CN103493735A - Light regulation and control method for tissue culture and multiplication of camptotheca acuminata decaisne calluses - Google Patents
Light regulation and control method for tissue culture and multiplication of camptotheca acuminata decaisne calluses Download PDFInfo
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Abstract
The invention discloses a light regulation and control method for tissue culture and multiplication of camptotheca acuminata calluses, which sieves an optimal culture medium formula for establishing a camptotheca acuminate clone. A result shows that an optimal induction culture medium of stems contains MS, 0.1mg/L of KT and 4mg/L of 2,4-D; the optimal multiplication conditions are as follows: B5, 0.5mg/L of KT, 1.0mg/L of NAA (Naphthalene Acetic Acid) and 0.5mg/L of the 2,4-D; leaves represent poor inductivity. The calluses are multiplied under the conditions of red light, green light, blue light, red-blue light and white light; by analyzing the increment of calluses in unit, the red light and the green light have an acceleration effect on the growth of the calluses, the blue light has the inhibition effect on the growth of the calluses, and the effects of the white light and the red-blue light are not obvious; the acceleration effect of the green light is the most significant.
Description
Technical field
The invention belongs to the plant cultivation technical field, relate to the light regulate and control method that a kind of camplotheca acuminata callus is cultivated and bred.
Background technology
Camplotheca acuminata (Campototheca acuminate Decaisne) is the Nyssaceae deciduous tree, China's endemic tree.1966, Monroe doctor E.WALL of the U.S. separated and obtains camptothecine (Compothecine, be called for short CPT) from the secondary metabolite of camplotheca acuminata.1985, the discovery CPT such as Hisang can block the synthetic of topoisomerase I and have anticarcinogenic effect.At present, existing multiple take CPT as the synthetic camptothecin derivative of precursor for clinical treatment oophoroma, colon cancer etc., in addition, CPT also has stronger inhibitory action to HIV virus.Along with continuous excavation, the checking of the functions such as CPT is antitumor, antiviral, the whole world rapidly increases the demand of CPT.From the nineties in 20th century, the countries such as the U.S., Japan, Canada and Britain actively drop into a large amount of manpower and materials and carry out camplotheca acuminata introducing and planting and CPT research and development, and camplotheca acuminata becomes Chinese yew the 2nd important woody anti-cancer medicinal plants afterwards.But in camplotheca acuminata, CPT content is low, rely on merely from plant the demand that CPT can not satisfying the market of extracting, utilize plant cell large-scale culture technology production high value secondary metabolite to obtain success in multiple medicinal plant, equally, existing correlative study confirmation, cultivating production CPT by the camplotheca acuminata callus is a kind of effective approach.
Light is as an important envirment factor, the important regulating action to growing of plant.Gu Qing etc. have studied illumination to camplotheca acuminata callus physio-biochemical characteristics and the synthetic impact of camptothecine, result shows, compare with dark cultivation, cultivate the physiological metabolism that can promote the camplotheca acuminata callus under light, and the synthetic and accumulation of camptothecine is had to certain facilitation.At present, fluorescent lamp is the principal light source that tissue is cultivated, but its energy consumption is high, and the life-span is short, mercurous, easily causes environmental pollution, and light quality is single, can not meet plant and cultivate the demand difference of different vegetative stages to light quality at tissue.Light-Emitting Diode (LED) is the luminous semiconductor electronic original paper of a kind of energy, pass through chemical modification method, adjust band structure and the energy gap of material, can realize that the red, yellow, green and blue orange is multicolor luminous, meet plant and cultivate the demand difference of different phase to light quality at tissue, simultaneously, the LED energy consumption reduces 80% with the incandescent lamp of effect, without poisonous metal mercury, can reach 100,000 hours service life, " national basic public services system " 12 " planning " is pointed out to support energetically to develop the LED industry, to substitute in a large number traditional fluorescent lamp in all trades and professions wide popularization and application at planning period LED lamp.
Summary of the invention
In order to address the above problem, the present invention proposes a kind of Optimal Medium and LED group training light source regulate and control method that can effectively improve Camptotheca acuminata callus induction rate and the rate of increase.
The light regulate and control method that a kind of camplotheca acuminata callus is cultivated and bred comprises the following steps:
1) the camplotheca acuminata spring of choosing is sent out to young sprout and cut into segment, rinse well with running water, with liquid detergent solution, soak 15min, repeatedly rinse under flowing water;
2) after rinsing well with flowing water, in 75% ethanolic solution, soak 1min, put immediately in people's 0.1% mercuric chloride solution the 5min that sterilizes, be placed on aseptic filter paper after aseptic water washing 5 times and dry standby inoculation and use;
3) blade is cut into to 5mm * 5mm, stem segment with axillary bud is cut into the segment of 2mm, the inducing culture MS+0.1mg/L KT+4mg/L2 that access prepares respectively under aseptic condition, 4-D is upper, and every preparation 1L medium adds 5.5g agar and 30g sucrose, adjusts pH to 5.9, inducing culture is divided and installs in blake bottle, every bottle of 40mL, in 121 ℃ of lower sterilizing 15min, 25 ± 2 ℃ of cultivation temperature, intensity of illumination 1000～1500lx, the dark cultivation one week, then proceed to illumination cultivation, every day light application time 12h;
4) callus induced is transferred to subculture on new subculture medium and cultivates, 30d subculture 1 time, and subculture medium is B5+0.5mg/L KT+1.0mg/L NAA+0.5mg/L2,4-D, 5.5g/L agar and 30g/L sucrose, pH is 5.9;
5) the 30d subculture is 1 time, continuously, after subculture 5 times, selects loose frangible callus and carries out cultivating under LED light.
Further preferably, adopt the light quality of the micro-subculture of green glow step 5).
The LED light source regulate and control method of the raising Camptotheca acuminata callus rate of increase provided by the invention, it is in the callus multiplicative stage, it is that 500～580nm, light quantity are at 105 μ molm that callus is transferred to spectral wavelength
-2s
-1under the LED green glow of left and right, cultivate.
Optimal Medium of the present invention and LED group training light regulate and control method can improve camplotheca acuminata callus induction rate and the rate of increase effectively, provide technique guarantee for obtaining a large amount of CPT extraction raw material camplotheca acuminata callus, in addition, the present invention can also reduce energy consumption, reduce production costs, environmental contamination reduction.
Embodiment
Following examples are used for the present invention is described, but are not used for limiting the scope of the invention.
Embodiment 1
The tender stem segments that the camplotheca acuminata of take gives birth to then without damage by disease and insect is explant.After the stem section is plucked and cleaned, the 1min that sterilizes in 75% ethanolic solution, put immediately in people's 0.1% mercuric chloride solution and continue sterilization 5min, aseptic water washing 3～4 times, and the aseptic filter paper suck dry moisture, standby.
The stem section is cut into to the segment of 2～3mm, connect respectively in the following 4 kinds of callus inducing mediums of people, 15 bottles of every kind of culture medium inoculateds, every bottle graft enters 5 explants, the dark cultivation after one week of inoculation proceeds under light and cultivates, cultivation temperature (25 ± 2) ℃, intensity of illumination 1000～1500lx, light application time 12h/ days, light source is fluorescent lamp.Test 3 repetitions.
①MS+0.1mg／L?KT+4mg/L2，4-D
②MS+0.5mg／L?KT+4mg／L2，4-D
③MS+2mg／L6-BA+2mg/L?NAA+2mg／L2，4-D
④MS+0.5mg／L6-BA+2mg／L?NAA
Above 4 kinds of medium agars are 5.5g/L, sucrose 30g/L, and pH is 5.9.
Inoculate and observe the growing state of Camptotheca acuminata explant on above-mentioned 4 kinds of medium rear every day.The 5th day, 3. the explant on number medium started to occur brownization, and the 8th day, 4. the explant on number medium also started to occur brownization, after 20 days 3. number and 4. the Brown rate on number medium surpass 90%.And 2. 1. number number medium has higher callus of induce rate to Camptotheca acuminata, the 10th day, 1. at first callus appears in the explant on number medium, and 2. the explant on number medium started to occur callus at the 12nd day, the 35th day, 1. number the callus induction rate on and 2. number medium is added up, and the result 1. callus induction rate mean value of number medium is 92.3%, 2. number be 85.4%.1. number and 2. visible, number medium is the suitable inducing culture of Camptotheca acuminata callus.
Embodiment 2
The callus of inducing on 1. number and 2. number medium is carried out respectively to the subculture cultivation, and subculture medium used is:
④MS+2.0mg／L6-BA+0.5mg/LNAA
⑤MS+0.5mg／L?KT+4.0mg/LNAA
(⑥B5+0.5mg／L?KT+1.0mg／LNAA+0.5mg／L2，4-D
The subculture condition of culture is cultivation temperature (25 ± 2) ℃, intensity of illumination 1000～15001x, and light application time 12h/ days, light source is fluorescent lamp.
1. number subculture is cultivated and within 30 days, to be observed afterwards statistics and the 2. subculture cultivation situation of evoked callus on number medium, find: the callus subculture of 1. inducing on number medium grows fine on B5 (6. number) medium, callus short texture more than 85% is arranged, easily fragmentary, of light color and transparent, for good callus, and subculture some surface of callus on MS (4. and 5. number) medium is water stain shape, some is densification and poor growth too, is unfavorable for the continued growth of callus; The callus subculture of 2. inducing on number medium is out of condition, finds that it serious fine and close, the sclerosis of callus all occurs in any subculture medium, is round bead shape, is not suitable for carrying out the subculture cultivation.
Consider just generation and subculture cultivation results, the best callus inducing medium of Camptotheca acuminata is 1. number medium: MS+0.1mg/L KT+4mg/L2,4-D, best subculture medium is 6. number medium: B5+0.5mg/L KT+1.0mg/LNAA+0.5mg/L2,4-D.
Embodiment 3
Subculture is cultivated in the propagation of transferring of the good callus on 6. number medium, 6. the switching medium still adopts number, the culture dish that the switching container is specification 90*15mm, after switching, culture dish is placed under fluorescent lamp and 5 different light medium LED light sources and cultivated, each light is transferred 9 culture dishes, repeats for 3 times.Regulate the distance of LED lamp control software and light source and culture dish, make light quantity all at 105 μ molm
-2s
-1left and right, the photoperiod is 16hd
-1, cultivate 30 days.Culturing room's relative moisture 70% ± 5%, temperature (24 ± 1) ℃.
Wherein, light source is the LED lamp purchased from Hangzhou Han Hui Electro-optical Technology, INC. (US) 62 Martin Road, Concord, Massachusetts 017.Concrete technical parameter is in Table 1.
The important technological parameters of table 1 different light medium LED light source
Cultivate after 30 days, observe the morphological feature of callus under different light medium, find that the callus of cultivating under blue light and red blue light is aubergine or yellow, and serious fine and close, sclerosis; Callus under ruddiness is faint yellow, but loose not; Callus density under white light and fluorescent lamp is approximate, but in the majority with aubergine under fluorescent lamp, under white light, take faint yellow as main; Callus short texture under green glow, be faint yellow or white, and growing way is good.
Propagation multiple and the brown rate of callus under the statistics different light medium, in Table 2.As seen from Table 2, the callus propagation multiple of cultivating under the LED green glow is the highest, be 3.502, LED ruddiness takes second place, be 2.951, all the other light qualities are processed all lower than CK, carry out variance analysis (table 3), find extremely remarkable (0.001≤P≤0.01 of callus propagation multiple difference under each light quality, difference is extremely remarkable), therefore, adopt the Tukey method to carry out multiple ratio (table 4), find to process 4 (LED green glows) and process 3 (LED ruddiness) difference not remarkable, but and CK (fluorescent lamp) etc. other process significant differences, simultaneously, process 3 except with process 5 significant differences, with all the other, process all without significant difference, in addition, callus propagation multiple between all the other processing is all without significant difference, visible, with the conventional light source fluorescent lamp, compare, the LED green glow has significant facilitation to callus growth, and other light quality effects are not obvious.
As seen from Table 2, the callus browning rate under the LED blue light is higher than CK, and all the other are processed all lower than CK, and minimum is the LED green glow, is 12.7%, and brown rate is carried out to variance analysis (table 5), finds that the brown rate difference between each processing is not remarkable.
Therefore, from morphological feature, propagation multiple and the brown rate of callus, consider, it is the LED green glow that camplotheca acuminata callus propagation is cultivated best light source.
The propagation multiple of callus and brown rate under table 2 different light medium
The propagation multiple variance analysis table of callus under table 3 different light medium
| Source of variation | Sum of squares | Degree of freedom | All square | The F value | The p value |
| Between processing | 5.8857 | 5 | 1.1771 | 7.315 | 0.0023 |
| In processing | 1.931 | 12 | 0.1609 | ? | ? |
| Total variation | 7.8167 | 17 | ? | ? | ? |
The propagation multiple significance test of callus under table 4 different light medium
| Process | Average | 4 | 3 | 1 | 6 | 2 | 5 |
| 4 | 3.635 | ? | 0.5668 | 0.0385 | 0.0207 | 0.0118 | 0.002 |
| 3 | 2.951 | 0.5507 | ? | 0.8504 | 0.3051 | 0.1883 | 0.0326 |
| 1 | 2.5737 | 0.928 | 0.3773 | ? | 0.8927 | 0.736 | 0.2101 |
| 6 | 2.2307 | 1.271 | 0.7203 | 0.343 | ? | 0.9993 | 0.7187 |
| 2 | 2.1207 | 1.381 | 0.8303 | 0.453 | 0.1?1 | ? | 0.8807 |
| 5 | 1.7673 | 1.7343 | 1.1837 | 0.8063 | 0.4633 | 0.3533 | ? |
Tukey method multiple ratio is (lower triangle is equal value difference, and upper triangle is significance level)
The brown rate variance analysis table of callus under table 5 different light medium
| Source of variation | Sum of squares | Degree of freedom | All square | The F value | The p value |
| Between processing | 69.2771 | 5 | 13.8554 | 0.714 | 0.6247 |
| In processing | 232.8092 | 12 | 19.4008 | ? | ? |
| Total variation | 302.0863 | 17 | ? | ? | ? |
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (2)
1. the light regulate and control method that the camplotheca acuminata callus is cultivated and bred, is characterized in that, comprises the following steps:
1) the camplotheca acuminata spring of choosing is sent out to young sprout and cut into segment, rinse well with running water, with liquid detergent solution, soak 15min, repeatedly rinse under flowing water;
2) after rinsing well with flowing water, in 75% ethanolic solution, soak 1min, put immediately in people's 0.1% mercuric chloride solution the 5min that sterilizes, be placed on aseptic filter paper after aseptic water washing 5 times and dry standby inoculation and use;
3) blade is cut into to 5mm * 5mm, stem segment with axillary bud is cut into the segment of 2mm, the inducing culture MS+0.1mg/L KT+4mg/L2 that access prepares respectively under aseptic condition, 4-D is upper, and every preparation 1L medium adds 5.5g agar and 30g sucrose, adjusts pH to 5.9, inducing culture is divided and installs in blake bottle, every bottle of 40mL, in 121 ℃ of lower sterilizing 15min, 25 ± 2 ℃ of cultivation temperature, intensity of illumination 1000～1500lx, the dark cultivation one week, then proceed to illumination cultivation, every day light application time 12h;
4) callus induced is transferred to subculture on new subculture medium and cultivates, 30d subculture 1 time, and subculture medium is B5+0.5mg/L KT+1.0mg/L NAA+0.5mg/L2,4-D, 5.5g/L agar and 30g/L sucrose, pH is 5.9;
5) the 30d subculture is 1 time, continuously, after subculture 5 times, selects loose frangible callus and carries out cultivating under LED light.
2. the light regulate and control method that camplotheca acuminata callus according to claim 1 is cultivated and bred, is characterized in that step 5) the middle light quality that adopts the micro-subculture of green glow.
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| CN110199875A (en) * | 2019-05-28 | 2019-09-06 | 浙江农林大学 | A kind of abductive approach and its culture medium of camplotheca acuminata Cotyledon Callus |
| CN110800612A (en) * | 2019-12-06 | 2020-02-18 | 大连工业大学 | Efficient and low-consumption camptotheca acuminata tissue culture and transplantation method |
| CN112655554A (en) * | 2020-12-16 | 2021-04-16 | 云南省农业科学院园艺作物研究所 | Method for accelerating induction of apple callus and growth based on LED light quality |
| CN114982634A (en) * | 2022-06-14 | 2022-09-02 | 江苏青好景观园艺有限公司 | Cultivation method of ilex verticillata tissue culture seedlings |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114982634A (en) * | 2022-06-14 | 2022-09-02 | 江苏青好景观园艺有限公司 | Cultivation method of ilex verticillata tissue culture seedlings |
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