CN103704132A - Inducing method of Pogostemon cablin polyploid - Google Patents
Inducing method of Pogostemon cablin polyploid Download PDFInfo
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- CN103704132A CN103704132A CN201310657063.XA CN201310657063A CN103704132A CN 103704132 A CN103704132 A CN 103704132A CN 201310657063 A CN201310657063 A CN 201310657063A CN 103704132 A CN103704132 A CN 103704132A
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Abstract
The invention discloses an inducing method of a Pogostemon cablin polyploid. The invention for the first time employs regenerated multiple shoots of Pogostemon cablin callus as an induction material, and uses a colchicine infusion method to conduct polyploid induction on stem apexes of the multiple shoots; and the induced stem apexes are subjected to dedifferentiation and redifferentiation to obtain a polyploid plant. The induction material is combined with the induction method employed by the invention to significantly increase the inductivity of the Pogostemon cablin polyploidy up to 63.3%, realize survival rate of 100%, and reduce the incidence of chimera to merely 10%. The Pogostemon cablin polyploid obtained by the invention has good stability, and maintains similar characters as a parental generation after 2 generations of asexual cutting propagation. The Pogostemon cablin tetraploid obtained by the invention has leaf area and stem much larger than those of a diploid, and has more branches. The invention is conducive to the improvement of Pogostemon cablin oil yield, promotes quality improvement of the Pogostemon cablin oil, and achieves high yield and good quality of Pogostemon cablin, so as to meet the requirements of quality of Pogostemon cablin medicinal material and related products by people.
Description
Technical field
The present invention relates to cultured in vitro and colchicine and process the method in conjunction with seed selection polyploid, be specifically related to the abductive approach of Pogostemon cablin polyploid, belong to polyploid breeding technical field.
Background technology
Pogostemon cablin (
pogostemon cablin(Blanco) Benth), for Labiatae thorn stamen grass belongs to herbaceos perennial, by ancient Chinese medicine doctor, be considered as the summer-heat and damp key medicine in season, clinical practice is extensive, and curative effect is better, is famous genuine southern medicine.In addition, the important source material of the patchouli oil extracting from Pogostemon cablin or medical industry and light industry industry, can be used for preparing various formulations, cosmetic skin care product, fixastive and agrochemical.Pogostemon cablin is introduced a fine variety after China's South Subtropical Area of China plantation, because the ecological conditions such as weather are different, introduces rear rare the blossoming and having seeds of China's cultivation, has sometimes opened flower, but Anther Abortion, so adopt the modes of reproduction of cuttage more.Due to the long-term cottage propagation that adopts, cause that deterioration of variety, plant strain growth are slow, uneven, damage by disease and insect incidence is high, a little less than resistance, cause output and quality to decline, in the urgent need to carrying out breeding of new variety.
Plant polyploid has the advantages that the huge property of organ, resistance strengthen, and the increase of organ can not only improve the output of the medicinal material that corresponding site is used as medicine, and increases the quality of medicinal material, the corresponding increase of its active constituent content, and resistance also strengthens.Pogostemon cablin medicinal part is above-ground plant parts, and its total volatile oil content is mainly distributed in the blade, stem of plant, and it is carried out to polyploid breeding is the important channel of improving its quality.
Polyploid is mainly derived from spontaneous mutation and induced mutations, and wherein artificial mutation comprises physics and chemistry method.In mutagenesis, colchicine is one of medicament of commonly using the most, and success induces polyploid and promotes the use of in numerous crops, gardening plant and some medicinal plants.Common chemical mutagen has colchicin and weed killer herbicide, as the happy spirit of prodiamine, oryzalin, formyl amiprophos, oxygen etc., wherein colchicin be find so far to double effect best, most popular chromosome doubling agent.Application tissue culture technique is cultivated the new field that polyploid is tissue culture technique development, and it is the combination of tissue culture technique and colchicin chemical induction technology.Mutagenesis and the combination of organizing cultivation, make to have incorporated in multiploid induction the advantage of tissue cultivation, experimental condition is easy to control, and can shorten breeding time, because tissue cultivation can realize Fast-propagation, can a large amount of vegetable material of single treatment during multiploid induction, accelerated greatly breeding process, can obtain more polyploid material, due to selected materials, children is tender mostly in addition, and is in the histocyte of mitogenetic state, is easily subject to the impact of colchicin, induction success rate is higher, and polyploid purity is high.Conventional induction mode has infusion process, drop method and mixed training method, and above three kinds of methods cut both ways, and for the induction of different vegetable materials, has also produced the different effects that doubles.Colchicin is a kind of toxic reagent, and dealing with improperly can damage plant, and the concentration of colchicine treatment and time, its valid density was 0.01%～1% because of the difference difference to some extent of the method for induction and material, general 0.2%～0.4% most widely used.
The main original position revulsion that adopts of the early stage research of Polyploid Induction Methods, processes at non-in vitro the seed of test plant, the seedling growing, stem apex etc. with the mutagen of variable concentrations, thus induce chromosome redoublement.The method of processing has infusion method, injection, semar technique and investment etc.Although these methods can obtain polyploid, often there is the phenomenon that chimera probability is high.Along with the development of biotechnology, cultured in vitro double method starts prevailing in recent years, and from having been reported, the main path of the method has two: the one, in tissue culture procedures, by colchicine solution, directly process culture materials; The 2nd, with the solid culture medium that contains colchicine, material is cultivated to a period of time, then proceed to differential medium, then differentiate whole plant by mutant.Double cultivation in vitro because experimental condition is easy to control, the variation plant back mutation phenomenon causing affected by environment reduces, and result of the test repeatability is strong.Yet one of key issue of multiploid induction is to occur chimera, although chimera is a kind of more common phenomenon in induction polyploid, the improper polyploid cell ratio that often causes of material selection is few, is replaced very soon by dliploid.
Summary of the invention
In order to address the above problem, the present invention, by choosing specific induced material, improving induction and cultural method, has set up the abductive approach of the Pogostemon cablin polyploid that a kind of inductivity is high, chimera incidence is low.
The object of the present invention is to provide a kind of abductive approach of Pogostemon cablin polyploid.
The technical solution used in the present invention is:
An abductive approach for Pogostemon cablin polyploid, is characterized in that: comprise the following steps:
1) induction of callus: get the blade of Pogostemon cablin, sterilization, cleans, and under aseptic condition, is cut into 1～2cm
2small pieces, be seeded in callus medium, carry out callus and cultivate 12～14 days, obtain callus;
2) induction of Multiple Buds: under aseptic condition, callus is transferred in Multiple Buds medium and is cultivated 26～30 days, obtain Multiple Buds;
3) colchicine is processed: under aseptic condition, the long Multiple Buds stem apex of clip 0.5～1cm is soaked in 0.3～0.5gL
-1colchicine solution in, 24～72h vibrates under dark condition;
4) renewal cultivation: under aseptic condition, the Multiple Buds stem apex sterile water wash clean after colchicine is processed, is inoculated in callus medium and cultivates after 12～14 days, is inoculated in Multiple Buds medium and cultivates 26～30 days, obtains polyploid Multiple Buds;
5) propagation is cultivated: polyploid Multiple Buds is inoculated in new Multiple Buds medium and breeds and cultivate 20～25 days;
6) polyploid is identified: the stem apex of getting the rear Multiple Buds of propagation cultivation carries out Chromosome Number Observation, observes 5 above division phase chromosome numbers simultaneously and double on a tip of a root, and can confirm this variant is polyploid;
7) culture of rootage: under aseptic condition, the strain that is accredited as polyploid is cut from base portion, be transferred in root media and cultivate 8～15 days, obtain the seedling of taking root;
8) hardening: take out the seedling of taking root, be transferred in the basin that vermiculite and sand are housed, cultivate 28～40 days in indoor hardening;
9) transplant seedlings: after hardening, basin seedling is moved on to outdoor, or seedling is transplanted to from basin to field, in screening, have the cloudy canopy of 70～85% sunshade rates, cultivate after 8～12 days, remove cloudy canopy, carry out field planting management.
Further, above-mentioned all callus medium comprise MS minimal medium, 0.1～0.3mgL
-16-BA, 0.4～0.6 mgL
-1nAA, 30～32gL
-1sucrose and 6.0～7.0 gL
-1agar.
Further, above-mentioned all Multiple Buds medium comprise MS basal medium, 0.1～0.3mgL
-16-BA, 30～32gL
-1sucrose and 6.0～7.0 gL
-1agar.
Further, above-mentioned all root medias comprise 1/2MS basal medium, 0.1～0.3 mgL
-1iBA, 30～32gL
-1sucrose and 6.0～7.0 gL
-1agar.
Further, the condition that above-mentioned all callus are cultivated is: 28～30 ℃ of temperature, intensity of illumination are that 1000～1200Lx, periodicity of illumination 8～12h illumination/12～16h are dark.
Further, above-mentioned all Multiple Buds condition of culture, culture of rootage condition, hardening condition of culture are independently: 25 ℃～28 ℃ of temperature, intensity of illumination are that 1000～1200Lx, periodicity of illumination 8～12h illumination/12～16h are dark.
Further, the volume ratio of the vermiculite described in step 8) and sand is 1:(1～1.5).
The invention has the beneficial effects as follows:
It is induced material that the Multiple Buds of Pogostemon cablin callus regeneration is take in the present invention first, utilize colchicine infusion method that the stem apex of Multiple Buds is carried out to multiploid induction, again the stem apex after induction is dedifferented and broken up, obtain polyploid plant, induction result shows, induction and cultural method that the induced material that the present invention chooses adopts in conjunction with the present invention, significantly improved the inductivity (up to 63.33%) of Pogostemon cablin polyploid and reduced chimeric incidence (being only 10%).
The Pogostemon cablin polyploid survival rate 100% that the present invention obtains, and good stability, through asexual cottage propagation, after 2 generations, its proterties is still similar the present age to induction.
The Pogostemon cablin plant that the present invention obtains is huge compared with dliploid, its leaf area, stem are many greatly compared with dliploid, and branch amount is also more, its active ingredient patchouli oil content is also significantly higher than dliploid, contribute to improve the output that patchouli oil is extracted, promote the improvement of patchouli oil quality, realize the object of Pogostemon cablin good quality and high output, meet the demand of people to Pogostemon cablin medicinal material and Related product quality thereof.
Accompanying drawing explanation
Fig. 1 is Pogostemon cablin dliploid and tetraploid potted plant;
Fig. 2 Pogostemon cablin field cultivation plant, the Pogostemon cablin liploid plant that A is field cultivation, the Pogostemon cablin tetraploid plant that B is field cultivation;
Fig. 3 Pogostemon cablin chromosome observation figure, A is the diplontic chromosome of Pogostemon cablin, and 2n=2 * 14=28, and B is the tetraploid chromosome of Pogostemon cablin, 4n=4 * 14=56.
Embodiment
Below in conjunction with specific embodiment, the present invention is further illustrated, but be not limited to this.
embodiment 1:
1) induction of callus: the mature leaf of healthy plant of take is material is washed after 15s in 70% alcohol, then with 0.1% mercuric chloride sterilization 8～10min, sterile water washing 4 times, is cut into 1～2cm by the blade of processing
2small pieces, be seeded to callus medium (containing MS basal medium, 0.2mgL
-16-BA, 0.5 mgL
-1nAA, 30～32gL
-1sucrose and 6.0～7.0 gL
-1agar) in, at 28～30 ℃, intensity of illumination is 1000～1200Lx, under the condition of periodicity of illumination 10h illumination/24h dark, cultivates 12～14 days, obtains callus;
2) inducing clumping bud: callus is transferred to Multiple Buds medium (containing MS basal medium, 0.1 mgL
-16-BA, 30～32gL
-1sucrose and 6.0～7.0 gL
-1agar) cultivate about 4 weeks, obtain a large amount of Multiple Buds, condition of culture is 25 ℃～28 ℃ of temperature, intensity of illumination 1000～1200Lx, periodicity of illumination 8～12h illumination/12～16h;
3) colchicine is processed: under aseptic condition, the long Multiple Buds stem apex of clip 0.5～1cm is immersed in 0.4 gL
-1in the colchicine of concentration, shaken cultivation 24～72h under room temperature dark condition;
4) renewal cultivation: under aseptic condition, after processing, Multiple Buds stem apex is clean with aseptic water washing, is inoculated in callus medium, cultivates and within about 2 weeks, obtains callus, the same step 1) of condition of culture; Callus is forwarded to Multiple Buds medium again, cultivates about 4 weeks, obtain a large amount of Multiple Buds, condition of culture is with step 2);
5) propagation is cultivated: after renewal cultivation, by the Multiple Buds comparison of processed group and control group, the larger Multiple Buds of Feature change such as screening plant height degree, stem rugosity, interval, leaf area, vane thickness, stomatal frequency, pore opening are inoculated in new Multiple Buds medium breeds and cultivates 20～25 days, and condition of culture is with step 2);
6) polyploid is identified: after propagation is cultivated, select the stable variation strain of outward appearance table shape, by its stem apex compressing tablet, observe chromosome number, on a stem apex, observe 5 above division phase chromosome numbers simultaneously and double (as shown in Figure 3), can determine that this variant is polyploid, the stem apex number of processing in step 3) of take is radix, and the inductivity of statistics polyploid is 63.33%;
7) culture of rootage: by being accredited as the strain of polyploid, cut from base portion, be transferred to root media (containing 1/2MS basal medium, 0.2 mgL
-1iBA, 30～32gL
-1sucrose and 6.0～7.0 gL
-1agar) root induction, cultivates 8～15 days, obtains the seedling of taking root, and condition of culture is with step 2);
8) in vermiculite hardening: take out the seedling of taking root, wash away medium, be transferred to that to contain volume ratio be 1:(1～1.5) and the basin of sand, carry out hardening, hardening is cultivated about 30 days, and condition of culture is with step 2);
9) transplant seedlings: after hardening, basin seedling is moved to outdoor, or seedling is transplanted to field from basin, cover the cloudy canopy of 70～80% sunshade rates, cultivate about 10 days, remove cloudy canopy, carry out normal field planting management, growth of seedling healthy and strong (as depicted in figs. 1 and 2), the successful polyploid of all inductions is all survived, and survival rate reaches 100%.
The effect of the effect of abductive approach in embodiment and other conventional abductive approachs is compared.
In process of the present invention, also choose seedling stem apex, ripe plant stem apex, callus and carried out Pogostemon cablin multiploid induction as induced material, its multiploid induction effect and embodiment 1 compare, and comparative result is as shown in table 1.
The multiploid induction effect comparison of the different Pogostemon cablin induced materials of table 1
Correction data in table 1 shows, choose the effect that effect that Multiple Buds carries out Pogostemon cablin multiploid induction as induced material is significantly better than other three kinds of induced materials, its inductivity can reach 63.33%, the Pogostemon cablin polyploid survival rate 100% that induction obtains, chimera incidence is low, is 10%.
Pogostemon cablin polyploid prepared in embodiment is made to follow-up Detection of Stability.
By the polyploid of inducing in embodiment 1 through asexual cottage propagation after 2 generations, its offspring being carried out to the tree characteristics indexs such as chromosome number statistics, guard cell's counting, chloroplast counting, pore opening and density measure detects, testing result all shows similar the present age to induction, still keep its polyploid proterties, illustrate that the Pogostemon cablin polyploid that the inventive method induction generates has good stability.
Claims (7)
1. an abductive approach for Pogostemon cablin polyploid, is characterized in that: comprise the following steps:
1) induction of callus: get the blade of Pogostemon cablin, sterilization, cleans, and under aseptic condition, is cut into 1～2cm
2small pieces, be seeded in callus medium and cultivate 12～14 days, obtain callus;
2) induction of Multiple Buds: under aseptic condition, callus is transferred in Multiple Buds medium and is cultivated 26～30 days, obtain Multiple Buds;
3) colchicine is processed: under aseptic condition, the long Multiple Buds stem apex of clip 0.5～1cm is soaked in 0.3～0.5gL
-1colchicine solution in, 24～72h vibrates under dark condition;
4) renewal cultivation: under aseptic condition, the Multiple Buds stem apex sterile water wash clean after colchicine is processed, is inoculated in callus medium and cultivates after 12～14 days, is inoculated in Multiple Buds medium and cultivates 26～30 days, obtains polyploid Multiple Buds;
5) propagation is cultivated: polyploid Multiple Buds is inoculated in new Multiple Buds medium and breeds and cultivate 20～25 days;
6) polyploid is identified: the stem apex of getting the rear Multiple Buds of propagation cultivation carries out Chromosome Number Observation, observes 5 above division phase chromosome numbers simultaneously and double on a tip of a root, and can confirm this variant is polyploid;
7) culture of rootage: under aseptic condition, the strain that is accredited as polyploid is cut from base portion, be transferred in root media and cultivate 8～15 days, obtain the seedling of taking root;
8) hardening: take out the seedling of taking root, be transferred in the basin that vermiculite and sand are housed, cultivate 28～40 days in indoor hardening;
9) transplant seedlings: after hardening, basin seedling is moved on to outdoor, or seedling is transplanted to from basin to field, in screening, have the cloudy canopy of 70～85% sunshade rates, cultivate after 8～12 days, remove cloudy canopy, carry out field planting management.
2. a kind of abductive approach of Pogostemon cablin polyploid according to claim 1, is characterized in that: described callus medium comprises MS minimal medium, 0.1～0.3mgL
-16-BA, 0.4～0.6 mgL
-1nAA, 30～32gL
-1sucrose and 6.0～7.0 gL
-1agar.
3. a kind of abductive approach of Pogostemon cablin polyploid according to claim 1, is characterized in that: described Multiple Buds medium comprises MS basal medium, 0.1～0.3mgL
-16-BA, 30～32gL
-1sucrose and 6.0～7.0 gL
-1agar.
4. a kind of abductive approach of Pogostemon cablin polyploid according to claim 1, is characterized in that: described root media comprises 1/2MS basal medium, 0.1～0.3 mgL
-1iBA, 30～32gL
-1sucrose and 6.0～7.0 gL
-1agar.
5. a kind of abductive approach of Pogostemon cablin polyploid according to claim 1, is characterized in that: the condition that described callus is cultivated is: 28～30 ℃ of temperature, intensity of illumination are that 1000～1200Lx, periodicity of illumination 8～12h illumination/12～16h are dark.
6. a kind of abductive approach of Pogostemon cablin polyploid according to claim 1, is characterized in that: Multiple Buds condition of culture, culture of rootage condition, hardening condition of culture are independently: 25 ℃～28 ℃ of temperature, intensity of illumination are that 1000～1200Lx, periodicity of illumination 8～12h illumination/12～16h are dark.
7. according to a kind of abductive approach of Pogostemon cablin polyploid according to claim 1, it is characterized in that: the vermiculite described in step 8) and the volume ratio of sand are 1:(1～1.5).
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107027622A (en) * | 2017-01-20 | 2017-08-11 | 广东药科大学 | A kind of Herba Andrographitis Polyploid Induction Methods |
| CN111990260A (en) * | 2020-09-22 | 2020-11-27 | 广东药科大学 | Tissue culture rapid propagation method of ketone type patchouli |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JPH07123878A (en) * | 1993-11-08 | 1995-05-16 | Kao Corp | Autotetraploid patchouli plant and production of patchouli essential oil using the same |
| CN102703422A (en) * | 2012-06-04 | 2012-10-03 | 广东省中药研究所 | Culture method for new pogostemon cablin breed |
-
2013
- 2013-12-05 CN CN201310657063.XA patent/CN103704132A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07123878A (en) * | 1993-11-08 | 1995-05-16 | Kao Corp | Autotetraploid patchouli plant and production of patchouli essential oil using the same |
| CN102703422A (en) * | 2012-06-04 | 2012-10-03 | 广东省中药研究所 | Culture method for new pogostemon cablin breed |
Non-Patent Citations (1)
| Title |
|---|
| YUWU,MINGLI: "Induction of tetraploid plants of Pogostemon cablin (Blanco)and its quality evaluation", 《PHARMACOGNOSY JOURNAL》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107027622A (en) * | 2017-01-20 | 2017-08-11 | 广东药科大学 | A kind of Herba Andrographitis Polyploid Induction Methods |
| CN111990260A (en) * | 2020-09-22 | 2020-11-27 | 广东药科大学 | Tissue culture rapid propagation method of ketone type patchouli |
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