CN110771512B - Efficient induction method of rabdosia lophanthide callus - Google Patents
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- 241001183967 Isodon Species 0.000 title claims abstract description 48
- 230000006698 induction Effects 0.000 title claims abstract description 39
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- 229920001817 Agar Polymers 0.000 claims abstract description 9
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 9
- 229930006000 Sucrose Natural products 0.000 claims abstract description 9
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- 238000012258 culturing Methods 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 238000012136 culture method Methods 0.000 claims description 3
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims description 3
- 230000000249 desinfective effect Effects 0.000 claims description 2
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- 241000344723 Iris orientalis Mill., 1768 Species 0.000 description 1
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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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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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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- Biotechnology (AREA)
- Developmental Biology & Embryology (AREA)
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a high-efficiency induction method of rabdosia lophanthide callus, which takes rabdosia lophanthide hypocotyl parts as explants for inducing callus. Sterilizing mature rabdosia lophanthide seeds, inoculating the rabdosia lophanthide seeds into a culture medium to germinate radicles, cutting hypocotyls and radicle parts which grow to be 0.5-1 cm, putting the hypocotyls and the radicle parts into an induction culture medium, and inducing callus on the hypocotyls. The optimal culture medium is MS +1.0 mg/L6-BA +1.5mg/L2,4-D +30g/L sucrose +7g/L agar, the induction rate reaches 95.34% in 30 days, and the method lays a solid foundation for optimizing a tissue culture system and establishing a genetic transformation system.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a high-efficiency induction method of rabdosia lophanthide callus.
Background
Rabdosia lophanthide (Iris sanguinea) is a perennial herb flower of Iris, Iridaceae, also known as Iris orientalis. The plant height is about 50-100 cm, the rootstock is strong, the linear leaves are blue and small, and the plant has a special flower shape, generally 2-3 flowers; the flowering period is 6-7 months, and 7-9 months are fruit periods. The flower has bright and rich colors, handsome plant shape, strong cold resistance and high ornamental value, has extremely high market potential when used as landscaping and flower arrangement, has relatively wide application in gardens, and is mainly applied to aspects of ornamental vegetation, green land sheet planting, grassland spot decorating, pattern flower beds and the like under forests; the rabdosia lophanthide can be used as potted flowers for indoor small-environment home decoration, and can also be used as a flower arrangement material. At present, the rabdosia lophanthide is not well developed and utilized in China, the wild environment is damaged along with the development, and the wild resources are less and less.
Tissue culture is widely used because it allows for efficient propagation of plants and can improve plant quality (Shimizu, k.et al.). Boltenkov and e.v.zarmebo take rabi seed as explant, the relationship of the explant to hormone content change during dedifferentiation and redifferentiation process was studied, the induction rate was not counted, and the root was not formed (e.v.boltenkov and e.v.zarmebo, 2005). The annual Yuxin et al use the roots of rabdosia lophanthide, dormant rhizomes with buds and germinated buds as explants to carry out adventitious bud induction, and the conclusion is that the proper explants are rhizomes with buds and the induction rate is 88.57%. At present, the rabdosia lophanthide related research is less, most of the rabdosia lophanthide related research is physiological research, and the research on the aspects of tissue culture, mass propagation and the like is very deficient.
Although the induction of rabdosia lophanthide callus is developed in related researches at present, the induction rate is not high, and aiming at the problem, the experience of predecessors is summarized, experimental materials, conditions, culture media and the like are optimized, the rabdosia lophanthide callus induction method is provided, the induction rate is improved, and the establishment of a regeneration system of a rabdosia lophanthide callus approach is facilitated.
Disclosure of Invention
The method can induce and culture a large amount of calli which grow robustly, and can form adventitious buds through redifferentiation under proper conditions so as to form plants, thereby realizing the rapid propagation of the panrya seedlings and laying a foundation for earlier stage work of flower and color genetic research.
The invention provides a high-efficiency induction method of rabdosia lophanthide callus, which has the technical scheme that:
seed selection and disinfection: selecting ripe rabdosia lophanthide seeds with cracked seed pods, and sterilizing the rabdosia lophanthide seeds;
induction and growth of embryonic axis: inoculating the sterilized rabdosia lophanthide seeds into solid culture medium MS, 0.5 mg/L6-BA, 30g/L sucrose and 3.5g/L agar;
induction and generation of callus: and cutting off the part of the embryonic axis and the embryonic root with the total growth length of 0.5-1 cm, putting the part into an induction culture medium, and putting the part into solid culture medium MS +1.0 mg/L6-BA +1.5mg/L2,4-D +30g/L sucrose +7g/L agar. Subculturing for 15 days for 1 time, and subculturing for two times continuously, wherein yellow callus will be generated on the hypocotyl, and the obtained callus with yellow color is selected finally;
the pH values of all the culture media are adjusted to 5.8-6.0, the culture temperature of a culture room is 25 +/-1 ℃, the illumination time is 14h/d, and the illumination intensity is 25 mu mol/(m)2.s)；
Further, the disinfection treatment in the step (1) is to soak rabdosia lophanthide seeds in hot water at 80 ℃ for 1 day, sterilize the rabdosia lophanthide seeds with 75% alcohol for 30s, and disinfect the rabdosia lophanthide seeds with 4% NaClO solution for 20 min;
further, the step (3) of putting the hypocotyl and radicle part with the total growth length of 0.5-1 cm into an induction culture medium refers to putting the robust hypocotyl and radicle with the length of 0.5-1 cm, which are just grown on the seed, into the induction culture medium;
further, the callus with yellow color obtained in the step (3) is the callus with yellow color, obvious particles and loose production from the hypocotyl part;
the invention adopts the tissue culture technology, and has the beneficial effects in the callus induction process: no browning phenomenon exists in the induction process, callus is generated without bacterial infection, the induction rate is as high as 95.34%, and a new way is provided for realizing the rapid propagation of the rabdosia lophanthide seedlings.
FIG. 1 is schematic diagram of induction callus culture of rabdosia lophanthide
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.
The efficient induction culture method for the rabdosia lophanthide callus provided by the embodiment comprises the following steps:
selecting and disinfecting seeds: selecting ripe rabdosia lophanthide seeds with split pods, putting the rabdosia lophanthide seeds into a conical flask, adding a proper amount of water and a detergent, continuously shaking for 10min to remove impurities, then washing the rabdosia lophanthide seeds for 1h under running water, soaking the rabdosia lophanthide seeds in tap water with the temperature of 80 ℃ for 1d after no foam exists, then putting the rabdosia lophanthide seeds into the sterilized conical flask again in a sterile operating platform, sterilizing the rabdosia lophanthide for 30s by using 75% alcohol, washing the rabdosia lophanthide for 3 times by using sterile water, sterilizing the rabdosia lophanthide for 20min by using 4% NaClO, washing the rabdosia lophanthide seeds for 5 times and soaking the rabdosia lophanthide seeds for 10 min;
induction and growth of embryonic axis: inoculating sterilized rabdosia lophanthide seeds into a solid culture medium MS +0.5 mg/L6-BA +30g/L sucrose +3.5g/L agar by using forceps, wherein the pH value of the culture medium is 5.8-6.0, the inoculation density is 20 grains/dish, the culture temperature of a culture room is 25 +/-1 ℃, the illumination time is 14h/d, and the illumination intensity is 25 mu mol/(m)2S), when the seeds are cultured for about 11 days, the radicles of the seeds germinate, the parts with the embryonic axes and with the lengths of 0.5-1 cm are cut successively, and the germination is finished after 30 days;
induction and generation of callus: putting the cut part with the embryonic axis and the growth of 0.5-1 cm into an induction culture medium by using forceps, putting the induction culture medium into agar of a solid culture medium MS +1.0 mg/L6-BA +1.5mg/L2,4-D +30g/L sucrose +7g/L, wherein the pH value of the culture medium is 5.8-6.0, the inoculation density is 15 per dish, the culture temperature of a culture chamber is 25 +/-1 ℃, the illumination time is 14h/D, and the illumination intensity is 25 mu mol/(m mol)2S), culturing for 15 days for 1 subculture, continuously subculturing for 2 times, and finally selecting the obtained callus which is yellow and strong in growth, wherein the browning rate is 0%, the pollution rate is 0%, and the induction rate is as high as 95.34%.
Induction Medium selection experiment
Primarily selecting the hormone concentration suitable for the rabdosia lophanthide induced callus according to published data, adding 6-BA, NAA and 2,4-D with different concentrations into an MS culture medium, simultaneously adding 7g/L of agar and 30g/L of cane sugar, and adjusting the pH to 5.8-6.0. At the inoculation time, 30 embryonic axes are inoculated in each treatment, 3 times of treatment are repeated, and data statistics are carried out after 30d of culture, and the details are shown in a table 1.
TABLE 1 comparative experiments with different hormone combinations
It can be seen that when the concentration of 6-BA is 1mg/L, the callus state cultured by matching with other two hormones is better, the induction effect of 6-BA and 2,4-D on the callus is larger, and the NAA effect is minimum;
the result shows that the induction rate is highest and reaches 95.34 percent in the culture medium MS +1.0 mg/L6-BA +1.5mg/L2,4-D +30g/L sucrose +7g/L agar.
The above description is only a preferred embodiment of the present invention, and any person skilled in the art may modify the present invention or modify it to an equivalent technical solution by using the technical solution described above.
Claims (4)
1. A high-efficiency induction culture method of rabdosia lophanthide callus is characterized by comprising the following steps:
(1) selecting and disinfecting seeds: selecting ripe and full-grained rabdosia lophanthide seeds with cracked seed pods, and sterilizing the rabdosia lophanthide seeds;
(2) induction and growth of embryonic axis: inoculating the sterilized rabdosia lophanthide seeds into solid culture medium MS, 0.5 mg/L6-BA, 30g/L sucrose and 3.5g/L agar;
(3) induction and generation of callus: cutting the radicle and hypocotyl part growing to 0.5-1 cm, putting the radicle and hypocotyl part into an induction culture medium MS +1.0 mg/L6-BA +1.5mg/L2,4-D +30g/L sucrose +7g/L agar, culturing for 1 subculture for 15 days, continuously subculturing for 2 times, and finally selecting the obtained callus with yellow color;
the pH values of all the culture media are adjusted to 5.8-6.0, the culture temperature of a culture room is 25 +/-1 ℃, the illumination time is 14h/d, and the illumination intensity is 25 mu mol/(m mol)2·s )。
2. The method for efficiently inducing and culturing the rabdosia lophanthide callus according to claim 1, wherein the sterilization treatment in the step (1) is to soak rabdosia lophanthide seeds in hot water at 80 ℃ for 1 day, sterilize the rabdosia lophanthide seeds with 75% alcohol for 30s, and sterilize the rabdosia lophanthide seeds with 4% NaClO solution for 20 min.
3. The efficient induction culture method of rabdosia lophanthide callus according to claim 1, wherein the step (3) of placing the radicle and hypocotyl part into the induction culture medium is to place a robust radicle and hypocotyl with a length of 0.5-1 cm, which are cut from a seed, into the induction culture medium.
4. The method for efficiently inducing and culturing the rabdosia lophanthide callus according to claim 1, wherein the callus with yellow color obtained in the step (3) is the callus with yellow color, distinct and loose particles and grown from hypocotyls.
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|---|---|---|---|---|
| CN101816287A (en) * | 2010-05-05 | 2010-09-01 | 南京大学 | Method for culturing Iris pseudacorus callus |
| CN104996302A (en) * | 2015-08-20 | 2015-10-28 | 浙江大学 | Method for remarkably improving callus inductivity of iris pseudacorus L. |
| CN108496800A (en) * | 2018-04-10 | 2018-09-07 | 黑龙江省科学院自然与生态研究所 | Chinese small iris high frequency regenerating system method is established based on callus induction |
| CN109452170A (en) * | 2018-11-07 | 2019-03-12 | 东北林业大学 | A kind of method of sword-like iris seed root evoked callus culture |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI323641B (en) * | 2006-12-29 | 2010-04-21 | Tatung Co | Tissues with rich flavonoids of neomarica gracilis and belamcanda chinensis and culture methods for the same |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101816287A (en) * | 2010-05-05 | 2010-09-01 | 南京大学 | Method for culturing Iris pseudacorus callus |
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| CN108496800A (en) * | 2018-04-10 | 2018-09-07 | 黑龙江省科学院自然与生态研究所 | Chinese small iris high frequency regenerating system method is established based on callus induction |
| CN109452170A (en) * | 2018-11-07 | 2019-03-12 | 东北林业大学 | A kind of method of sword-like iris seed root evoked callus culture |
Non-Patent Citations (3)
| Title |
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| "Establishment of an efficient in vitro propagation system for Iris sanguinea";Ling Wang等;《Scientific Reports》;20181120;第8卷(第1期);第1-8页 * |
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