CN104221865B - A kind of method of rubus coreanus tissue culture and rapid proliferation - Google Patents
A kind of method of rubus coreanus tissue culture and rapid proliferation Download PDFInfo
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Abstract
The invention discloses a kind of method of rubus coreanus tissue culture and rapid proliferation, comprise the following steps successively: the sterilization of (1) rubus coreanus stem section and inoculation: get the new stem of the rubus coreanus extracted out then, the moderate lignification stem section being cut into band axillalry bud is inoculated; (2) induction of Multiple Buds; (3) propagation of indefinite bud; (4) culture of rootage of regeneration plant; (5) acclimatization and transplants.The medium of heterogeneity and proportioning is have employed in above-mentioned steps.Adopt method of the present invention, rubus coreanus plant can be obtained rapidly, be beneficial to industrialization and produce.Adopt the inventive method, the adventitious bud induction frequency of rubus coreanus Multiple Buds is up to 92.5%, and adventitious bud proliferation multiple can reach 7.2, and plantlet of transplant survival rate can reach more than 95%.
Description
Technical field
The invention belongs to technical field of plant culture, be specifically related to the method for rubus coreanus tissue culture and rapid proliferation.
Background technology
Rubus coreanus (
rubuscoreanusmiq.), have another name called Radix Rubi Multibracteati, the black husky certain kind of berries, vegetable seeds bubble, later dragon, two is careless, two is busy, for rose family rubus defoliation small arbor, be distributed in the provinces such as Shaanxi, Gansu, Jiangsu, Anhui, Zhejiang, Jiangxi, Henan, Hubei, Sichuan and Guizhou.Be used as medicine with fruit, leaf, root and stem, rattan the given birth to adventive root that lands.Fruit is warm in nature, and taste is sweet, sour, and tool invigorates the kidney and stops nocturnal emission effect, cures mainly impotence, seminal emission, the enuresis, is also used as medicine as raspberry; Root, adventive root are cool in nature, bitter, and tool regulating menstruation and activating blood, hemostasis and pain-relieving effect, cure mainly traumatic injury, fracture, irregular menstruation and traumatism and bleeding.Leaf is cool in nature, bitter, puckery, tool dispelling pathogenic wind for improving eyesight, effect of removing dampness and detoxicating, cures mainly that the eye of wind is shed tears, arthralgia due to wind-dampness and dog bite.Except medicinal, fruit is direct-edible or make wine.Some kinds belonged to together at present have been reported, but the tissue cultures of rubus coreanus and Fast-propagation there is not been reported.
The tissue-culturing rapid propagation belonging to various plants together has been reported, but the report of R.sumatranus Miq tissue culture and rapid proliferation there is not yet.The minimal medium overwhelming majority that rubus adopts is MS medium or modified MS medium, is seldom other medium, and as Rubus laciniatus employing is improvement NN69 medium.The explant adopted is mainly stem section, and by the mode that direct induced bundle is sprouted or first evoked callus breaks up again, and the main employing such as blade, petiole is direct evoking adventive bud.The hormone combinations that the direct induced bundle of stem section is sprouted is generally the combination of 6-BA and NAA or the combination of 6-BA and IBA, also has the combination induction stem section of 6-BA and NAA to produce callus and the report of differentiation (by raspberry); The combination of the combination of what blade directly produced that indefinite bud mainly adopts is BA and IAA, the combination of BA and NAA, the combination of TDZ and NAA and ZT and NAA.Main minimal medium of taking root is 1/2 concentration, and that mainly adopt is NAA, adopts IBA in addition, also has and adopts NAA and IAA used in combination, the report of regenerated root under also having external use 0.5g/LIBA to soak.
Summary of the invention
Technical problem to be solved by this invention is: the deficiency existed for prior art, provides a kind of survival rate high, is beneficial to the method realizing the rubus coreanus tissue culture and rapid proliferation that industrialization is produced.
For realizing the object of the present invention, be achieved by the following technical solutions: a kind of method of rubus coreanus tissue culture and rapid proliferation, comprises the following steps successively:
(1) sterilization of rubus coreanus stem section and inoculation
Get the new stem of the rubus coreanus extracted out then, be cut into the stem section of band axillalry bud, be divided into slightly according to degree of lignification, moderate and height, first softly scrub under running water with brush, add appropriate liquid detergent solution and soak 2-4min, after running water 0.5h, in super-clean bench, stem section is proceeded in the alcoholic solution of 70% and soak 2min, with aseptic water washing 3 times, immersing dropping has 5 Tween-80s, and mass fraction is the HgCl of 0.1%
2soaking disinfection 11-17min in solution, then 3 times are fully embathed with sterile water, be inoculated into normal polar orientation and be added with TDZ0.5mg/L, in the WPM minimal medium of 6-BA2.0mg/L, sucrose addition in this medium is 30g/L, agar addition is 7.0g/L, pH is 5.8-6.0, and medium is autoclave sterilization 20min at 121 DEG C in advance.The medium connecting explant is first placed in dark 10h, subsequently illumination cultivation; Condition of culture is: cultivation temperature is 25 ± 1 DEG C, proceeds in dark and cultivate 10h after every illumination cultivation 14h; Intensity of illumination 30-40 μm of ol/ (m
2s);
(2) induction of Multiple Buds
Choose the moderate lignification stem section in new stem, first softly scrub under running water with brush, add appropriate liquid detergent solution and soak 2-4min, after running water 0.5h, in super-clean bench, stem section is proceeded in the alcoholic solution of 70% and soak 2min, with aseptic water washing 3 times, immerse the 0.1%HgCl dripping and have 5 Tween-80s
2soaking disinfection 13-14min in solution, then 3 times are fully embathed with sterile water, be inoculated into normal polar orientation and be added with TDZ0-2.0mg/L, in the WPM minimal medium of 6-BA0-5.0mg/L, IAA0-5.0mg/L, CH0-1.0mg/L, sucrose addition in this medium is 30g/L, agar addition is 7g/L, pH is 5.8-6.0, autoclave sterilization 20min at medium is placed in 121 DEG C.The medium connecting explant is first placed in dark 10h, subsequently illumination cultivation, and condition of culture is: cultivation temperature is (25 ± 1) DEG C, and light application time is that 14h light/10h is dark, intensity of illumination 30-40 μm of ol/ (m
2s);
(3) propagation of indefinite bud
The indefinite bud clump that previous step induction obtains is cut into the sprout tuber of 2-3 strain indefinite bud, be inoculated into and be added with TDZ0-2.0mg/L, 6-BA0-5.0mg/L, IAA0-5.0mg/L, cultivate in the WPM minimal medium of CH0-1.0mg/L, condition of culture is: cultivation temperature is (25 ± 1) DEG C, and light application time is that 14h light/10h is dark, intensity of illumination 30-40 μm of ol/ (m
2s);
(4) culture of rootage of regeneration plant
Cut blade light green, grow vigorous and that 2 ~ 3cm is high indefinite bud, be inserted into 12.5%-100%WPM, NAA0-1.0mg/L, in the root media of IAA0-2.0mg/L, AC0-2.0mg, the sucrose added in medium is 10-50g/L, agar addition is 7.0g/L, pH5.8-6.0;
(5) acclimatization and transplants
The height of seedling of plantlet in vitro to be taken root grows to more than 5cm, when root length is more than 5cm, unclamps the bottle cap of tissue culture bottle, in bottle, injects a small amount of clear water, prevent medium dry and cracked, but first do not remove bottle cap, the later half bottle cap of moving away of 12h, after 12h, remove bottle cap, allow light directly shine regenerated plant culture 2d.Then carefully medium is smashed to pieces, take out regeneration plant, carefully residual agar is cleaned with water, plant is colonizated in perlite, peat weighs is than for 1:(1-5) mixed-matrix in, water permeable and cover with heat and moisture preserving with transparent sealed polyethylene plastic, indoor temperature controls between 24-28 DEG C.
Preferably: in described step (1), the stem section of inoculation is lignification moderate stem section, and disinfecting time is 13-14min.
Preferably: the WPM medium in described step (2) is added with TDZ0.5mg/L, 6-BA2.0mg/L, IAA0.5mg/L, CH0.1-0.2mg/L.
Preferably: in described step (3), WPM medium is added with TDZ0.3mg/L, 6-BA1.0mg/L, IAA1.0mg/L, CH0.1-0.2mg/L.
Preferably: the root media in described step (4) is 50-100%WPM, NAA0.1mg/L, IAA0.2mg/L, AC0.1-0.2mg/L.
Preferably: the perlite of mixed-matrix in described step (5), peat weighs is than being 1:5.
Compared with prior art, the invention has the beneficial effects as follows: adopt method of the present invention, rubus coreanus plant can be obtained rapidly, be beneficial to industrialization and produce.Adopt the inventive method, the adventitious bud induction frequency of rubus coreanus Multiple Buds is up to 92.5%, and adventitious bud proliferation multiple can reach 7.2, and plantlet of transplant survival rate can reach more than 95%.
Embodiment
embodiment 1
A method for rubus coreanus tissue culture and rapid proliferation, comprises the following steps successively:
(1) sterilization of rubus coreanus stem section and inoculation
Get the new stem of the rubus coreanus extracted out then, be cut into the stem section of band axillalry bud, choose the stem section that degree of lignification is moderate, first softly scrub under running water with toothbrush, add appropriate liquid detergent solution and soak 2-4min, after running water 0.5h, in super-clean bench, stem section is proceeded in the alcoholic solution of 70% and soak 2min, with aseptic water washing 3 times, immerse the 0.1%HgCl dripping and have 5 Tween-80s
2soaking disinfection 13-14min in solution, then 3 times are fully embathed with sterile water, be inoculated into normal polar orientation and be added with TDZ0.5mg/L, in the WPM minimal medium of 6-BA2.0mg/L, sucrose addition in this medium is 30g/L, agar addition is 7.0g/L, pH is 5.8-6.0, autoclave sterilization 20min at medium is placed in 121 DEG C.The medium connecting explant is first placed in dark 10h, subsequently illumination cultivation, and condition of culture is: cultivation temperature is (25 ± 1) DEG C, and light application time is that 14h light/10h is dark, intensity of illumination 30-40 μm of ol/ (m
2s).After 30d, statistics finds, pollution rate is lower than 10%, and explant survival rate is more than 80%.
Step (1) is preliminary experiment, induces in order to choose best Multiple Buds.
(2) induction of Multiple Buds
Choose the moderate lignification stem section in the new stem extracted out then, first softly scrub under running water with toothbrush, add appropriate liquid detergent solution and soak 2-4min, after running water 0.5h, in super-clean bench, stem section is proceeded in the alcoholic solution of 70% and soak 2min, with aseptic water washing 3 times, immerse the 0.1%HgCl dripping and have 5 Tween-80s
2soaking disinfection 13-14min in solution, then 3 times are fully embathed with sterile water, be inoculated into normal polar orientation and be added with TDZ0.5mg/L, in the WPM minimal medium of 6-BA2.0mg/L, IAA0.5mg/L, CH0.1-0.2mg/L, sucrose addition in this medium is 30g/L, agar addition is 7g/L, pH is 5.8-6.0, autoclave sterilization 20min at medium is placed in 121 DEG C.The medium connecting explant is first placed in dark 10h, subsequently illumination cultivation, and condition of culture is: cultivation temperature is (25 ± 1) DEG C, and light application time is that 14h light/10h is dark, intensity of illumination 30-40 μm of ol/ (m
2s).After 30d, statistics finds, adventitious bud induction frequency is 92.5%, and indefinite bud number is 5.4, and indefinite bud-leaf look dark green, and well-grown, plant height and stem are slightly moderate.
(3) propagation of indefinite bud
The indefinite bud clump that previous step induction obtains is cut into the sprout tuber of 2-3 strain indefinite bud, be inoculated into and be added with TDZ0.3mg/L, 6-BA1.0mg/L, IAA1.0mg/L, cultivate in the WPM minimal medium of CH0.1-0.2mg/L, condition of culture is: cultivation temperature is (25 ± 1) DEG C, and light application time is that 14h light/10h is dark, intensity of illumination 30-40 μm of ol/ (m
2s).After 30d, statistics finds, proliferation times is 7.2, and without vitrifying seedling, plant is sturdy, and growth is very fast.
(4) culture of rootage of regeneration plant
Cut blade light green, grow vigorous and that 2 ~ 3cm is high indefinite bud, be inserted into 50-100%WPM, NAA0.1mg/L, in the root media of IAA0.2mg/L, AC0.1-0.2mg/L, the sucrose added in medium is 10-50g/L, agar addition is 7.0g/L, pH5.8-6.0.During 30d, statistics finds, rooting rate is more than 95%, and radical 4.6, root is slightly moderate, is suitable for acclimatization and transplants.
Wherein 50-100%WPM refers to the concentration of WPM minimal medium in medium, and the concentration of other material is unaffected.50%WPM just refers to that all substances consumption in WPM minimal medium formula is original 1/2.
(5) acclimatization and transplants
The height of seedling of plantlet in vitro to be taken root grows to more than 5cm, when root length is more than 5cm, unclamps the bottle cap of tissue culture bottle, in bottle, injects a small amount of clear water, prevent medium dry and cracked, but first do not remove bottle cap, the later half bottle cap of moving away of 12h, after 12h, remove bottle cap, allow light directly shine regenerated plant culture 2d.Then carefully medium is smashed to pieces, take out regeneration plant, carefully residual agar is cleaned with water, plant is colonizated in perlite, peat weighs is than in the mixed-matrix for 1:5, water permeable and cover with heat and moisture preserving with transparent sealed polyethylene plastic, indoor temperature controls between 24-28 DEG C.Treat that young leaves grows, can open film and foliar spray keeps moistening, adapt to can transplant large Tanaka after 3-5d, transplanting survival rate can reach more than 95%.
Initialism implication involved in literary composition:
AC active carbon
6-BA6-benzyl aminoadenine
CH caseinhydrolysate
IAA heteroauxin
NAA methyl α-naphthyl acetate
TDZ Thidiazuron (Thidiazuron)
embodiment 2
The difference of the present embodiment and embodiment 1 is step (1): the sterilization of rubus coreanus stem section and inoculation: get the new stem of the rubus coreanus extracted out then, be cut into the stem section of band axillalry bud, be divided into slightly according to degree of lignification, moderate and height, first softly scrub under running water with toothbrush, add appropriate liquid detergent solution and soak 2-4min, after running water 0.5h, in super-clean bench, stem section is proceeded in the alcoholic solution of 70% and soak 2min, with aseptic water washing 3 times, immerse the 0.1%HgCl dripping and have 5 Tween-80s
2soaking disinfection 11-17min in solution, then 3 times are fully embathed with sterile water, be inoculated into normal polar orientation and be added with TDZ0.5mg/L, in the WPM minimal medium of 6-BA2.0mg/L, sucrose addition in this medium is 30g/L, agar addition is 7.0g/L, pH is 5.8-6.0, autoclave sterilization 20min at medium is placed in 121 DEG C.The medium connecting explant is first placed in dark 10h, subsequently illumination cultivation, and condition of culture is: cultivation temperature is (25 ± 1) DEG C, and light application time is that 14h light/10h is dark, intensity of illumination 30-40 μm of ol/ (m
2s).After 30d, statistics finds, pollution rate is lower than 10%, and explant survival rate is more than 80%, and wherein lignification moderate stem section > lignification slight stem section > lignification severe stem section, drips the 0.1%HgCl having 5 Tween-80s
2in solution, soaking disinfection 13-14min is best disinfecting time, has not only sterilized and has damaged explant completely but also not, pollution-free, explant survival rate 95%.
The explant that table 1 different parts is drawn materials with disinfecting time on pollution rate and the impact becoming 9 motility rates
| Different parts | Different disinfecting time (min) | Pollution rate (%) | Survival rate (%) |
| Stem section that lignification is slight | 11-12 | 8 | 75 |
| Lignification moderate stem section | 13-14 | 0 | 95 |
| Lignification severe stem section | 16-17 | 0 | 55 |
| Lignification moderate stem section | 16-17 | 0 | 70 |
embodiment 3
The difference of the present embodiment and embodiment 1 is step (2): the induction of Multiple Buds: choose the moderate lignification stem section in the new stem extracted out then, first softly scrub under running water with toothbrush, add appropriate liquid detergent solution and soak 2-4min, after running water 0.5h, in super-clean bench, stem section is proceeded in the alcoholic solution of 70% and soak 2min, with aseptic water washing 3 times, immerse the 0.1%HgCl dripping and have 5 Tween-80s
2soaking disinfection 13-14min in solution, then 3 times are fully embathed with sterile water, be inoculated into normal polar orientation and be added with TDZ0-2.0mg/L, in the WPM minimal medium of 6-BA0-5.0mg/L, IAA0-5.0mg/L, CH0-1.0mg/L, sucrose addition in this medium is 30g/L, agar addition is 7g/L, pH is 5.8-6.0, autoclave sterilization 20min at medium is placed in 121 DEG C.The medium connecting explant is first placed in dark 10h, subsequently illumination cultivation, and condition of culture is: cultivation temperature is (25 ± 1) DEG C, and light application time is that 14h light/10h is dark, intensity of illumination 30-40 μm of ol/ (m
2s).After 30d, statistics finds, adventitious bud induction frequency is between 35%-92.5%, and indefinite bud number is between 1.4-5.8, wherein with WPM, TDZ0.5mg/L, 6-BA2.0mg/L, IAA0.5mg/L, CH0.1-0.2mg/L best results, adventitious bud induction frequency is 92.5%, and indefinite bud number is 5.4, and indefinite bud-leaf look dark green, well-grown, plant height and stem are slightly moderate.Result also shows, 6-BA is more weak for the inducing effect of Multiple Buds, and TDZ is comparatively strong, and both combined effects are better; IAA there is no impact for the induction of indefinite bud and indefinite bud number, but can promote the growth of indefinite bud; Caseinhydrolysate slightly can promote the growth of indefinite bud, there is no impact to the induction of indefinite bud and the quantity of indefinite bud.The basic element of cell division (TDZ, 6-BA) and the growth hormone (IAA) of high concentration all easily cause vitrified generation.
Table 2 different disposal is on the impact of inducing clumping bud
| Medium | Adventitious bud induction frequency (%) | Indefinite bud number | Growing state |
| WPM，6-BA2.0，IAA 1.0，CH 0.1 | 35 | 1.4 | Indefinite bud-leaf look pale green, growth still can |
| WPM，TDZ 0.5，6-BA 2.0，IAA 0.5，CH 0.1 | 92.5 | 5.4 | Indefinite bud-leaf look dark green, well-grown |
| WPM，TDZ 2.0，6-BA 5.0，IAA 0.5，CH 0.1 | 92.5 | 5.8 | Indefinite bud vitrifying is serious, the water stain shape of blade |
| WPM，TDZ 0.5，CH 0.1 | 80 | 4.8 | Indefinite bud-leaf look pale green, growth still can |
embodiment 4
The difference of the present embodiment and embodiment 1 is step (3): the propagation of indefinite bud: induce the indefinite bud clump obtained to be cut into the sprout tuber of 2-3 strain indefinite bud step (2), be inoculated into and be added with TDZ0-2.0mg/L, 6-BA0-5.0mg/L, IAA0-5.0mg/L, cultivate in the WPM minimal medium of CH0-1.0mg/L, condition of culture is: cultivation temperature is (25 ± 1) DEG C, and light application time is that 14h light/10h is dark, intensity of illumination 30-40 μm of ol/ (m
2s).After 30d, statistics finds, in each medium, the proliferation times of indefinite bud is between 4.5-7.8, and high growth hormone and the basic element of cell division all easily produce vitrification phenomenon, and the medium of putting up the best performance is WPM, TDZ0.3mg/L, 6-BA1.0mg/L, IAA1.0mg/L, CH0.1-0.2mg/L, its proliferation times is 7.2, and without vitrifying seedling, plant is sturdy, and growth is very fast.Improve the elongation growth that IAA concentration can promote indefinite bud, but have nothing to do to propagation multiplying power, the generation of the rate of increase is mainly the basic element of cell division (6-BA, TDZ), and the basic element of cell division of high concentration or growth hormone all easily cause the vitrifying of indefinite bud.
Table 3 different disposal is on the impact of adventitious bud proliferation
| Medium | Proliferation times | Growing state |
| WPM，6-BA2.0，IAA 1.0，CH 0.1 | 4.5 | Indefinite bud-leaf look pale green, growth still can |
| WPM，TDZ 0.3，6-BA 1.0，IAA 1.0，CH 0.1 | 7.2 | Indefinite bud-leaf look dark green, well-grown |
| WPM，TDZ 2.0，6-BA 5.0，IAA 1.0，CH 0.1 | 7.8 | Indefinite bud vitrifying is serious, the water stain shape of blade |
embodiment 5
The difference of the present embodiment and embodiment 1 is step (4): the culture of rootage of regeneration plant: cut blade light green, grow vigorous and that 2 ~ 3cm is high indefinite bud, be inserted into 12.5%-100%WPM, NAA0-1.0mg/L, IAA0-2.0mg/L, in the root media of AC0-2.0mg, the sucrose added in medium is 10-50g/L, and agar addition is 7.0g/L, pH5.8-6.0.During 30d statistics find, rooting rate at 45-97.5%, rootage duration 6-12 the earliest, d radical is between 2.3-5.2, illustrate and more easily take root, the concentration of WPM minimal medium is for taking root substantially without impact, and the regeneration plant growth of taking root under only too low concentration is weak; NAA and IAA to tool mutual promoting action of taking root, and increases with concentration, and rooting rate improves, but the auxin concentration of high concentration easily causes that root is thicker to shorten, so short very difficult water suction and nutrition, and growth easily dies down; Active carbon is also little for the Rooting effect of indefinite bud, and have slight facilitation, but the active carbon of high concentration has inhibition, regeneration plant growth is weak; Reduce sucrose concentration for taking root substantially without impact, low concentration sucrose can hinder the growth potential of regeneration plant.Best is combined as 50-100%WPM, NAA0.1mg/L, IAA0.2mg/L, AC0.1-0.2mg/L, and sucrose concentration is 20-30g/L, and its rooting rate can reach more than 95%, and radical 4.6, root is slightly moderate, is suitable for acclimatization and transplants.
Table 4 different disposal is on the impact of inducing clumping bud
| Medium | The root of hair time the earliest | Rooting rate (%) | Radical | Growing state |
| 12.5%WPM，AC 0.1 | 12 | 45 | 2.3 | Root is thin, long, Ye Sehuang |
| 50%WPM，NAA 0.1，IAA 0.2，AC 0.1 | 6 | 97.5 | 4.6 | Root is thick and root is long moderate, and leaf look dark green |
| WPM，NAA 1.0，IAA 2.0，AC 0.1 | 8 | 90 | 5.2 | Root is thick, short, Ye Sehuang |
embodiment 6
The difference of the present embodiment and embodiment 1 is step (5): acclimatization and transplants: the height of seedling of plantlet in vitro to be taken root grows to more than 5cm, when root length is more than 5cm, unclamp the bottle cap of tissue culture bottle, in bottle, inject a small amount of clear water, prevent medium dry and cracked, but first do not remove bottle cap, the later half bottle cap of moving away of 12h, after 12h, remove bottle cap, allow light directly shine regenerated plant culture 2d.Then carefully medium is smashed to pieces, take out regeneration plant, carefully residual agar is cleaned with water, plant is colonizated in perlite, peat weighs is than for 1:(1-5) mixed-matrix in, water permeable and cover with heat and moisture preserving with transparent sealed polyethylene plastic, indoor temperature controls between 24-28 DEG C.Treat that young leaves grows, can open film and foliar spray keeps moistening, adapt to can transplant large Tanaka after 3-5d, transplanting survival rate can reach more than 95%.
The different cultivation matrix of table 5 is on the impact of transplanting survival rate
| Matrix | Survival rate (%) |
| Little silt | 59 |
| Perlite, peat (1:5) | 98 |
| Perlite, peat (5:1) | 84 |
Comparative example 1 to 6 is known, and embodiment 1 is most preferred embodiment, and each step of embodiment 1 all have employed the optimal culture condition described in embodiment 2 to 6, can obtain the highest rubus coreanus plant yield.
Claims (1)
1. a method for rubus coreanus tissue culture and rapid proliferation, is characterized in that comprising the following steps successively:
(1) sterilization of rubus coreanus stem section and inoculation
Get the new stem of the rubus coreanus extracted out then, be cut into the stem section of band axillalry bud, choose the stem section that degree of lignification is moderate, first softly scrub under running water with toothbrush, add appropriate liquid detergent solution and soak 2-4min, after running water 0.5h, in super-clean bench, stem section is proceeded in the alcoholic solution of 70% and soak 2min, with aseptic water washing 3 times, immerse the 0.1%HgCl dripping and have 5 Tween-80s
2soaking disinfection 13-14min in solution, then 3 times are fully embathed with sterile water, be inoculated into normal polar orientation and be added with TDZ0.5mg/L, in the WPM minimal medium of 6-BA2.0mg/L, sucrose addition in this medium is 30g/L, agar addition is 7.0g/L, pH is 5.8-6.0, autoclave sterilization 20min at medium is placed in 121 DEG C;
The medium connecting explant is first placed in dark 10h, subsequently illumination cultivation, and condition of culture is: cultivation temperature is (25 ± 1) DEG C, and light application time is that 14h light/10h is dark, intensity of illumination 30-40 μm of ol/ (m
2s);
After 30d, statistics finds, pollution rate is lower than 10%, and explant survival rate is more than 80%;
(2) induction of Multiple Buds
Choose the moderate lignification stem section in the new stem extracted out then, first softly scrub under running water with toothbrush, add appropriate liquid detergent solution and soak 2-4min, after running water 0.5h, in super-clean bench, stem section is proceeded in the alcoholic solution of 70% and soak 2min, with aseptic water washing 3 times, immerse the 0.1%HgCl dripping and have 5 Tween-80s
2soaking disinfection 13-14min in solution, then 3 times are fully embathed with sterile water, be inoculated into normal polar orientation and be added with TDZ0.5mg/L, in the WPM minimal medium of 6-BA2.0mg/L, IAA0.5mg/L, CH0.1-0.2mg/L, sucrose addition in this medium is 30g/L, agar addition is 7g/L, pH is 5.8-6.0, autoclave sterilization 20min at medium is placed in 121 DEG C;
The medium connecting explant is first placed in dark 10h, subsequently illumination cultivation, and condition of culture is: cultivation temperature is (25 ± 1) DEG C, and light application time is that 14h light/10h is dark, intensity of illumination 30-40 μm of ol/ (m
2s);
After 30d, statistics finds, adventitious bud induction frequency is 92.5%, and indefinite bud number is 5.4, and indefinite bud-leaf look dark green, well-grown, and plant height and stem are slightly moderate;
(3) propagation of indefinite bud
The indefinite bud clump that previous step induction obtains is cut into the sprout tuber of 2-3 strain indefinite bud, be inoculated into and be added with TDZ0.3mg/L, 6-BA1.0mg/L, IAA1.0mg/L, cultivate in the WPM minimal medium of CH0.1-0.2mg/L, condition of culture is: cultivation temperature is (25 ± 1) DEG C, and light application time is that 14h light/10h is dark, intensity of illumination 30-40 μm of ol/ (m
2s);
After 30d, statistics finds, proliferation times is 7.2, and without vitrifying seedling, plant is sturdy, and growth is very fast;
(4) culture of rootage of regeneration plant
Cut blade light green, grow vigorous and that 2 ~ 3cm is high indefinite bud, be inserted into 50-100%WPM, NAA0.1mg/L, in the root media of IAA0.2mg/L, AC0.1-0.2mg/L, the sucrose added in medium is 10-50g/L, agar addition is 7.0g/L, pH5.8-6.0;
During 30d statistics find, rooting rate more than 95%, radical 4.6, root is slightly moderate, is suitable for acclimatization and transplants;
Wherein 50-100%WPM refers to the concentration of WPM minimal medium in medium;
50%WPM just refers to that all substances consumption in WPM minimal medium formula is original 1/2;
(5) acclimatization and transplants
The height of seedling of plantlet in vitro to be taken root grows to more than 5cm, when root length is more than 5cm, unclamps the bottle cap of tissue culture bottle, in bottle, injects a small amount of clear water, prevent medium dry and cracked, but first do not remove bottle cap, the later half bottle cap of moving away of 12h, after 12h, remove bottle cap, allow light directly shine regenerated plant culture 2d;
Then carefully medium is smashed to pieces, take out regeneration plant, carefully residual agar is cleaned with water, plant is colonizated in perlite, peat weighs is than in the mixed-matrix for 1:5, water permeable and cover with heat and moisture preserving with transparent sealed polyethylene plastic, indoor temperature controls between 24-28 DEG C;
Treat that young leaves grows, open film and foliar spray keeps moistening, adapt to transplant large Tanaka after 3-5d.
Priority Applications (1)
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| 树莓和黑莓茎段组织培养研究初报;王丽玲等;《经济林研究》;20020930;第20卷(第3期);第24-25页 * |
| 树莓的组织培养技术;董丽芬等;《西北林学院学报》;20031231;第18卷(第2期);第42-43页 * |
| 树莓组织培养技术;崔雪艳等;《现代农业科技》;20091231(第12期);第42，45页 * |
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