CN106106142A - A kind of method inducing Herba Andrographis polyploid - Google Patents
A kind of method inducing Herba Andrographis polyploid Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 70
- 241000746375 Andrographis Species 0.000 title claims abstract description 68
- 208000020584 Polyploidy Diseases 0.000 title claims abstract description 34
- 230000001939 inductive effect Effects 0.000 title claims abstract description 9
- IAKHMKGGTNLKSZ-INIZCTEOSA-N (S)-colchicine Chemical compound C1([C@@H](NC(C)=O)CC2)=CC(=O)C(OC)=CC=C1C1=C2C=C(OC)C(OC)=C1OC IAKHMKGGTNLKSZ-INIZCTEOSA-N 0.000 claims abstract description 106
- 229960001338 colchicine Drugs 0.000 claims abstract description 53
- 230000008569 process Effects 0.000 claims abstract description 36
- 230000006698 induction Effects 0.000 claims abstract description 22
- 241000196324 Embryophyta Species 0.000 claims abstract description 15
- 240000002853 Nelumbo nucifera Species 0.000 claims abstract description 8
- 235000006508 Nelumbo nucifera Nutrition 0.000 claims abstract description 8
- 235000006510 Nelumbo pentapetala Nutrition 0.000 claims abstract description 8
- 238000009395 breeding Methods 0.000 claims abstract description 6
- 230000001488 breeding effect Effects 0.000 claims abstract description 6
- 239000000126 substance Substances 0.000 claims abstract description 4
- 238000010924 continuous production Methods 0.000 claims description 3
- 210000000056 organ Anatomy 0.000 abstract description 5
- 230000006866 deterioration Effects 0.000 abstract description 4
- 235000016709 nutrition Nutrition 0.000 abstract description 3
- 230000035764 nutrition Effects 0.000 abstract description 3
- 238000013461 design Methods 0.000 abstract description 2
- 208000035199 Tetraploidy Diseases 0.000 description 20
- 239000000243 solution Substances 0.000 description 16
- 210000000349 chromosome Anatomy 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 5
- 235000013399 edible fruits Nutrition 0.000 description 5
- 238000012545 processing Methods 0.000 description 5
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 210000003763 chloroplast Anatomy 0.000 description 2
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- 230000003203 everyday effect Effects 0.000 description 2
- 230000001976 improved effect Effects 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 244000118350 Andrographis paniculata Species 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
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Classifications
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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
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
- A01H1/08—Methods for producing changes in chromosome number
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- Health & Medical Sciences (AREA)
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- Molecular Biology (AREA)
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- Developmental Biology & Embryology (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses Colchicine application in terms of Herba Andrographis breeding and induction Herba Andrographis polyploid.Also disclose a kind of method inducing heart lotus polyploid, be when Herba Andrographis seedling length paramount 0.8～1.2cm, utilize 10～30 μ L 500～2000mg/L Colchicine to seedling apical point process 2～6 times, chemical induction produce polyploid plant.The inventive method can improve the kind of Herba Andrographis, improve yield and the quality of Herba Andrographis, the nutrition organs giantisms such as root, stem, leaf, effective medicinal components content increase, annidation and resistance strengthen, efficiently solve the various problems such as the solution variety deterioration of Herba Andrographis, yield and Quality Down, provide theoretical foundation and technical foundation for the sustainable use of Herba Andrographis germ plasm resource and the Industry Promotion of Herba Andrographis standardized planting.And method is reasonable in design, credible result, there is stronger practicality and generalization.
Description
Technical field
The invention belongs to technical field of biological breeding.More particularly, to a kind of method inducing Herba Andrographis polyploid.
Background technology
Herba Andrographis derives from acanthaceous plant Herba Andrographis Andrographis paniculata (Burm.f.) Nees., with
Aerial parts is used as medicine, and calls Herba Andrographitis, cuts Magnoliacoco, Herba vallisneriae Spiralis, Fructus Canarii albi lotus, for Guangdong genuine medicinal materials.Originate in Philippine, India,
The torrid areas such as Sri Lanka, Thailand, is introduced the cultivation of the ground such as Guangdong, Fujian by overseas Chinese the fifties in last century, after gradually move northward, existing
In Hunan, Hubei, Jiangxi, Sichuan, Shaanxi, the ground such as Beijing have establishing in large scale.Herba Andrographis is cold in nature, bitter in the mouth, have heat-clearing and toxic substances removing,
The effect such as removing heat from blood, detumescence, can antiinflammatory, antibacterial, antiviral, anticancer, anti-cardiovascular disease.
The situations such as geographical environment, water and soil, weather and the production management due to various places are different, and long-term artificial culture lacks
The management of systematic science, causes the phenomenons such as Herba Andrographis variety deterioration, yield decline, active constituent content reduction serious.
Therefore, carry out high yield and high quality, pharmaceutical ingredient content height new germ plasm nurturing research extremely urgent, be also market compel
It is essential and asks.
Summary of the invention
The technical problem to be solved in the present invention is to overcome Herba Andrographis variety deterioration in prior art, yield and Quality Down
Problem, it is provided that a kind of method inducing Herba Andrographis polyploid, makes Herba Andrographis kind be improved, and yield and quality are improved.
It is an object of the invention to provide Colchicine application in terms of Herba Andrographis breeding.
Another object of the present invention is to provide Colchicine application in terms of induction Herba Andrographis polyploid.
Another object of the present invention is to provide a kind of method inducing Herba Andrographis polyploid.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
Colchicine application in terms of Herba Andrographis breeding.
Colchicine application in terms of induction Herba Andrographis polyploid.
A kind of method inducing heart lotus polyploid, is to utilize Colchicine to click on Herba Andrographis chitting piece or growth of seedling
Row chemical induction produces polyploid plant.
It is further preferred that the method for described induction heart lotus polyploid be colchicine solution point is dropped in chitting piece or
In 0.8～1.2cm high seedling apical point, every morning 6:00 and 18:00 in afternoon respectively processes once, coprocessing 2～6 times.
It is highly preferred that the concentration of described Colchicine is 500～2000mg/L, use 10～30 μ L every time.
It is highly preferred that the concentration of described Colchicine is 2000mg/L, use 20 μ L every time.
It is highly preferred that the number of processes of described Colchicine is 2 times, 4 times or 6 times.
It is highly preferred that the method for described induction heart lotus polyploid is to be dropped in by 20 μ L 2000mg/L colchicine solution points
On chitting piece or 0.8～1.2cm high seedling apical point, every morning 6:00 and 18:00 in afternoon respectively processes once, co-continuous
Process 3 days.
More specifically, as one preferably can embodiment, the method for described induction heart lotus polyploid is to work as Herba Andrographis
During seedling length paramount 0.8～1.2cm, utilize Colchicine point to drop in seedling apical point according to below scheme and process 6 times, chemistry
Induction generation polyploid plant:
First day: utilize 20 μ L 2000mg/L colchicine solutions the morning 6:00 and 18:00 in afternoon respectively process once；
Second day: utilize 20 μ L 1800mg/L colchicine solutions the morning 6:00 and 18:00 in afternoon respectively process once；
3rd day: utilize 20 μ L 2000mg/L colchicine solutions the morning 6:00 and 18:00 in afternoon respectively process once.
The present invention have studied condition and the method for colchicine-induced Herba Andrographis polyploid first, for further breeding work
Lay the foundation；Use directly to drip and ooze method, Herba Andrographis seedling apical point is processed, it is possible to obtain polyploid plant.
The present invention also uses the multiple methods such as identification of morphology method, cytological Identification method, Chromosome number observation identification method to carry out
Polyploid detects, and result of study has the strongest cogency.
Result shows, the organ such as Herba Andrographis polyploid leaf, cane, fruit and seed has " huge property " feature, is conducive to
Improve Herba Andrographis yield.Herba Andrographis somatic double, the active constituent content compared with normal that on chromosome, gene regulation produces
Diploid is high, highly beneficial for improving quality of medicinal material.Tetraploid Herba Andrographis plant is more sturdy than diploid cane, lodging tolerance
Strengthen.
In sum, there is stronger science, innovation and practicality in the inventive method.
The method have the advantages that
The inventive method can improve the kind of Herba Andrographis, improves yield and the quality of Herba Andrographis, has stronger practicality
Property and generalization, it is demonstrated experimentally that the method is reasonable in design, credible result, and there is promotional value.
It addition, Herba Andrographis polyploid plant is due to chromosome doubling in cell, show as the nutrition organs such as root, stem, leaf huge
Type, effective medicinal components content increase, annidation and resistance strengthen.Therefore, the cultivation of Herba Andrographis polyploid is to solve
One of effective ways of various problems such as the certainly variety deterioration of Herba Andrographis, yield and Quality Down, for Herba Andrographis germ plasm resource
The Industry Promotion of sustainable use and Herba Andrographis standardized planting provides theoretical foundation and technical foundation.
Accompanying drawing explanation
Fig. 1 is that Herba Andrographis diploid is compared with tetraploid leaf；In figure: A. diploid；B. tetraploid.
Fig. 2 is that Herba Andrographis diploid is compared with tetraploid fruit, seed；In figure: A. tetraploid；B. diploid.
Fig. 3 is that Herba Andrographis diploid is compared with tetraploid pore；In figure: A. diploid；B. tetraploid.
Fig. 4 is that Herba Andrographis diploid compares (1000 ×) with tetraploid chromosomes；In figure: A. diploid；B. tetraploid.
Detailed description of the invention
Further illustrate the present invention below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
Limit in any form.Unless stated otherwise, the present invention uses reagent, method and apparatus are the examination of the art routine
Agent, method and apparatus.
Unless stated otherwise, following example agents useful for same and material are commercial.
The impact on Herba Andrographis seed of the embodiment 1 Colchicine concentration
1, the Colchicine utilizing variable concentrations gradient (100～3000mg/L) processes Herba Andrographis seed, and research is different dense
The degree Colchicine impact on Herba Andrographis seed germination.
2, shown in result, along with the rising of Colchicine concentration, the sprouting to Herba Andrographis seed has certain positive role,
When Colchicine concentration is more than 1000mg/L, this positive role has certain weakening, when Colchicine concentration is more than
During 2000mg/L, then the sprouting to Herba Andrographis seed has inhibitory action, and can cause the death of Herba Andrographis seed.
Therefore, in follow-up test, the concentration of Colchicine is not higher than 2000mg/L.
The determination of embodiment 2 Herba Andrographis multiploid induction time
Inventor shows through substantial amounts of preliminary study, utilizes Colchicine to process punching in intraday different time sections
Lotus seedling, the effect of multiploid induction has significant difference.
Result shows, for the multiploid induction of Herba Andrographis seedling, every morning 5:00～7:00 or 17:00 in afternoon～
Processing during 19:00, multiploid induction effect is significantly higher than other times section.
Study display further, the most once locate during 5:00～7:00 and 17:00 in afternoon～19:00 every morning
Reason, more preferable than the multiploid induction effect after every day single treatment.
Therefore, follow-up test selects every morning 6:00,18:00 in afternoon respectively to process scheme once.
Embodiment 3 Herba Andrographis multiploid induction condition optimizing
1, use directly to drip and ooze method, colchicine solution point is dropped in Herba Andrographis seedling apical point, every day at the upper and lower noon
(6:00,18:00) respectively processes 1 time, each 20 μ L, concentration be set to 0mg/L (drop distilled water), 500mg/L, 1000mg/L,
4 horizontal processings of 2000mg/L, number of processes is 2 times, 4 times, 63 levels.It is repeated 3 times, statistical data.
2, the results are shown in Table 1.
Herba Andrographis polyploid plant inducing effect is compared (n=3) by table 1 variable concentrations Colchicine and number of processes
Note: 1. P < 0.05, from 0,500,1000mg/L processes different number of times and compares.
Known by table 1, from 0,500,1000mg/L process different number of times and compare, the colchicine-induced of 2000mg/L 6 times
Morphological variation rate, tetraploid variant number, tetraploid add multiplying power and significantly increase (P < 0.05).
Embodiment 4 Herba Andrographis polyploid is identified
1, morphological characteristic is observed
(1) the Herba Andrographis plant of 5 strain Different Ploidy is chosen at random respectively, length of blade, blade width thick to its plant height, stem
The morphological indexs such as degree, leaf area measure, and every strain takes 6 blades at random.Repeated observation 3 times, statistical data.
(2) the results are shown in Table 2 and accompanying drawing 1～2 shown in.
Table 2 compares (n=3) by Herba Andrographis diploid with tetraploid morphological index
Note: 1. P < 0.05,2. P < 0.01, compares with diploid.
From table 2, Fig. 1, comparing with diploid, tetraploid plant height is the shortest in diploid (P < 0.05), tetraploid
Stem is thick, length of blade, width of blade, blade area, fruit length, seed diameter pole are noticeably greater than diploid (P < 0.01),
Polyploid has the features such as plant is short and small, stem is thick, blade is thick, leaf area is big, fruit length, seed are big, and leaf color relatively two times
Body is dark green.
2, histological characterization
(1) tear take plant base portion upwards the 2nd～3 blades lower epidermis make interim water load, with I-KI weak solution dye 3
～5min, under 400 × power microscope, randomly select 10 visuals field, add up the stomatal number under each visual field, calculate stomatal frequency
(individual/visual field), measures the length and width of guard cell simultaneously；And randomly selecting 30 cells, statistics guard cell's leaf is green
Body number.Repeated measures 3 times, statistical data.
(2) the results are shown in Table shown in 3.
Table 3 Herba Andrographis diploid compares (n=3) with tetraploid Leaf lower epidermis feature
Note: 1. P < 0.05,2. P < 0.01, compares with diploid.
By table 3, Fig. 2,3 knowing, comparing with diploid, tetraploid pore length is noticeably greater than diploid (P < 0.05)；Four
The stomatal frequency of times body is significantly less than diploid (P < 0.05)；Tetraploid chloroplast number pole be significantly more than diploid (P <
0.01).Tetraploid blade lower epidermis cell pore length, width, density, chloroplast number be respectively diplontic 130%,
107%, 56%, 200%.
3, Chromosome number is identified
(1) taking the tip of a root to be placed in the EP pipe of 0.5ml, the colchicine solution adding 2g/L carries out pretreatment, treatment temperature
It is 4 DEG C, after 1.5h, material distilled water is cleaned 3 times, washes 5min every time, use filter paper suck dry moisture, be placed in the EP pipe of 0.5ml
In, add the fixing 24h of Ka Nuoshi fixative (dehydrated alcohol: glacial acetic acid=3:1), clean 3 times with distilled water, blot water with filter paper
Add appropriate 0.075mol/L KCL aqueous solution after Fen and carry out hyposmosis, clean 3 times with distilled water after 1.5h, wash 5min every time, wash
After Jing, dissociate with the hydrochloric acid of 1mol/L, water-bath 15min, dye by improvement carbolic acid magenta, 30min, tabletting mirror
Inspection, Taking Pictures recording.
(2) result is shown in Fig. 4.
Known by Fig. 4, diploid chromosome 2X ≈ 50, tetraploid chromosomes 4X ≈ 100, the plant dyeing after induction doubles
Body number is 2 times of former liploid plant, is autotetraploid.
In sum, the seedling apical point of the Colchicine process Herba Andrographis of employing 2000mg/L 6 times, generation can be induced
Herba Andrographis polyploid, inductivity is 12.5%, and induction gained Herba Andrographis plant is tetraploid, and the organ such as leaf, fruit, seed is equal
Hugeization, Chromosome number doubles.
The method using the present invention, induces Herba Andrographis polyploid, and nutrition organs shows giantism, and chromosome number adds
Times, it is applied to produce there is stronger practicality and generalization.
Embodiment 5 Herba Andrographis multiploid induction process optimization
1, based on the studies above, the induced processes of Herba Andrographis polyploid is optimized by inventor further, use with
Lower three kinds of schemes process the Herba Andrographis seedling of about 1cm, and the seedling after processing is carried out polyploid qualification.
Three kinds of processing schemes:
(1) the optimum process condition that the studies above of the present invention obtains, it may be assumed that utilize 20 μ L 2000mg/L colchicine solutions
The morning 6:00 and 18:00 in afternoon respectively process once；Continuous processing three days.
(2) Colchicine " low-high-low " concentration processes, i.e.
First day: utilize 20 μ L 1800mg/L colchicine solutions the morning 6:00 and 18:00 in afternoon respectively process once；
Second day: utilize 20 μ L 2000mg/L colchicine solutions the morning 6:00 and 18:00 in afternoon respectively process once；
3rd day: utilize 20 μ L 1800mg/L colchicine solutions the morning 6:00 and 18:00 in afternoon respectively process once.
(3) Colchicine " high-low-high " concentration processes, it may be assumed that
First day: utilize 20 μ L 2000mg/L colchicine solutions the morning 6:00 and 18:00 in afternoon respectively process once；
Second day: utilize 20 μ L 1800mg/L colchicine solutions the morning 6:00 and 18:00 in afternoon respectively process once；
3rd day: utilize 20 μ L 2000mg/L colchicine solutions the morning 6:00 and 18:00 in afternoon respectively process once.
2, polyploid qualification result shows, the inductivity of scheme (1) is 12.5%, and the inductivity of scheme (2) is 10%, side
The inductivity of case (3) is 16.3%.
Result shows, uses scheme (3), uses the Colchicine of suitably " high-low-high " concentration successively even in i.e. three days
Continuous process Herba Andrographis seedling, multiploid induction best results.
Claims (10)
1. Colchicine application in terms of Herba Andrographis breeding.
2. Colchicine application in terms of induction Herba Andrographis polyploid.
3. the method inducing heart lotus polyploid, it is characterised in that be to utilize Colchicine to Herba Andrographis chitting piece or children
Seedling growing point carries out chemical induction and produces polyploid plant.
Method the most according to claim 3, it is characterised in that be that colchicine solution point is dropped in chitting piece or 0.8
～in the high seedling apical point of 1.2cm, every morning 6:00 and 18:00 in afternoon respectively processes once, coprocessing 2～6 times.
Method the most according to claim 4, it is characterised in that the concentration of described Colchicine is 500～2000mg/L, often
Secondary use 10～30 μ L.
Method the most according to claim 4, it is characterised in that described Colchicine uses 10～30 μ L every time.
Method the most according to claim 4, it is characterised in that the concentration of described Colchicine is 2000 mg/L, makes every time
With 20 μ L.
Method the most according to claim 4, it is characterised in that the number of processes of described Colchicine is 2 times, 4 times or 6
Secondary.
Method the most according to claim 4, it is characterised in that be that 20 μ L 2000mg/L colchicine solution points are dropped in
On chitting piece or 0.8～1.2cm high seedling apical point, every morning 6:00 and 18:00 in afternoon respectively processes once, co-continuous
Process 3 days.
Method the most according to claim 4, it is characterised in that be when Herba Andrographis seedling length paramount 0.8～1.2cm, press
Utilize Colchicine point to drop in seedling apical point according to below scheme to process 6 times, chemical induction generation polyploid plant:
First day: utilize 20 μ L 2000mg/L colchicine solutions the morning 6:00 and 18:00 in afternoon respectively process once；
Second day: utilize 20 μ L 1800mg/L colchicine solutions the morning 6:00 and 18:00 in afternoon respectively process once；
3rd day: utilize 20 μ L 2000mg/L colchicine solutions the morning 6:00 and 18:00 in afternoon respectively process once.
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| CN107027622A (en) * | 2017-01-20 | 2017-08-11 | 广东药科大学 | A kind of Herba Andrographitis Polyploid Induction Methods |
| CN106922524A (en) * | 2017-05-08 | 2017-07-07 | 河北科技师范学院 | The authentication method of breed cucumber method and polyploid plant based on induction polyploid |
| CN106993531A (en) * | 2017-05-08 | 2017-08-01 | 河北科技师范学院 | The authentication method of breed cucumber method and polyploid plant based on induction polyploid |
| CN110063161A (en) * | 2019-03-20 | 2019-07-30 | 广州中医药大学(广州中医药研究院) | A method of promote Herba Andrographitis to grow using sesbania Azorhizobium caulinadans and improves its active constituent content |
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