CN104756867B - The method for building up of camellia azalea Callus of Leaf regenerating system - Google Patents
The method for building up of camellia azalea Callus of Leaf regenerating system Download PDFInfo
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Abstract
The method for building up of camellia azalea Callus of Leaf regenerating system, belongs to biological technical field.It is comprised the following steps：1）The acquisition of camellia azalea aseptic seedling；2）The callus induction and subculture of blade；3）The differentiation of callus adventitious bud and plant regeneration；4）Regeneration plant is taken root and rooting culture.The present invention carries out callus induction using camellia azalea tests for sterility, on the basis of state adjustment is carried out to callus, adventitious buds differentiation is carried out using the MS culture mediums of specific hormone combination, " filter-bridge rooting method " is finally utilized to solve the technical barrier that camellia is difficult to take root, so as to realize the induction of camellia azalea Callus of Leaf and the regeneration of plant, cultivation cycle is short, and breeding coefficient is high, is that camellia molecular breeding is laid a good foundation.
Description
Technical field
The invention belongs to biological technical field, and in particular to the foundation side of camellia azalea Callus of Leaf regenerating system
Method.
Background technology
Camellia azalea（Camellia azalea）, also referred to as rhododendron leaves camellia, Zhang Shi Camellias or referred to as cuckoo
Tea, is the exclusive Camellia Plants kind of China.Its pattern is gorgeous, and with blooming throughout the year, the peculiar proterties of summer full blossom, be not
The excellent Woody flower in gardens that can be obtained more；It is in more than the 280 camellia original seed for being found at present it is a small number of can see whole years bloom
Section Camellia preciousness original seed, compensate for Camellia Section Camellia species summer without the blank that the flowers are in blossom.Its NATURAL DISTRIBUTION is only limited to
In Guangdong Province Yangchun City E Huang Zhang Nature Reserve, existing wild ancestor quantity was extremely rare, in quilt in 2004《Chinese species are red
Color name is recorded》Critical species is classified as, is described as " plant giant panda ".
Breeding about camellia azalea at present is still carried out in traditional mode such as artificial cuttage, grafting, and these modes are received
It is limited to the factors such as scion growth conditions, operating technology level and grafting season and causes it to expand numerous limited amount, in addition
Wild ornamental resources are very deficient, therefore in order to protect in maximum efficiency and exploitation camellia azalea, it is necessary to find a kind of more high
The approach of effect, such as Plant Tissue Breeding.At present it is numerous research respectively with the flower pesticide of camellia azalea, newly take out tender tip, petiole and
Blade has carried out the induction of callus for explant, and obtains callus induction, but be showed no adventitious bud differentiation and
The report of plant regeneration.
The content of the invention
For the problem that prior art is present, a kind of camellia azalea blade callus is provided it is an object of the invention to design
The technical scheme of the method for building up of regeneration system.
The method for building up of described camellia azalea Callus of Leaf regenerating system, it is characterised in that including following step
Suddenly：
1）The acquisition of camellia azalea aseptic seedling
A, the selection of explant and pretreatment
Mid-March, clip camellia azalea tender tip stem section is standby after being disinfected after removal blade；
B, stem section Initial culture and proliferation of propagation
The stem section of sanitized is cut to the stem section with least one axillary bud, stem section training Initial culture base is inoculated in
Middle culture, culture grows the tender shoots of 2~4cm to stem section, then is cut into single bud, is inoculated in stem section squamous subculture
Cultivated in base, culture obtains the budling that grows thickly of 3~4cm shapings, the every 50 ± 5d of the budling that grows thickly carries out a squamous subculture, obtain big
Amount camellia azalea aseptic seedling；
Described stem section training Initial culture base is MS+25~35g/L sucrose+6~7g/L+0.1~0.3mg/L of agar NAA
+ 4~6mg/L6-BA+0.04~0.06%PVP+45~55mL/L CW；
Described stem section subculture medium is MS+35~45g/L sucrose+6~7g/L+0.1~0.3mg/L of agar NAA+1
~3mg/L6-BA+1~3mg/L2-ip+45~55mL/L CW；
2）The callus induction and squamous subculture of blade
The good shaping aseptic seedling of growth conditions is chosen, blade is cut, is inoculated in calli induction media, culture 35~
Go out to grow faint yellow, fine grained callus by paddle cutout and vein after 45d to be forwarded in callus subculture medium, often
45 ± 5d transfers once, carries out squamous subculture；
Described calli induction media is+0.4~0.6mg/L2,4-D+ of MS+25~35g/L sucrose+6~7g/L agar
45~55mL/L CW；
Described callus subculture medium is+0.4~0.6mg/L of MS+25~35g/L sucrose+6~7g/L agar
TDZ+90~110ml/L CW；
3）The differentiation of callus adventitious bud and plant regeneration
By after 2~3 squamous subcultures, choosing yellow green, fine grained callus, transfer in adventitious buds differentiation culture medium
In, 35 ± 5d is cultivated, obtain being molded budling；
Described adventitious buds differentiation culture medium is+0.1~0.15mg/L of MS+25~35g/L sucrose+6~7g/L agar
NAA+8~12mg/L6-BA+90~110mL/L CW+450~550 mL/L proline+90~110mL/L glutamine；
4）Regeneration plant is taken root and rooting culture
Budling to be formed is long to 2~3cm, can obtain what is taken root using " filter-bridge rooting method ", after dim light culture 50d
Regeneration plant.
The method for building up of described camellia azalea Callus of Leaf regenerating system, it is characterised in that described step 1）
The alcohol surface sterilization of volume ratio 75% 30 seconds is used after middle camellia azalea tender tip stem section removal blade, quality volume is then used by
Than the HgCl for 0.1%2Stem section is layered on the plate containing filter paper by immersion 8 minutes, aseptic water washing 5~6 times, cleaning after finishing
In dry up moisture.
The method for building up of described camellia azalea Callus of Leaf regenerating system, it is characterised in that described step 1）
Middle Initial culture condition is to cultivate 7d under the conditions of light intensity 100Lx, after move to and cultivate under the conditions of 2500~3000Lx of light intensity.
The method for building up of described camellia azalea Callus of Leaf regenerating system, it is characterised in that described step 1）
Middle squamous subculture condition is 2500~3000Lx of illumination, light application time is 12h/d, cultivation temperature is 25 ± 2 DEG C.
The method for building up of described camellia azalea Callus of Leaf regenerating system, it is characterised in that described step 2）
With 3）Middle condition of culture is 2500~3000Lx of illumination, light application time is 12h/d, cultivation temperature is 25 ± 2 DEG C.
The method for building up of described camellia azalea Callus of Leaf regenerating system, it is characterised in that described step 4）
Middle filter-bridge rooting method is inoculated to taking root first to carry out concentration 500mg/LIBA solution immersion 60min to shaping budling base portion
In inducing culture.
The method for building up of described camellia azalea Callus of Leaf regenerating system, it is characterised in that described step 4）
Middle rooting induction culture medium is MV+15~25g/L sucrose.
Invention selection large-scale breeding first obtains aseptic seedling, then therefrom clip aseptic blade carries out callus as explant
Tissue induction work, in such During Callus Induction, the selection of its explant is not just by sampling season and sterilization method
Influence；Aseptic stem section can act also as explant simultaneously carries out other experiment works.
The present invention is in the induction of callus and squamous subculture to callus inducing medium and callus subculture
Medium optimization, the culture of the culture medium matched by both specific PGRs can obtain yellow green, be rich in water profit gloss and be in
Fine grain callus, this kind of callus growth be vigorous, with certain adventitious buds differentiation potentiality, beneficial to callus not
The differentiation of normal bud.
The present invention uses " filter-bridge rooting method ", and the regeneration plant taken root can be obtained after dim light culture 50d, overcomes woody
The difficulty of flower plant rooting induction.
The present invention carries out callus induction using camellia azalea tests for sterility, and state tune is being carried out to callus
On the basis of whole, adventitious buds differentiation is carried out using the MS culture mediums of specific hormone combination, finally utilize " filter-bridge rooting method " solution
Certainly camellia is difficult to the technical barrier taken root, so as to realize the induction of camellia azalea Callus of Leaf and the regeneration of plant, culture
Cycle is short, breeding coefficient is high, is that camellia molecular breeding is laid a good foundation.
Specific embodiment
Further illustrate the present invention with reference to embodiments.
Embodiment 1：
The acquisition of 1 camellia azalea aseptic seedling
（1）The selection of explant and pretreatment
Mid-March, clip camellia azalea tender tip stem section, with the alcohol surface sterilization 30 of volume ratio 75% after removal blade
Second, it is then used by the HgCl that mass volume ratio is 0.1%2Immersion 8 minutes, aseptic water washing 5~6 times is cleaned stem after finishing
Section is layered on and moisture is dried up in the plate containing filter paper；It is standby；
（2）Stem section Initial culture and proliferation of propagation
The stem section of sanitized is cut to the stem section with least one axillary bud, stem section training Initial culture base MS+ is inoculated in
30g/L sucrose+6.5g/L agar+0.2mg/L（NAA）+5.0mg/L6-BA+0.05%PVP（Account for MS gross weights）+50mL/L
（CW）（Coconut Juice, coconut water）In, being positioned under the conditions of light intensity 100Lx carries out dim light culture 7d, then moves to light intensity
Under the conditions of 2500~3000Lx, stem section grows the tender shoots of 3cm or so after culture 50d, then is cut into single bud, is inoculated in stem section
Subculture medium MS+40g/L sucrose+6.5g/L agar+0.2mg/L（NAA）+2.0mg/L6-BA+2.0mg/L2-ip+50mL/L
（CW）（Coconut Juice, coconut water）In, 2500~3000Lx of illumination, light application time are 12h/d, cultivation temperature is 25 ± 2 DEG C
（Following condition of culture is all identical in the case of unaccounted）Under the conditions of carry out fast breeding expand it is numerous, culture 70d after obtain 3
The budling that grows thickly of~4cm shapings, the every 50 ± 5d of the budling that grows thickly carries out a squamous subculture, obtains a large amount of camellia azaleas aseptic
Seedling.
The callus induction and squamous subculture of 2 blades
The good shaping aseptic seedling of growth conditions is chosen, blade is cut, calli induction media MS+30g/L sugarcanes are inoculated in
Sugar+6.5g/L agar+0.5mg/L2,4-D+50mL/L（CW）（Coconut Juice, coconut water）In, blade is cut after culture 40d
Mouth and vein go out to grow faint yellow, fine grained callus and are forwarded to callus subculture medium MS+30g/L sucrose+6.5g/
The mg/L of L agar+0.5（TDZ）+100ml/L（CW）（Coconut Juice, coconut water）In, every 45 ± 5d switchings are once.Through excessive
After secondary squamous subculture, flaxen callus gradually becomes yellow green callus, and surface is rich in water profit gloss, in fine grained
Shape.Callus induction rate is 94.05%.
The differentiation of 3 callus adventitious buds and plant regeneration
By after 2~3 squamous subcultures, choosing yellow green, fine grained callus, transfer in adventitious buds differentiation culture medium
MS+30g/L sucrose+6.5g/L agar+0.125mg/L（NAA）+10mg/L6-BA+100 mL/L（CW）+ 500 mL/L proline
In+100 mL/L glutamine, after 35 ± 5d of culture, fine grained callus differentiates light green bud point；Subsequent light green bud
Point is gradually molded, and some adventitious bud base portions also grow with the radicula of white fluff.Differentiation ration of adventitious buds 87.12%.
4 regeneration plants are taken root and rooting culture
Budling to be formed is long to 3cm or so, using " filter-bridge rooting method ", first carries out high concentration to its base portion
(500mg/L) IBA solution soaks 60min, inoculates into the culture of rootage bottle of MV+20 g/L sucrose, after dim light culture 50d
Young root is successfully induced, opposite radical bud bar carries out rooting culture after main young root degree of lignification is higher, visible after 7d successfully to move
The seedling terminal bud germination of cultivation, grows brownish red tender leaf.
MS minimal mediums component list in the present invention：
MV fluid nutrient mediums component list in the present invention
Stem section training Initial culture base uses MS+25g/L sucrose+6g/L agar+0.1mg/L in the embodiment（NAA）+4mg/
L6-BA+0.04%PVP+45mL/L（CW）, or using MS+35g/L sucrose+7g/L agar+0.3mg/L（NAA）+6mg/L6-BA+
0.06%PVP+55mL/L（CW）；
Stem section subculture medium uses MS+35g/L sucrose+6g/L agar+0.1mg/L（NAA）+1mg/L6-BA+1mg/
L2-ip+45mL/L（CW）, or using MS+45g/L sucrose+7g/L agar+0.3mg/L（NAA）+3mg/L6-BA+3mg/L2-ip
+55mL/L（CW）；
Calli induction media uses MS+25g/L sucrose+6g/L agar+0.4mg/L2,4-D+45mL/L（CW）, or adopt
With MS+35g/L sucrose+7g/L agar+0.6mg/L2,4-D+55mL/L（CW）；
Callus subculture medium uses MS+25g/L sucrose+6g/L agar+0.4mg/L（TDZ）+90ml/L（CW）,
Or using MS+35g/L sucrose+7g/L agar+0.6mg/L（TDZ）+110ml/L（CW）；
Adventitious buds differentiation culture medium uses MS+25g/L sucrose+6g/L agar+0.1mg/L（NAA）+8mg/L6-BA+
90mL/L（CW）+ 450 mL/L proline+90mL/L glutamine, or using MS+35g/L sucrose+7g/L agar+0.15mg/L
（NAA）+12mg/L6-BA+110mL/L（CW）+ 550 mL/L proline+110mL/L glutamine, finally can also reach and real
Apply the identical technique effect of example 1.
Claims (5)
1. the method for building up of camellia azalea Callus of Leaf regenerating system, it is characterised in that comprise the following steps：
1）The acquisition of camellia azalea aseptic seedling
A, the selection of explant and pretreatment
Mid-March, clip camellia azalea tender tip stem section is standby after being disinfected after removal blade；
B, stem section Initial culture and proliferation of propagation
The stem section of sanitized is cut to the stem section with least one axillary bud, stem section training Initial culture base is inoculated in
Middle culture, culture grows the tender shoots of 2~4cm to stem section, then is cut into single bud, is inoculated in stem section subculture medium
Culture, culture obtains the budling that grows thickly of 3~4cm shapings, and the every 50 ± 5d of the budling that grows thickly carries out a squamous subculture, obtains a large amount of Du
Cuckoo Camellia aseptic seedling；
Described stem section training Initial culture base be MS+25~35g/L sucrose+6~7g/L+0.1~0.3mg/L of agar NAA+4~
6mg/L6-BA+0.04~0.06%PVP+45~55mL/L CW；
Described stem section subculture medium be MS+35~45g/L sucrose+6~7g/L+0.1~0.3mg/L of agar NAA+1~
3mg/L 6-BA+1~3mg/L2-ip+45~55mL/L CW；
Initial culture condition is to cultivate 7d under the conditions of light intensity 100Lx, after move to and cultivate under the conditions of 2500~3000Lx of light intensity；Subculture
Condition of culture is 2500~3000Lx of illumination, light application time is 12h/d, cultivation temperature is 25 ± 2 DEG C；
2）The callus induction and squamous subculture of blade
The good shaping aseptic seedling of growth conditions is chosen, blade is cut, is inoculated in calli induction media, cultivate 35~45d
Faint yellow, the fine grained callus that will be grown at paddle cutout and vein afterwards are forwarded in callus subculture medium, often
45 ± 5d transfers once, carries out squamous subculture；
Described calli induction media be+0.4~0.6mg/L2,4-D+45 of MS+25~35g/L sucrose+6~7g/L agar~
55mL/L CW；
Described callus subculture medium is MS+25~35g/L sucrose+6~7g/L+0.4~0.6mg/L of agar TDZ+
90~110ml/L CW；
3）The differentiation of callus adventitious bud and plant regeneration
By after 2~3 squamous subcultures, choosing yellow green, fine grained callus, transfer in adventitious buds differentiation culture medium,
35 ± 5d of culture, obtains being molded budling；
Described adventitious buds differentiation culture medium is MS+25~35g/L sucrose+6~7g/L+0.1~0.15mg/L of agar NAA+8
~12mg/L6-BA+90~110mL/L CW+450~550 mL/L proline+90~110mL/L glutamine；
4）Regeneration plant is taken root and rooting culture
Budling to be formed is long to 2~3cm, using " filter-bridge rooting method ", the regeneration taken root can be obtained after dim light culture 50d
Plant.
2. the method for building up of camellia azalea Callus of Leaf regenerating system as claimed in claim 1, it is characterised in that institute
The step of stating 1）The alcohol surface sterilization of volume ratio 75% 30 seconds is used after middle camellia azalea tender tip stem section removal blade, is then made
With the HgCl that mass volume ratio is 0.1%2Immersion 8 minutes, aseptic water washing 5~6 times, cleaning finish after by stem section be layered on containing
Moisture is dried up in the plate of filter paper.
3. the method for building up of camellia azalea Callus of Leaf regenerating system as claimed in claim 1, it is characterised in that institute
The step of stating 2）With 3）Middle condition of culture is 2500~3000Lx of illumination, light application time is 12h/d, cultivation temperature is 25 ± 2 DEG C.
4. the method for building up of camellia azalea Callus of Leaf regenerating system as claimed in claim 1, it is characterised in that institute
The step of stating 4）Middle filter-bridge rooting method soaks 60min first to carry out concentration 500mg/LIBA solution to shaping budling base portion, then
It is seeded in rooting induction culture medium.
5. the method for building up of camellia azalea Callus of Leaf regenerating system as claimed in claim 4, it is characterised in that institute
The step of stating 4）Middle rooting induction culture medium is MV+15~25g/L sucrose.
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| CN105104194B (en) * | 2015-08-20 | 2017-06-23 | 中央民族大学 | Promote Anji white tea callus proliferation and the method for improving wherein polyphenol content |
| CN105830917B (en) * | 2016-03-24 | 2017-11-17 | 江苏省农业科学院 | The method of Chinese azalea leaf regeneration plant |
| CN107135948B (en) * | 2017-06-02 | 2019-07-12 | 广西壮族自治区南宁良凤江国家森林公园 | A kind of method that Camellia nitidissima spray cultivates tissue-cultured seedling under sunlight conditions |
| CN109275569A (en) * | 2018-11-27 | 2019-01-29 | 广西玉林市华睿茶业有限公司 | A kind of method for building up of tealeaves regenerating system |
| CN110604057B (en) * | 2019-10-17 | 2021-04-06 | 中国林业科学研究院亚热带林业研究所 | Regeneration method of camellia oleifera regeneration system |
| CN110604060B (en) * | 2019-10-17 | 2021-05-14 | 中国林业科学研究院亚热带林业研究所 | Method for obtaining regeneration plant by in vitro culture of Zhejiang red camellia anther |
| CN111972292A (en) * | 2020-09-08 | 2020-11-24 | 陕西理工大学 | Purple bud tea cultivation method |
| CN117546778B (en) * | 2023-12-15 | 2024-08-27 | 安徽农业大学 | Tea tree leaf adventitious bud induction method |
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