322.4A CN111280056A (en) 2020-03-02 2020-03-02 Subculture breeding method of stingless pepper tissue culture seedlings
PCT/CN2020/134255 WO2021174933A1 (en) 2020-03-02 2020-12-07 Subculture breeding method for thorn-free zanthoxylum bungeanum maxim tissue culture seedlings
NL2027681A NL2027681B1 (en) 2020-03-02 2021-03-02 In vitro propagation method of tissue culture seedlings of zanthoxylum armatum

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Application publication date: 20200616