CN104429956A - Method for test-tube rooting of cunninghamia lanceolata tissue culture seedlings by using compound LED light source - Google Patents
Method for test-tube rooting of cunninghamia lanceolata tissue culture seedlings by using compound LED light source Download PDFInfo
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Abstract
The invention discloses a method for test-tube rooting of cunninghamia lanceolata tissue culture seedlings by using a compound LED light source. The method comprises the following steps: inoculating sub-culturing bud seedlings, which are cultured for 30 days and 3-4 cm in height, into a rooting medium prepared from 1/4 MS as a minimal medium and adding IBA with the concentration 0.7-1.1 mg/L, NAA with the concentration of 0.2-0.6 mg/L, ABT with the concentration of 0-0.2 mg/L, 1.5% of saccharose and 0.55% of agar; putting the rooting medium under an LED lamp with different light quality ratios for culturing and obtaining test-tube rooting seedlings. The method has the advantages as follows: compared with a fluorescent lamp, the compound LED lamp is used as the tissue culture light source, the test-tube rooting culture effect of cunninghamia lanceolata is better, the primary rooting time is shortened to 10 days, the rooting rate is 91% above, and 10 root systems can be achieved at most, so that the problems of long cunninghamia lanceolata tissue culture seedling test-tube rooting growth cycle, low rooting rate, poor root system quality and the like are effectively solved, and the production cost is reduced.
Description
Technical field
The present invention relates to plant biotechnology field.A kind of specifically method of carrying out taking root in China fir plantlet in vitro bottle with composite LED light source.
Background technology
China fir Cunninghamia Lanceolata (Lamb.) Hook. (Taxodiaceae Taxodiaceae, Cunninghamia Cunninghamia), its growth is fast, output is high, wood property good, purposes is many, the important reproducting tree species of south China, distribution is wider, be distributed between ° 03 ' N of ° 53 ' E and 19 ° 30 ' N ~ 34,101 ° of 30 ' E ~ 121, throughout 17 provinces and regions of Subtropic of China.Current domestic own through there being the research of many excellent Chinese fir clonal tissue cultures, mainly concentrate on the aspects such as explant cultivation, callus tissue culture and induction, gene engineering, medium preparation, in recent years, about the rapid propagation in vitro of China fir and genetic improvement become study hotspot, carry out large quantifier elimination, and achieve certain achievement.China fir plantlet in vitro culture of rootage is still a difficulty of also not capturing, and there is the problems such as rooting rate is low, poor growth, root system quality are not good, hampers the factorial seedling growth of China fir, commercial operation always.
LED is as the solid-state cold light source of new generation of semiconductor, have that structure is simple, lightweight, specular removal, low energy consumption, the life-span is long and respond the features such as fast, in recent years, LED has been used successfully to the fields such as artificially feed, plant tissue culture, genetic breeding, plant factor and astroponics, and expand to the various fields of agricultural and biological industry, so far, LED light source is successfully applied to the aspect such as cultivation, group training of various plants, and horticultural crop comprises romaine lettuce, pepper, cucumber, tomato etc.; Group training aspect comprises sweet potato, potato, cotton, chrysanthemum etc.And the application of LED light source in forestry is still less.
Have much about the research of taking root in China fir plantlet in vitro bottle at present, but all concentrate in more traditional method such as adjustment minimal medium and growth regulator etc.
Chinese patent CN101480166A discloses a kind of rooting method for tissue culture of Chinese fir, cut China fir proliferation and subculture seedling, be inoculated in China fir root media to carry out tissue cultures and take root, China fir root media is by modified MS medium, ABT#0.4 ~ 0.8mg/L, IBA 0.1 ~ 0.5mg/L and root sun dilution 2 ~ 6mg/L forms, this kind of method is adopted to cultivate, the rootage duration of China fir foreshortens to 16 ~ 22 days, after cultivating through 22 days, the highest rooting rate reaches 96%, its result display China fir culture of rootage stage is responsive to hormon concentration proportioning, and there is significant difference in seedling growth situation, the ABT# of high concentration, though IBA and the root sun have higher rooting rate, but part nursery stock occurs burning seedling, white seedling phenomenon.
Chinese patent CN103229723A discloses a kind of China fir rooting of vitro seedling abductive approach, cut the proliferation and subculture seedling of 1 ~ 2cm, be inoculated in pre-root media and cultivate, pre-root media for minimal medium, comprises NAA0.02 ~ 0.08mg/L with 1/2 MS ~ MS.After 15 ~ 25 days pre-culture of rootage, then carry out culture of rootage, root media for minimal medium, comprises IBA 0.10 ~ 0.25mg/L, NAA 0.05 ~ 0.08mg/L with 1/2 MS.The rooting rate of the China fir test-tube plantlet of this kind of method can reach 88 ~ 96%, and root system is sprouted relatively more neat, but pre-culture of rootage adds production process, and rootage duration extends relatively.
Chinese patent CN102726299A discloses the China fir two step method tissue culture and rapid propagation method based on LED light source, what employing light intensity was adjustable is red, blue led combined light source step-by-step processing stage by stage, first use monochromatic ruddiness as China fir group training light source, improve the growth rate of plantlet in vitro, then ultra-blue-light is re-used as light source, can it be effectively promoted to take root and strong sprout, the method can improve growth rate and the nursery stock robustness of Chinese Fir Seedling to a certain extent, shorten growth cycle and rootage duration, and increase root system number and improving activity of root system within the specific limits, but do not point out concrete growth cycle and initial rootage duration, root system number, it cultivates operation relative complex simultaneously.
Summary of the invention
The present invention is directed to the deficiency that above-mentioned technology exists, a kind of method that composite LED light source carries out taking root in China fir plantlet in vitro bottle is proposed, fundamentally solve China fir group training process to take root difficult problem, reducing production cost simultaneously, providing foundation for realizing China fir plantlet in vitro factorial seedling growth.Use composite LED light source to process seedling of taking root in China fir tissue culture bottle, not only effectively can improve rooting rate, radical and root system quality, also effectively reduce production cost.
Technical scheme of the present invention is: carry out the method for taking root in China fir plantlet in vitro bottle with composite LED light source, comprise the steps:
(1) nursery stock squamous subculture: the proliferation and subculture seedling cutting 1 ~ 2cm, is inoculated in subculture medium;
(2) culture of rootage: until proliferation and subculture seedling in step (1) after 30 days cultivate, the seedling choosing high 3 ~ 4cm is transferred in root media;
(3) light treatment: tested by subculture under nursery stock is placed in fluorescent lamp and cultivate, light application time is 12h/d; Take root under experiment plantlet in vitro is placed in the LED light source of red, blue, purple, green different ratio and carry out culture of rootage, light application time is 12 ~ 24h/d;
Described subculture medium take MS as minimal medium, also adds IBA 0.2 ~ 0.3mg/L, 6-BA 0.6 ~ 0.8mg/L, KT 1 ~ 2mg/L, sucrose 3.0% and agar 0.55%, medium pH 6.0;
Described root media take 1/4MS as minimal medium, also adds IBA 0.7 ~ 1.1mg/L, NAA 0.2 ~ 0.6mg/L, ABT 0 ~ 0.2mg/L, sucrose 1.5% and agar 0.55%, medium pH 6.0;
The illumination used of described squamous subculture stage is fluorescent lamp, intensity of illumination 1800 ~ 2000lux; Red-light LED crest is 660 ± 20nm, blue light crest is 450 ± 20nm, purple light crest is 400 ± 20nm, green glow crest is 550 ± 20nm, the LED light source that the culture of rootage stage adopts is: red blue proportioning light R:B=2 ~ 10:1, red royal purple proportioning light R:B:P=4 ~ 8:1:1, red royal purple green proportioning light R:B:P:G=6 ~ 8:1:1:1, intensity of illumination 1200 ~ 2000lux; Cultivation temperature is 23 ~ 26 DEG C.
Compared with prior art, outstanding advantages of the present invention is:
The present invention carries out squamous subculture under fluorescent light, then directly moves access root media, does not excise callus; On the basis of traditional plant tissue cultures, use novel composite LED light source, effectively can solve that the rooting rate existed in tissue culture technology is in the past low, the problem such as slowly of taking root.
The present invention determines the best working concentration of the optimal proportion of red, blue, purple, green glow, optimum illumination time and growth regulator, draws the combination of the proportioning of optimal LED light quality and the best working concentration of light application time and growth regulator thereof.
The method can start to take root after inoculation for about 10 days, root media with 1/4 MS for minimal medium, also add IBA0.7mg/L, NAA 0.2mg/L, ABT 0.2mg/L, sucrose 1.5% and agar 0.55%, pH 6.0, when utilizing composite LED light source R:B:P:G=6:1:1:1 process simultaneously, substantially increase rooting rate and the root system quality of China fir plantlet in vitro, its rooting rate is more than 91%.
According to plant photosynthesis absorption Spectrum characteristic, plant is the most responsive to 400 ~ 510nm royal purple light section, 610 ~ 720nm blood orange light, the reaction of 720 ~ 780nm far-red light, and wherein absorbable wavelength mainly concentrates on royal purple light section crest be 450nm and blood orange light section crest is 660nm.Ruddiness is the required light quality of crop normal growth, is played a role in regulation and control photomorphogenesis by phytochrome; Blue light is the Light quality of ruddiness for crop genetics necessity, and under red blue complex light condition, Photosynthetic Rate is higher; Purple light, green glow are important photosynthetic active radiations, and research finds, the basis of red blue light is added purple light, green glow etc. and can increase the indexs such as spinach petiole length.
It is red that experiment seedling of taking root in China fir tissue culture bottle is placed in LED light source proportioning: blue: purple: to cultivate under the light quality of green=6:1:1:1, effectively can improve nursery stock rooting rate, root system quality, and plant strain growth is compared with healthy under other light quality.
Accompanying drawing explanation
Fig. 1 is tested number 4 and red in embodiment 1: blue: purple: picture of taking root under green=6:1:1:1.
Fig. 2 is tested number 2 and red in embodiment 2: picture of taking root under indigo plant=8:1.
Embodiment
Below in conjunction with embodiment, technical scheme of the present invention is further illustrated.
Embodiment 1
Method of carrying out taking root in China fir plantlet in vitro bottle with composite LED light source of the present invention, comprises the following steps:
(1) nursery stock squamous subculture: the proliferation and subculture seedling cutting 1 ~ 2cm, is inoculated in subculture medium.
(2) culture of rootage: after proliferation and subculture seedling in step (1) was cultivated through 30 days, seedling is transferred in root media.
(3) light treatment: tested by subculture under nursery stock is placed in fluorescent lamp and cultivate, light application time is 12h/d; Take root under experiment plantlet in vitro is placed in the LED light source of red, blue, purple, green different ratio and carry out culture of rootage, light application time is 12h/d.
Described subculture medium take MS as minimal medium, and also have IBA0.3mg/L, 6-BA0.6mg/L, KT2mg/L sucrose 1.5% and agar 0.55%, the pH value of medium is 6.0;
Described root media take 1/4MS as minimal medium, and also have IBA0.7 ~ 1.1mg/L, NAA0.2 ~ 0.6mg/L, ABT 0 ~ 0.2mg/L sucrose 1.5% and agar 0.55%, the pH value of medium is 6.0;
The illumination that the squamous subculture stage adopts is fluorescent lamp, intensity of illumination 1800 ~ 2000lux; Red-light LED (crest is 660 ± 20nm), blue light (450 ± 20nm), purple light (400 ± 20nm), green glow (550 ± 20nm), the LED light source that the culture of rootage stage adopts is divided into following 10 kinds: red blue proportioning light (R:B=2:1,4:1,6:1,8:1,10:1), red royal purple proportioning light (R:B:P=4:1:1,6:1:1 and 8:1:1), the green proportioning light (R:B:P:G=6:1:1:1 and 8:1:1:1) of red royal purple, intensity of illumination 1200 ~ 2000lux; Cultivation temperature is 23 ~ 26 DEG C.
Table 1 is the use list of growth regulator under the process of 12h exposure experiments to light, under this embodiment, China fir group trains the relative growth index of seedling of taking root in table 2, result shows: the situation of taking root of seedling of taking root in China fir plantlet in vitro bottle under different culture media formula and different light medium is not quite similar, rooting rate, mean elements, the indexs such as average root is long difference is larger, to make a living foundation basal culture medium at 1/4 MS, also has IBA 0.7mg/L, NAA 0.2mg/L, ABT 0.2mg/L, sucrose 1.5% and agar 0.55%, pH 6.0, utilize composite LED light source R:B:P:G=6:1:1:1 process simultaneously, when light application time is 12h/d, the rooting rate of China fir plantlet in vitro, mean elements, average root is long is respectively 91%, 3.1 bar, 0.9cm, IBA is described, NAA, ABT is with the use of more useful to the root induction of China fir, and on the basis of red blue light, add purple light, green glow composite light source when its proportioning is R:B:P:G=6:1:1:1, the most favourable to taking root of China fir, may be all light required for plant growth due to ruddiness, blue light, purple light, green glow, its acting in conjunction effectively can promote the growth of root system.
The use list of the lower growth regulator of table 1 12h LED illumination test process
The lower Chinese Fir Seedling of table 2 12h LED illumination test process is taken root situation statistical form
Embodiment 2
Another example utilizing composite LED light source to carry out the method for taking root in China fir plantlet in vitro bottle of the present invention, beyond different with control technology condition divided by lower operating procedure, other steps and condition are with embodiment 1.
(1) light treatment: carry out culture of rootage under experiment plantlet in vitro of taking root is placed in the LED light source of red, blue, purple, green different ratio, light application time is 24h/d.
(2) root media take 1/4MS as minimal medium, and also have IBA 0 ~ 1.0mg/L, NAA 0 ~ 1.0mg/L, ABT0 ~ 1.0mg/L, sucrose 1.5% and agar 0.55%, the pH value of medium is 6.0;
(3) illumination that adopts of squamous subculture stage is fluorescent lamp, intensity of illumination 1800 ~ 2000lux; Red-light LED (crest is 660 ± 20nm), blue light (450 ± 20nm), purple light (400 ± 20nm), green glow (550 ± 20nm), the LED light source that the culture of rootage stage adopts is divided into following 10 kinds: red blue proportioning light (R:B=2:1,4:1,6:1,8:1,10:1), red royal purple proportioning light (R:B:P=4:1:1,6:1:1 and 8:1:1), the green proportioning light (R:B:P:G=6:1:1:1 and 8:1:1:1) of red royal purple, intensity of illumination 1200 ~ 2000lux; Cultivation temperature is 23 ~ 26 DEG C.
Table 3 is the use list of growth regulator under the process of 24h exposure experiments to light, under this embodiment, China fir group trains the relative growth index of seedling of taking root in table 4, result shows: the situation of taking root of seedling of taking root in China fir plantlet in vitro bottle under different culture media formula and different light medium is not quite similar, rooting rate, mean elements, the indexs such as average root is long difference is larger, to make a living foundation basal culture medium at 1/4 MS, also has IBA 0.9mg/L, NAA 0.4mg/L, sucrose 1.5% and agar 0.55%, pH 6.0, utilize composite LED light source R:B=8:1 process simultaneously, when light application time is 24h/d, the rooting rate of China fir plantlet in vitro, mean elements, average root is long is respectively 90%, 1.2 bar, 1.5cm, IBA is described, NAA with the use of, during R:B=8:1, the most favourable to taking root of China fir, and give 24h/d illumination Chinese Fir Seedling and take root more early, within 10th day, have after inoculation and take root, illustrate that simple red blue light is more conducive to promoting taking root of Chinese Fir Seedling.
The use list of the lower growth regulator of table 3 24h LED illumination test process
The lower Chinese Fir Seedling of table 4 24h LED illumination test process is taken root situation statistical form
Claims (2)
1. carry out the method for taking root in China fir plantlet in vitro bottle with composite LED light source, it is characterized in that, comprise the steps:
(1) nursery stock squamous subculture: the proliferation and subculture seedling cutting 1 ~ 2cm, is inoculated in subculture medium;
(2) culture of rootage: until proliferation and subculture seedling in step (1) after 30 days cultivate, the seedling choosing high 3 ~ 4cm is transferred in root media;
(3) light treatment: tested by subculture under nursery stock is placed in fluorescent lamp and cultivate, light application time is 12h/d; Take root under experiment plantlet in vitro is placed in the LED light source of red, blue, purple, green different ratio and carry out culture of rootage, light application time is 12 ~ 24h/d;
Described subculture medium take MS as minimal medium, also adds IBA 0.2 ~ 0.3mg/L, 6-BA 0.6 ~ 0.8mg/L, KT 1 ~ 2mg/L, sucrose 3.0% and agar 0.55%, medium pH 6.0;
Described root media take 1/4MS as minimal medium, also adds IBA 0.7 ~ 1.1mg/L, NAA 0.2 ~ 0.6mg/L, ABT 0 ~ 0.2mg/L, sucrose 1.5% and agar 0.55%, medium pH 6.0;
The illumination used of described squamous subculture stage is fluorescent lamp, intensity of illumination 1800 ~ 2000lux; Red-light LED crest is 660 ± 20nm, blue light crest is 450 ± 20nm, purple light crest is 400 ± 20nm, green glow crest is 550 ± 20nm, the LED light source that the culture of rootage stage adopts is: red blue proportioning light R:B=2 ~ 10:1, red royal purple proportioning light R:B:P=4 ~ 8:1:1, red royal purple green proportioning light R:B:P:G=6 ~ 8:1:1:1, intensity of illumination 1200 ~ 2000lux; Cultivation temperature is 23 ~ 26 DEG C.
2. method of carrying out taking root in China fir plantlet in vitro bottle with composite LED light source according to claim 1, it is characterized in that, described culture of rootage stage light treatment is that to be placed in LED light source proportioning be red to experiment seedling by taking root in China fir tissue culture bottle: blue: purple: to cultivate under the light quality of green=6:1:1:1.
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| CN105660416A (en) * | 2016-03-16 | 2016-06-15 | 蒋凡 | Root induction method for Chinese fir test-tube plantlets |
| CN105830919A (en) * | 2016-03-30 | 2016-08-10 | 安徽科技学院 | Hibiscus coccineus tissue culture method |
| CN106922534A (en) * | 2017-03-27 | 2017-07-07 | 河池乐康生态农业科技有限公司 | A kind of method that tissue cultures quickly breed China fir |
| CN108370749A (en) * | 2018-03-21 | 2018-08-07 | 黑龙江中医药大学 | A method of for promoting wilsonii seedling to grow |
| CN109744146A (en) * | 2018-04-18 | 2019-05-14 | 蒋凡 | Rooting method in a kind of China fir tissue-cultured seedling bottle |
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Application publication date: 20150325 |