CN110547198A - Method for rapidly propagating tillandsia through seed tissue culture - Google Patents
Method for rapidly propagating tillandsia through seed tissue culture Download PDFInfo
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- CN110547198A CN110547198A CN201910903598.8A CN201910903598A CN110547198A CN 110547198 A CN110547198 A CN 110547198A CN 201910903598 A CN201910903598 A CN 201910903598A CN 110547198 A CN110547198 A CN 110547198A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention discloses a method for quickly propagating tillandsia through seed tissue culture, which comprises the following steps of 1) selecting the collection time of seed pods of the tillandsia; 2) sterilizing the seed pod; 3) pod treatment and embryogenic callus induction: subculturing the embryonic callus obtained in the step 3) to obtain embryonic callus and embryoid; 5) performing rapid propagation differentiation culture on the embryogenic callus; 6) strong seedling and rooting culture; 7) hardening, transplanting and planting aseptic seedlings. The method has simple operation, the embryo of the seed is easy to form embryogenic callus, when the embryo is transferred to a differentiation culture medium, a large number of cluster buds are easy to form, hundreds of small bud seedlings or plantlets with different sizes can be continuously formed within 3-4 months, the plantlets can be cultured in a strong seedling rooting culture medium for about 25 days to form strong seedlings with root stems and leaves, the seedlings are easy to survive outdoors after hardening, and the survival rate is as high as 100%.
Description
Technical Field
The invention relates to the field of plant breeding, in particular to a method for quickly propagating tillandsia through seed tissue culture.
Background
The air plants are various in types, different in plant shapes, various in posture, colorful in flower and leaf and beautiful in personality, the air plants are not only strong in ornamental value and good in decorative effect, but also have strong air purification functions of absorbing formaldehyde, phenylenes, CO 2 and the like, and are more popular as ornamental plants, potential commercial opportunities are unavailable due to the characteristics.
at present, the main reasons for restricting the popularization and application of the air pineapples are fewer seedlings, higher price and especially some rare provenances. The air plants can be propagated through seeds or by plants, but the seed propagation production difficulty is high, and the hybrid characters are easy to separate, so that the characters of the parent plants are difficult to maintain; the main factors influencing the seed propagation are that the requirements on the seeding and seedling raising technology and facilities are high, the difficulty is high, and particularly, the success rate of the used substrate for seeding and seedling raising is low if the substrate is not suitable for seeding and seedling raising; secondly, whether the seeds germinate and the seedlings can grow normally is easily influenced by the change of environmental conditions such as temperature, humidity, ventilation and the like; in addition, the seedlings are very fragile in the initial stage and grow very slowly, and generally the seedlings within 2 years cannot be transplanted and sprayed with any chemical substances and fertilizers, otherwise the seedlings die in large quantities; therefore, the produced tillandsia are propagated mainly in a side bud division mode, but a side bud generally grows to one sub-plant on one side of a mother plant after being bloomed, the sub-plants are easily separated and survive when growing to 1/3-1/2 size of the mother plant, the number of the first sub-plants propagated by the division plant is limited, the second propagation coefficient is very low, and meanwhile, seedlings grow very slowly. In summary, neither seed propagation nor plant division propagation of the air plants can meet the production requirements, which directly leads to the difficulty in the commercial production and industrial development of the air plants, especially some rare and excellent germplasm resources.
disclosure of Invention
The purpose of the invention is as follows: the invention provides a method for quickly propagating air pineapples by seed tissue culture, aiming at the defects of the prior art of the air pineapples.
the technical scheme is as follows: in order to solve the technical problems, the invention provides a method for quickly propagating tillandsia through seed tissue culture, which comprises the following steps:
1) selecting the collection time of the air plant pods;
2) Sterilizing the seed pod;
3) pod treatment and embryogenic callus induction: cutting the disinfected seed pod at 1/2 of the front section of the seed pod by using scissors, inoculating the seeds on a primary induction callus culture medium, and culturing in dark or natural light until the seeds germinate and generate embryogenic callus;
4) Culturing the embryogenic callus: transferring the embryogenic callus obtained in the step 3) to a culture medium 1 for subculture induction of embryogenic callus to form development for dark culture for 50-58d, performing intermediate subculture once, then transferring to a culture medium 2 for subculture induction of embryogenic callus to form development, and performing natural light culture; obtaining embryonic callus and embryoid;
5) and (3) rapid propagation and differentiation culture of the embryogenic callus: then transferring the embryonic callus and the embryoid into a rapid propagation and differentiation culture medium of the embryonic callus, culturing under the light of 1500-2500LX, observing and counting the differentiation occurrence condition and proliferation multiple of the cluster buds after 28-30 days;
6) Strong seedling and rooting culture: transferring the bud seedling into a strong seedling rooting culture medium after 28-30 days, and growing the bud seedling into a normal plant with a plurality of stem roots;
7) Hardening, transplanting and planting aseptic seedlings.
Wherein the primary induction callus culture medium in the step 3) is MS +6-BA0.2-0.5mg/L + NAA0.2-0.5mg/L +2,4-D0.2-0.5 mg/L.
Wherein the culture medium 1 for inducing the formation and development of the embryogenic callus through the subculture in the step 4) is MS +2,4-D2.0mg/L +6-BA0.1mg/L + NAA0.5mg/L.
wherein the culture medium 2 for inducing the formation and development of the embryogenic callus through the subculture in the step 4) is MS +6-BA0.1mg/L + NAA0.5mg/L +2, 4-D0.5mg/L.
wherein the rapid propagation and differentiation culture medium of the embryogenic callus in the step 5) is MS +6-BA0.5mg/L + NAA0.1mg/L.
Wherein the rooting culture medium for the strong seedlings in the step 6) is Ms +6-BA0.1mg/L + IBA0.5mg/L.
wherein, preferably, the primary induction callus culture medium is MS +6-BA0.5mg/L + NAA0.2mg/L +2, 4-D0.2mg/L.
has the advantages that: compared with the prior art, the invention has the following advantages: the method has simple operation, the embryo of the seed is easy to form embryogenic callus, when the embryo is transferred to a differentiation culture medium, a large number of cluster buds are easy to form, hundreds of small bud seedlings or plantlets with different sizes can be continuously formed within 3-4 months, the plantlets can be cultured in a strong seedling rooting culture medium for about 25 days to form strong seedlings with root stems and leaves, the seedlings are easy to survive outdoors after hardening, and the survival rate is as high as 100%. Therefore, the technology of the invention not only solves the problems of difficult seed propagation, low propagation coefficient by plant division, slow growth and the like of the air plants, but also is beneficial to large-scale industrialized production of excellent rare germplasm, and provides an effective way for commercial production and industrial development of the air plants.
Drawings
FIG. 1 seed callus induction;
FIG. 2 formation of embryogenic callus;
FIG. 3 embryogenic callus culture;
FIG. 4 is a multiplication culture of multiple shoots;
FIG. 5 fast propagation and strong seedling culture;
FIG. 6 rooting, transplanting and hardening;
FIG. 7 is a diagram showing that it takes about 95 days for the Bekeley seed to induce embryogenesis to differentiate multiple shoots and to propagate and proliferate rapidly until the regeneration of the plant is formed;
FIG. 8 Harris seeds induce embryogenic callus to differentiate multiple shoots and rapid propagation and proliferation to regenerate the plants for about 90 days;
FIG. 9 shows that it takes about 90 days for the trichrome seeds to induce embryogenesis to grow into differentiated cluster buds and to propagate and proliferate rapidly until the plants are regenerated;
FIG. 10 is a diagram of the differentiation, rapid propagation and proliferation culture of aseptic seeds of Usnea barbata for inducing embryonic callus;
FIG. 11 shows the differentiation, rapid propagation, proliferation and culture of embryogenic callus and cluster buds of Han dynasty;
FIG. 12 Hami melon genipin (left) and Cola roll (right) induced differentiation and regeneration of multiple shoots from embryogenic callus;
FIG. 13 differentiation and rapid propagation proliferation culture of multiple shoots of Mexican genipin (left) and Deluyi genipin (right).
the specific implementation mode is as follows:
The invention is further illustrated by the following examples.
All the culture temperatures in the examples of the present invention were 20 to 28 ℃ and, although not specifically indicated, the culture medium pH was 5.8, sucrose was 30g/L, and agar was 6.5 g/L.
Example 1: collecting, sterilizing and treating seed pods of tillandsia
The varieties adopted by the invention are as follows: harris T.Harrisii, Floweringi T.intermedia, Berkeley T.brashy, trichromatic T.juncea, trichromatic flower (Irish blue) T.tricolor, willow T.balbisiana, Carbita T.capitata, Daltania T.pseudobailey, amber T.schideeana, Meidusa T.cabaut-medige, Fuji T.fetuioides Clump, Wanhan T.van-hysning, (rhinoceros horn T.Seleriana, Violet T.Aerathos, atmospheric flower T.Aerathic Hybrid, Araujei, Bejeri, Ferro-iron T.veloina, Kitaso T.Struth, sodium hypochlorite T.Stricta, Octopus T.Araujei, Baker T.Bergerri, Caryuri, Bluey et-2.20.7.7.7.7.10.1-10.1.7.10.1-10% by using water, and washing the surface of the sterilized water, and soaking the dried fish in the sterile liquid of 10.2.7-10.7.7.7.7.8.10-10.10-10% by using alcohol, washing with sterile water for at least 10 times, wiping water with sterilized absorbent paper, cutting with scissors at 1/2 at the front part of the seed pod, and picking out the seeds with yellowish-white crown hair at the top for use.
example 2: influence of different culture medium formulas and subculture on induction of embryogenic callus of seeds
1. influence of different medium formulations on seed germination and callus formation:
Inoculating the sterilized seeds on a culture medium for inducing seed germination and callus formation, wherein the culture medium comprises: MS +6-BA0.2-0.5mg/L + NAA0.2-0.5mg/L +2,4-D0.2-0.5mg/L, dark culture was performed, 10 grains were inoculated per flask, 10 flasks were inoculated in total, and the germination of the seeds and the growth of the callus were observed for about 30 days, as shown in Table 1 (T. juncea, trichrome, as an example).
TABLE 1 Effect of different Medium formulations on seed Germination and callus induction
As can be seen from table 1: the germination rate of all the formula seeds is 100 percent; the germination rates of the seeds of other varieties are 100%, but the growth and development states of the callus tissues are different and greatly different, the callus degrees are also different, wherein K01 is smaller and harder in callus compared with other formulas, most seeds are abnormal in germination, a large amount of yellow linear abnormal seedlings can be formed in the later period, and the K2 is relatively better (shown in figure 1), but the formula is better at the end, and the embryogenic callus tissue is easier to develop, and the materials are required to be transferred to a culture medium for embryogenic callus tissue formation for continuous observation.
2. The influence of subculture with different formulas on the formation and development of embryogenic callus:
Embryogenic callus formation is accomplished by the following two steps: 1. the above culture material was first transferred onto medium 1: namely MS +2,4-D2.0mg/L +6-BA0.1mg/L + NAA0.5mg/L, dark culturing for 50-58d, and carrying out secondary culture once; 2. then, transfer to medium 2: MS +6-BA0.1mg/L + NAA0.5mg/L +2,4-D0.5mg/L, cultured under natural light for about 28 days, at this time, some explants with the formula can be seen to form a large amount of light yellow or yellow embryonic callus with certain viscosity, especially K2 can be seen under a microscope to form an embryoid body structure (taking T.juncea as an example, see fig. 2 and fig. 3).
TABLE 2 Effect of different culture Medium formulations on the development of embryogenic callus of subculture materials
As can be seen from table 2: the K01 formula has almost no embryogenic callus, and analysis shows that mainly in the early germination stage, the seedlings suddenly cultured under 2,4-D (2.0mg/L) with higher concentration are poisoned and die; k02 is difficult to transform into normal embryogenic callus due to abnormal germination of initial seeds; the other formulas can form embryonic callus, but the development degree is greatly different, the best formula is K2, more normal embryonic callus and embryoid can be developed, and the method is favorable for quickly forming a large number of healthy cluster seedlings and plantlets. The embryogenic calli of other varieties developed to a similar extent as the trichromatic flowers.
example 3: and (3) rapid propagation, differentiation, culture and transplantation of seedlings:
1. And (3) rapid propagation and differentiation culture of the embryogenic callus:
The embryogenic calli obtained in example 2 above were transferred to culture medium: MS +6-BA0.5mg/L + NAA0.1mg/L, respectively in 1500 + 2500LX light culture for 14h/d, 28-30 days after statistics of multiple bud proliferation and embryonic callus differentiation (Table 3, with three color T.juncea as an example).
TABLE 3 Effect of different media formulations on the Rapid propagation differentiation of embryogenic callus
the analysis of the results in table 3 can show that: the best formula of the primary generation callus induction culture medium is MS +6-BA0.5mg/L + NAA0.2mg/L +2, 4-D0.2mg/L, the formula of the culture medium for inducing the formation and development of the embryogenic callus through subculture is MS +2,4-D2.0mg/L +6-BA0.1mg/L + NAA0.5mg/L dark culture and MS +6-BA0.1mg/L + NAA0.5mg/L +2,4-D0.5mg/L natural light culture respectively, and the culture medium is transferred into the embryogenic callus rapid propagation differentiation culture medium MS +6-BA0.5mg/L + NAA0.1mg/L, so that the multiplication times of the cluster buds can reach 23 times (figure 4), the buds grow healthily and have dark green leaf color, and the late stage rooting culture and seedling hardening and transplanting culture are facilitated.
2. Strong seedling and rapid propagation or rooting culture: dividing the cluster bud seedlings into single plants, transferring the single plants into strong seedlings for rapid propagation or rooting culture (figure 5), culturing the single plants under 2000-2500LX light for 14h/d, and growing 100% of small bud seedlings into normal robust plants with a plurality of roots, stems and leaves in about 28 days. The culture medium for strong seedling and rapid propagation comprises: ms +6-BA0.5mg/L + IBA0.2mg/L; the strong seedling rooting culture medium comprises: ms +6-BA0.1mg/L + IBA0.5mg/L; when the concentration of 6-BA in the culture medium is 0.5mg/L, IBA and the concentration is 0.2mg/L, the method is favorable for the young plant to continuously and rapidly propagate the seedling and grow more and more robustly (figure 6); when the concentration of 6-BA0.1mg/L, IBA in the culture medium reaches 0.5mg/L, a plant can generate a large amount of fibrous roots and grow robustly.
3. Transplanting and surviving of aseptic seedlings: and (3) uncovering the sealing film of the bottleneck of the plant growing normally and robustly for indoor hardening for 2-3d, then taking out the plantlets, cleaning the culture medium attached to the plants, scattering the culture medium in a hole tray of a greenhouse, appropriately shading, spraying water in time, keeping the seedlings in a moist and air-permeable state until the plantlets adapt to a new environment and start to grow normally, and counting the survival rate to 100% after 20 d.
Claims (6)
1. A method for quickly propagating tillandsia through seed tissue culture is characterized by comprising the following steps:
1) Selecting the collection time of the air plant pods;
2) Sterilizing the seed pod;
3) pod treatment and embryogenic callus induction: cutting the disinfected seed pod at 1/2 of the front section of the seed pod by using scissors, inoculating the seeds on a primary induction callus culture medium, and culturing in dark or natural light until the seeds germinate and generate embryogenic callus; the primary induced callus culture medium is MS +6-BA0.2-0.5mg/L + NAA0.2-0.5mg/L +2,4-D0.2-0.5 mg/L;
4) Culturing the embryogenic callus: transferring the embryogenic callus obtained in the step 3) to a culture medium 1 for subculture induction of embryogenic callus to form development for dark culture for 50-58d, performing intermediate subculture once, then transferring to a culture medium 2 for subculture induction of embryogenic callus to form development, and performing natural light culture; obtaining embryonic callus and embryoid;
5) and (3) rapid propagation and differentiation culture of the embryogenic callus: then transferring the embryonic callus and the embryoid into a rapid propagation and differentiation culture medium of the embryonic callus, culturing under the light of 1500-2500LX, observing and counting the differentiation occurrence condition and proliferation multiple of the cluster buds after 28-30 days;
6) strong seedling and rooting culture: transferring the bud seedling into a strong seedling rooting culture medium after 28-30 days, and growing the bud seedling into a normal plant with a plurality of stem roots;
7) hardening, transplanting and planting aseptic seedlings.
2. the method for rapid propagation of tillandsia through seed tissue culture as claimed in claim 1, wherein the primary callus induction medium in step 3) is MS +6-BA0.2-0.5mg/L + NAA0.2-0.5mg/L +2,4-D0.2-0.5 mg/L.
3. The method for rapidly propagating tillandsia through seed tissue culture as claimed in claim 1, wherein the culture medium 1 for inducing embryogenic callus formation and development through subculture in the step 4) is MS +2,4-D2.0mg/L +6-BA0.1mg/L + NAA0.5mg/L.
4. the method for rapidly propagating tillandsia through seed tissue culture according to claim 1, wherein the culture medium 2 for inducing the formation and development of embryogenic callus through the subculture in the step 4) is MS +6-BA0.1mg/L + NAA0.5mg/L +2, 4-D0.5mg/L.
5. The method for tissue culture and rapid propagation of tillandsia through seeds of claim 1, wherein the rapid propagation and differentiation medium of the embryogenic callus of step 5) is MS +6-BA0.5mg/L + NAA0.1mg/L.
6. The method for rapid propagation of tillandsia through seed tissue culture according to claim 1, wherein the rooting medium for strong seedlings in step 6) is Ms +6-ba0.1mg/L + iba 0.5mg/L.
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| CN201910903598.8A CN110547198B (en) | 2019-09-23 | 2019-09-23 | Method for rapidly propagating tillandsia through seed tissue culture |
| PCT/CN2020/102335 WO2021057206A1 (en) | 2019-09-23 | 2020-07-16 | Method for rapidly propagating tillandsia by means of seed tissue culture |
| ZA2022/02920A ZA202202920B (en) | 2019-09-23 | 2022-03-10 | Method for rapidly propagating tillandsia by means of seed tissue culture |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2021057206A1 (en) * | 2019-09-23 | 2021-04-01 | 江苏农林职业技术学院 | Method for rapidly propagating tillandsia by means of seed tissue culture |
| CN114568064A (en) * | 2022-03-01 | 2022-06-03 | 广西南亚热带农业科学研究所 | Pineapple seed breathable water-retention breeding method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114946660A (en) * | 2022-06-16 | 2022-08-30 | 天津博奥聚能生物科技有限公司 | Method for cultivating salvia miltiorrhiza tissue culture seedlings suitable for mechanical planting |
| CN114885842B (en) * | 2022-06-30 | 2023-07-28 | 中南林业科技大学 | Method for disinfecting and regenerating seed of Junzhen semen |
| CN115386532A (en) * | 2022-09-16 | 2022-11-25 | 西北农林科技大学 | Peach pulp callus induction subculture and genetic transformation method |
| CN115777538B (en) * | 2022-12-07 | 2023-08-29 | 河北省农林科学院棉花研究所（河北省农林科学院特种经济作物研究所） | Short-period cotton cultivation method |
| CN117426303B (en) * | 2023-12-04 | 2024-08-23 | 南京林业大学 | Asexual propagation method and transplanting method for air pineapple |
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| CN103461120A (en) * | 2013-08-22 | 2013-12-25 | 江苏农林职业技术学院 | Air plant tissue culture medium and method for tissue cultivating and quickly propagating air plant |
| CN104094844A (en) * | 2014-06-26 | 2014-10-15 | 江苏农林职业技术学院 | Tissue culture and inducing culture method for Tillandsia |
| CN109548652A (en) * | 2018-12-06 | 2019-04-02 | 深圳市仙湖植物园管理处（深圳市园林研究中心） | Tillandsia callus and its cultural method and application |
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| CN110547198B (en) * | 2019-09-23 | 2022-04-26 | 江苏农林职业技术学院 | Method for rapidly propagating tillandsia through seed tissue culture |
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| CN103461120A (en) * | 2013-08-22 | 2013-12-25 | 江苏农林职业技术学院 | Air plant tissue culture medium and method for tissue cultivating and quickly propagating air plant |
| CN104094844A (en) * | 2014-06-26 | 2014-10-15 | 江苏农林职业技术学院 | Tissue culture and inducing culture method for Tillandsia |
| CN109548652A (en) * | 2018-12-06 | 2019-04-02 | 深圳市仙湖植物园管理处（深圳市园林研究中心） | Tillandsia callus and its cultural method and application |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2021057206A1 (en) * | 2019-09-23 | 2021-04-01 | 江苏农林职业技术学院 | Method for rapidly propagating tillandsia by means of seed tissue culture |
| CN114568064A (en) * | 2022-03-01 | 2022-06-03 | 广西南亚热带农业科学研究所 | Pineapple seed breathable water-retention breeding method |
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| ZA202202920B (en) | 2022-05-25 |
| CN110547198B (en) | 2022-04-26 |
| WO2021057206A1 (en) | 2021-04-01 |
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