CN106106183A - A kind of tissue culture and rapid propagation method of tongue post lip post lettuce tongue - Google Patents
A kind of tissue culture and rapid propagation method of tongue post lip post lettuce tongue Download PDFInfo
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- CN106106183A CN106106183A CN201610651481.1A CN201610651481A CN106106183A CN 106106183 A CN106106183 A CN 106106183A CN 201610651481 A CN201610651481 A CN 201610651481A CN 106106183 A CN106106183 A CN 106106183A
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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
- A01H4/008—Methods for regeneration to complete plants
-
- 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
Abstract
The invention discloses the tissue cultivation rapid breeding method of a kind of tongue post lip post lettuce tongue, the steps such as preparation, initial culture, successive transfer culture, root culture and the transplanting domestication including outer implant, particularly as follows: take, young leaflet tablet is treated obtains outer implant, produce adventitious bud through initial culture and obtain shoot, again the blade of shoot is cut into small pieces and carries out successive transfer culture as outer implant, obtain healthy and strong plant, then plant is transferred and cultivate in root media, obtain the whole plant taken root, after obtain planting stock through domestication.The present invention improves the reproduction speed of seedling effectively and quickly and ensure that the breeding quality of seedling, a large amount of high quality seedling is provided as the resistance to cloudy flowers of indoor appreciation flowers and gardens for tongue post lip post lettuce tongue, breeding coefficient is high, and meanwhile, transplanting survival rate reaches more than 95%.
Description
Technical field
The present invention relates to plant tissue culture field, particularly to the tissue culture and rapid propagation method of a kind of tongue post lip post lettuce tongue.
Background technology
Tongue post lip post lettuce tongue (Chirita liguliformis W.T.Wang) is Gesneriaceae (Gesneriaceae) lip
Post lettuce tongue belongs to perennial herb.Tongue post lip post lettuce tongue has thick root stock, and blade base is raw, to life, long 2-11 centimetre, wide 4-9 millimeter,
By pubescence.Blade ground paper matter, oval.Inflorescence about 2,2-3 returns branch, and every inflorescence there are about 7 flowers；Peduncle length 20 centimetres,
Carried out pubescence, July at florescence；The narrow triangle of bract, is about 3.5 millimeters, wide 1.1 millimeters, by pubescence；The long 2.5-4 of bennet
Centimetre, by pubescence.Calyx 5 splits and reaches base portion, and it is linear that sliver drapes over one's shoulders needle-like, long 3-3.5 millimeter, wide 1 millimeter, outside by pubescence,
Inner face is without hair.Corolla lavender, is about 2.2 centimetres, outside dredge by pubescence, inner face under upper lip by pubescence；Tube length is about
1.5 centimetres, mouth diameters about 6 millimeters.Capsule is linear, is about 3 centimetres, wide 2 millimeters, by pubescence；Seed is oval, is about
0.25 millimeter.Produce southwestern Guizho (Anlong, volume are enjoyed).It is born in the dark and damp place of mountain valley sylvan life, height above sea level about 800 meters.
The pattern of tongue post lip post lettuce tongue is plain, and its blade is thick, the most resistance to the moon, it is easy to management, is developed as indoor appreciation
Flowers and the resistance to cloudy flowers of afforestation have higher market value, but wild resource is the most rare, it is difficult to meet on market
As being actually needed of ornamental flower and afforestation.Plant tissue culture is a kind of quick breeding technology, can be gardening
Market provides a large amount of high quality seedlings, it also avoid the destruction excavating wild tongue post lip post lettuce tongue to wild resource simultaneously.
Summary of the invention
The present invention provides the tissue culture and rapid propagation method of a kind of tongue post lip post lettuce tongue, is difficult to full solving wild tongue post lip post lettuce tongue
Being actually needed as ornamental flower and afforestation on foot market, improve effectively and rapidly seedling reproduction speed and
Ensure the breeding quality of seedling, provide a large amount of high-quality for tongue post lip post lettuce tongue as the resistance to cloudy flowers of indoor appreciation flowers and gardens
Seedling.
Technical scheme is as follows:
The tissue culture and rapid propagation method of a kind of tongue post lip post lettuce tongue, comprises the following steps:
(1) preparation of outer implant
Take the blade of tongue post lip post lettuce tongue, cleaned, sterilize and rinse, obtain outer implant；
(2) initial culture
Described outer implant is inoculated into the MS cultivation being added with 0.1～0.5mg/L 6-BA and 0.05～0.1mg/L NAA
Cultivating in base, there are a large amount of adventitious buds in the edge of described outer implant, continues cultivation to uncertain buds growth and becomes shoot；
(3) successive transfer culture
Described shoot is aseptically taken out, and is cut into small pieces by the blade of described shoot, then described fritter
It is inoculated into and is added with in the MS culture medium of 0.1～0.5mg/L 6-BA and 0.01～0.05mg/L NAA cultivation, described fritter
Adventitious bud occurs, continues to cultivate to adventitious bud and all become seedling；
(4) root culture
Described seedling is transferred to be added with in the MS root media of 0.1～0.3mg/L IBA and 15～30g/L sucrose
Cultivate, obtain Seedling of taking root；
(5) domestication is transplanted
Described Seedling of taking root removes in culture apparatus, transplants the container for plant growth in filling seedling medium and cultivates, and controls
Relative humidity, obtains planting stock.
Further preferably, the preparation of described step (1) outer implant includes the young leaflet tablet taking tongue post lip post lettuce tongue, with washing
After washing agent scouring, being placed in and rinse under water, be then placed under super-clean bench by blade, be cut into the blockage of the length of side about 1cm, described is little
Square successively use 70% alcohol washes, aseptic water washing, and move in 0.1% mercuric chloride solution added with a polysorbas20, take
Again by rinsed with sterile water after going out, obtain described outer implant.
Further preferably, the MS culture medium of described step (2) initial culture is added with 0.5mg/L 6-BA, 0.1mg/L
NAA, 30g/L sucrose and 6g/L agar, and pH is 5.8.
Further preferably, the condition of culture of described step (2) initial culture is for being 25 ± 2 DEG C in temperature, under first dark
Cultivate, the most again dislocation in photosynthetically active radiation be 40 μm ol m-2s-1, cultivate under light application time 12h.
Further preferably, described step (3) successive transfer culture also includes that, when adventitious bud all becomes seedling, timely plant division turns
Receive and the new MS culture medium being added with 0.1～0.5mg/L 6-BA and 0.01～0.05mg/L NAA continues cultivate, then
Carry out root culture again.
Further preferably, the MS culture medium of described step (3) successive transfer culture is added with 0.5mg/L 6-BA, 0.01mg/L
NAA, 30g/L sucrose and 6g/L agar, and pH is 5.8.
Further preferably, the condition of culture of described step (3) successive transfer culture and described step (4) root culture is all
Temperature 25 ± 2 DEG C, photosynthetically active radiation is 40 μm ol m-2s-1, cultivate under light application time 12h.
Further preferably, the MS culture medium of described step (4) root culture is added with 0.3mg/L IBA, 30g/L sugarcane
Sugar, 5.5g/L agar, and pH is 5.8.
Further preferably, described step (5) transplant the seedling medium of domestication be the peat composed of rotten mosses of volume ratio 3:1:1, Vermiculitum and
Perlite, and pH is 6.5.
Further preferably, described step (5) is transplanted domestication and is included removing described Seedling of taking root in culture bottle, washes away
It is attached to the culture medium solution of root, then transplants in the hole dish filling seedling medium, every cave one strain, it is positioned over seedbed
On, greenhouse sunshade after transplanting, maximum photosynthetically active radiation is not higher than 160 μm ol m-2s-1, and periodically spraying and moisturizing, temperature controls
Cultivate at 15～30 DEG C, transplant again after domestication.
Compared with prior art, beneficial effects of the present invention is as follows:
One, the tissue culture and rapid propagation method of a kind of tongue post lip post lettuce tongue of the present invention, improves the breeding of seedling effectively and rapidly
Speed and the breeding quality that ensure that seedling, provide as the resistance to cloudy flowers of indoor appreciation flowers and gardens for tongue post lip post lettuce tongue
A large amount of high quality seedlings, obtain numerous seedling by initial culture and successive transfer culture, and breeding coefficient is high, and through root culture
After, the transplanting survival rate of Seedling of taking root reaches more than 95%.
Two, the tissue culture and rapid propagation method of a kind of tongue post lip post lettuce tongue of the present invention, respectively to initial culture, successive transfer culture and life
The materials such as the hormone that the MS culture medium that root is cultivated is added and its content are optimized, and have obtained the cultivation of tissue-culturing rapid propagation each stage
Time defined medium, improve breeding coefficient and ensure that breeding quality.
Certainly, the either method implementing the present invention it is not absolutely required to reach all the above advantage simultaneously.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate this
Bright, rather than limit protection scope of the present invention.Those skilled in the art are according to changing that the present invention makes in actual applications
Enter and adjust, still falling within protection scope of the present invention.
The tissue culture and rapid propagation method of a kind of tongue post lip post lettuce tongue of the present invention, specifically includes following steps:
(1) preparation of outer implant
Time fine, introduce a fine variety and survive and the tongue post lip post lettuce tongue maternal plant of robust growth, be placed in ventilation, after one week, take children
Tender blade, after cleaning gently with liquid detergent, is placed under the tap water of flowing flushing 1 hour, is then positioned over by blade ultra-clean
Under platform, being cut into the blockage of the length of side about 1cm, first with 70% alcohol washes 10s, then 3 times (each 15s is left with aseptic water washing
Right), then move in 0.1% mercuric chloride solution added with a polysorbas20, vibrate 5-8 minute, finally use rinsed with sterile water 7-10 time,
Obtain described outer implant；
(2) initial culture
Described outer implant is inoculated into and is added with 0.5mg/L 6-BA, 0.1mg/L NAA, 30g/L sucrose and 6g/L agar,
And the MS culture medium that pH is 5.8 is cultivated, every bottle of about inoculation 5, culturing room's temperature is 25 ± 2 DEG C, first dark culturing one week, so
After again dislocation in photosynthetically active radiation (PAR) be 40 μm ol m-2s-1Group training frame on, light application time 12h, cultivate 19 days, described
The edge of outer implant a large amount of adventitious buds occur, bud is sturdy intensive, continues to cultivate to the 6th week, and uncertain buds growth is not completely but not
The shoot taken root；
(3) successive transfer culture
Described shoot is taken out under superclean bench aseptic condition, the blade of shoot is peeled and is cut into 0.5cm ×
The fritter of 0.5cm, then described fritter be inoculated into be added with 0.5mg/L 6-BA and 0.01mg/L NAA, 30g/L sucrose and
In 6g/L agar, and the MS culture medium that pH is 5.8 cultivate, every bottle graft kind 5, culturing room's temperature is 25 DEG C ± 2 DEG C, photosynthetic effectively
Radiation is 40 μm olm-2s-1, cultivate under light application time 12h, after cultivating 3 weeks, there is adventitious bud in described fritter, continues cultivation 3
In week, adventitious bud all becomes seedling, then plant division timely, be transferred to new be added with 0.5mg/L 6-BA, 0.01mg/L NAA,
The MS culture medium of 30g/L sucrose and 6g/L agar continues cultivate 3 weeks, it is thus achieved that healthy and strong plant；
(4) root culture
Described plant is transferred to be added with 0.3mg/L IBA, 30g/L sucrose and 5.5g/L agar, and pH is the MS of 5.8
Cultivating 25-30 days in root media, obtain complete Seedling of taking root of taking root, root length about 1cm, plant length to 3-4cm is high；
(5) domestication is transplanted
Described Seedling of taking root is removed in culture bottle, washes away the culture medium solution being attached to root, then transplant in dress
Completely have in 72 hole dishes of seedling medium, every cave one strain, it is positioned on seedbed, seedbed is equipped with spray in covering Small plastic shed, Small plastic shed
Mist device, regular spraying and moisturizing, described seedling medium is the peat composed of rotten mosses of volume ratio 3:1:1, Vermiculitum and perlite, and pH is 6.5,
Water permeable after filling hole tray；Greenhouse sunshade after transplanting, the photosynthetically active radiation in Small plastic shed is not higher than 160 μm ol m-2s-1,
Taming first week, the relative air humidity in Small plastic shed controls more than 95% by sprayer unit and shed, and shed thin film is complete
Closing, after one week, new root length progressively opens shed thin film after going out, and relative air humidity is maintained at more than 85%, maintains one
Week, the most again stronger ventilation area, air humidity progressively drops to 75%, and the temperature in greenhouse controls to cultivate at 15～30 DEG C,
After domesticating and cultivating 6 weeks, Plug seedling can be transplanted further in flowerpot.
In order to investigate at initial culture, successive transfer culture and root culture further, each culture medium is to tongue post lip post lettuce tongue
The impact of tissue-culturing rapid propagation, the present invention is also provided with some groups of experiments to optimize in the culture medium cultivating each stage.
When one, investigating initial culture, the culture medium prescription impact on Induce aerosor
Described outer implant is inoculated in following group of culture medium respectively cultivation, first group: 0.5mg/L 6-BA, 6g/L
Agar, 30g/L sucrose, and the MS culture medium that pH is 5.8；Second group: 0.5mg/L 6-BA, 0.1mg/L NAA, 6g/L agar,
30g/L sucrose, and the MS culture medium that pH is 5.8；3rd group: 0.5mg/L 6-BA, 0.1mg/L IBA, 6g/L agar, 30g/L
Sucrose, and the MS culture medium that pH is 5.8；4th group: 0.5mg/L 6-BA, 0.1mg/L IAA, 6g/L agar, 30g/L sucrose,
And the MS culture medium that pH is 5.8, often to organize and a culture bottle is set, each culture bottle all inoculates 5 outer implant, each culture bottle
Condition of culture be culturing room's temperature 25 ± 2 DEG C, first dark culturing one week, dislocation is in photosynthetically active radiation (PAR) the most again
It is 40 μm olm-2s-1Group training frame on, under light application time 12h cultivate, statistical result is as shown in Table 1.
Table one
Note: different letter (a, b) marks of the 3rd column data in table, there were significant differences in expression duncan's new multiple range method inspection (P <
0.05)
From the data analysis of table one it can be seen that first group was cultivated after 26 days, outer implant thickens, but has no obvious wound healing
Tissue, edge occurs that adventitious bud and bud are sturdy, but 19 adventitious buds only occurs in each outer implant；Cultivate after 19 days i.e. to go out for second group
Existing adventitious bud, raw 31 adventitious buds of each outer implant common property, sprout ratio is more uniform；The each outer implant of the 3rd group and the 4th group is produced
Raw adventitious bud number is respectively 25 and 23, and the time producing adventitious bud is longer than second group the most accordingly, therefore, and the present invention
When initial culture, culture medium is preferably selected and is added with 0.5mg/L 6-BA, 0.1mg/L NAA, 6g/L agar, 30g/L sucrose, and
PH is the MS culture medium of 5.8.
Two, the impact that tongue post lip post lettuce tongue successive transfer culture is bred by hormon horizontal combination is investigated
Being inoculated into respectively by described fritter in the culture medium that the group shown in table two is 1～10, the 1st group is matched group,
Being not added with the MS culture medium of any hormone, the 2nd～10 group is experimental group, the hormone added in MS culture medium such as table two secondary series
Shown in, being all also added into 6g/L agar, 30g/L sucrose, and pH in each of which group culture medium is 5.8, often organizes and arranges one
Culture bottle, each culture bottle all inoculates 5 fritters, and it is 25 DEG C ± 2 DEG C that the condition of culture of each culture bottle is culturing room's temperature,
Photosynthetically active radiation is 40 μm olm-2s-1, to cultivate under light application time 12h, statistical result is as shown in Table 2.
Table two
Note: different letter (a, b, c, d) marks of the 3rd column data in table, there were significant differences to represent duncan's new multiple range method inspection
(P<0.05)
From the data analysis of table two it can be seen that the concentration of 6-BA MS culture medium produces quantity to adventitious bud has significantly
Impact (P=0.005), and the concentration of NAA has no significant effect (P=0.247) to it, the reciprocal action of two kinds of hormones is also to it
Have a significant impact.The adventitious bud quantity that each fritter produces totally increases with the rising of 6-BA concentration, but 6-BA excessive concentration,
Bud is intensive and little, and sprout vitrification phenomenon is serious, and therefore, comprehensive growth coefficient and Seedling weight, the present invention trains at subculture
When supporting, preferably selecting the 6-BA being added with 0.5mg/L, 0.01mg/L NAA, 6g/L agar, 30g/L sucrose, and pH is the MS of 5.8
Culture medium.
Three, hormon level and the cane sugar content impact on tongue post lip post lettuce tongue root culture are investigated
Described seedling is inoculated in the culture medium that the group shown in table three is 1～6 respectively, has specifically investigated sucrose dense
Degree and the impact on seedling root culture of the IBA concentration, shown in second and third row of concrete each concentration such as table three, each of which group is cultivated
Base is all also added into 5.5g/L agar, and pH is 5.8, often organizes and arranges a culture bottle, each culture bottle all inoculate 5 little
Block, it is 25 DEG C ± 2 DEG C that the condition of culture of each culture bottle is culturing room's temperature, and photosynthetically active radiation is 40 μm olm-2s-1, light
Cultivating according under time 12h, statistical result is as shown in Table 3.
Table three
Note: different letter (a, b, c, d) marks of the 3rd column data in table, there were significant differences to represent duncan's new multiple range method inspection
(P<0.05)
From the data analysis of table three it can be seen that bottle Seedling plant height and radical are had a significant impact by the concentration of sucrose, IBA's is dense
Bottle seedling leaf number, radical, root length and plant height are all had a significant impact by degree, wherein under the concentration of same sucrose, there was added
During the culture medium culturing of 0.5mg/L IBA, radical is that at most root length is the longest, and plant is the highest, but the root of bottle Seedling is elongated and
Uneven, Seedling body is the most weak, therefore, under the concentration of same sucrose, preferably selects the culture medium of 0.3mg/L IBA；At identical IBA
Concentration under, add 30g/L sucrose cultivate time, bottle Seedling is the most healthy and the strongest, and root is uniform and sturdy, root length about 15mm, plant length is extremely
1.5-2cm it is high, it is easy to transplant.Therefore, the present invention, when root culture, preferably selects 0.3g/L IBA, 5.5g/L agar, 30g/L
Sucrose and the MS culture medium that pH is 5.8.
Present invention disclosed above preferred embodiment is only intended to help to illustrate the present invention.Preferred embodiment is the most detailed
Describe all of details, be also not intended to the detailed description of the invention that this invention is only described.Obviously, according to the content of this specification,
Can make many modifications and variations.These embodiments are chosen and specifically described to this specification, is to preferably explain the present invention
Principle and actual application so that skilled artisan can be best understood by and utilize the present invention.The present invention is only
Limited by claims and four corner thereof and equivalent.
Claims (10)
1. the tissue culture and rapid propagation method of a tongue post lip post lettuce tongue, it is characterised in that comprise the following steps:
(1) preparation of outer implant
Take the blade of tongue post lip post lettuce tongue, cleaned, sterilize and rinse, obtain outer implant；
(2) initial culture
Described outer implant is inoculated in the MS culture medium being added with 0.1～0.5mg/L 6-BA and 0.05～0.1mg/L NAA
Cultivating, there are a large amount of adventitious buds in the edge of described outer implant, continues cultivation to uncertain buds growth and becomes shoot；
(3) successive transfer culture
Described shoot is aseptically taken out, and is cut into small pieces by the blade of described shoot, then described fritter inoculation
Cultivating in the MS culture medium be added with 0.1～0.5mg/L 6-BA and 0.01～0.05mg/L NAA, described fritter occurs
Adventitious bud, continues to cultivate to adventitious bud and all becomes seedling；
(4) root culture
Described seedling is transferred to be added with in the MS root media of 0.1～0.3mg/L IBA and 15～30g/L sucrose training
Support, obtain Seedling of taking root；
(5) domestication is transplanted
Described Seedling of taking root removes in culture apparatus, transplants the container for plant growth in filling seedling medium and cultivates, and controls relatively
Humidity, obtains planting stock.
The tissue culture and rapid propagation method of a kind of tongue post lip post lettuce tongue the most according to claim 1, it is characterised in that described step
(1) preparation of outer implant includes the young leaflet tablet taking tongue post lip post lettuce tongue, after cleaning with detergent, is placed in and rinses under water, then
Blade is placed under super-clean bench, is cut into the blockage of the length of side about 1cm, described blockage successively use 70% alcohol washes, nothing
Bacterium water rinses, and moves in 0.1% mercuric chloride solution added with a polysorbas20, again by rinsed with sterile water after taking-up, obtains described
Outer implant.
The tissue culture and rapid propagation method of a kind of tongue post lip post lettuce tongue the most according to claim 1, it is characterised in that described step
(2) the MS culture medium of initial culture is added with 0.5mg/L 6-BA, 0.1mg/L NAA, 30g/L sucrose and 6g/L agar, and pH
It is 5.8.
The tissue culture and rapid propagation method of a kind of tongue post lip post lettuce tongue the most according to claim 1, it is characterised in that described step
(2) condition of culture of initial culture is for being 25 ± 2 DEG C in temperature, and the darkest lower cultivation, dislocation is in photosynthetically active radiation the most again
It is 40 μm ol m-2s-1, cultivate under light application time 12h.
The tissue culture and rapid propagation method of a kind of tongue post lip post lettuce tongue the most according to claim 1, it is characterised in that described step
(3) successive transfer culture also includes when adventitious bud all becomes seedling, and timely plant division is transferred to new be added with 0.1～0.5mg/L 6-
The MS culture medium of BA and 0.01～0.05mg/L NAA continues cultivate, carry out root culture the most again.
The tissue culture and rapid propagation method of a kind of tongue post lip post lettuce tongue, it is characterised in that described
The MS culture medium of step (3) successive transfer culture is added with 0.5mg/L 6-BA, 0.01mg/LNAA, 30g/L sucrose and 6g/L agar,
And pH is 5.8.
The tissue culture and rapid propagation method of a kind of tongue post lip post lettuce tongue the most according to claim 1, it is characterised in that described step
(3) condition of culture of successive transfer culture and described step (4) root culture is all to be 25 ± 2 DEG C in temperature, photosynthetically active radiation
It is 40 μm ol m-2s-1, cultivate under light application time 12h.
The tissue culture and rapid propagation method of a kind of tongue post lip post lettuce tongue the most according to claim 1, it is characterised in that described step
(4) the MS culture medium of root culture is added with 0.3mg/L IBA, 30g/L sucrose 5.5g/L agar, and pH is 5.8.
The tissue culture and rapid propagation method of a kind of tongue post lip post lettuce tongue the most according to claim 1, it is characterised in that described step
(5) seedling medium transplanting domestication is the peat composed of rotten mosses of volume ratio 3:1:1, Vermiculitum and perlite, and pH is 6.5.
The tissue culture and rapid propagation method of a kind of tongue post lip post lettuce tongue the most according to claim 1, it is characterised in that described step
Suddenly (5) are transplanted to tame and are included removing described Seedling of taking root in culture bottle, wash away the culture medium solution being attached to root, then
Transplant in the hole dish filling seedling medium, every cave one strain, it is positioned on seedbed, greenhouse sunshade after transplanting, maximum photosynthetic
Net long wave radiation is not higher than 160 μm ol m-2s-1, and periodically spraying and moisturizing, temperature controls to cultivate, after domestication again at 15～30 DEG C
Transplant.
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| CN115633638A (en) * | 2022-09-21 | 2023-01-24 | 广西科学院 | Tissue culture method of Lihua rocky sow moss |
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