CN102138526B - Tissue culture method of red-leaf sacred bamboo - Google Patents
Tissue culture method of red-leaf sacred bamboo Download PDFInfo
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Abstract
The invention discloses a tissue culture method of red-leaf sacred bamboo, which belongs to the technical field of plant cultivation. The method comprises the following steps of: (1) selecting a healthy plant with typical ornamental character performance to serve as a material from red-leaf sacred bamboo seedling plants, cutting new sprouts and removing pinnately compound leaves in the morning of a sunny day, and cleaning for later use; (2) soaking, disinfecting and flushing a cleaned branch with an alcohol solution and a mercury bichloride solution in sequence on an ultra-clean workbench; (3) stripping a stem apex in the branch, cutting a tip part and inoculating into a culture medium; (4) culturing for a certain period of time and transferring a plurality of indefinite buds formed by differentiation and proliferation of the stem apex into an enrichment medium for culturing; and (5) culturing for a certain period of time, cutting immature stems growing on the indefinite buds and culturing on a rooting medium to form a robust tissue culture rooted seedling. The tissue culture method has the advantages of high survival rate, high reproduction speed, multiplication coefficient of up to 7.3 and rooting rate of up to 87 percent. The obtained red-leaf sacred bamboo has high quality and wide market prospect.
Description
Technical field
The invention belongs to field of plant growing technology, be specially the method for tissue culture of red autumnal leaves heavenly bamboo.
Background technology
The red autumnal leaves heavenly bamboo is Berberidaceae Berberidaceae Nandina
NandinaCultivation new varieties, be that seed selection is out from the heavenly bamboo cultivated population in Senhe Seed Industry Co., Ltd., Zhejiang Pror..The red autumnal leaves heavenly bamboo is evergreen shrubs, and the high approximately 80cm of plant is grown thickly and few branch, is thick with leaves tree-like compactness; Internode is ultrashort, only have the about 5cm of length of 1/10,10 stipes of common heavenly bamboo, and common heavenly bamboo one is saved 8-10cm; Blade is two times three leaflet compound leaves, the blunt point of blade tip, base portion wedge shape, full edge, thin keratin, long 3-5cm, wide 1.5-2cm, common petiole 1.5-3cm.Red autumnal leaves heavenly bamboo happiness half is shady, also can normal growth under the high light, and happiness temperature climate, fertile, moistening well-drained soil; Winter direct sunlight to blade all redden, slightly meet cold air autumn, the leaf look just transfers cerise to by green, continues to the early spring in next year.
Red autumnal leaves heavenly bamboo whole strain leaf in winter look presents bright-coloured Chinese red, the implied meaning that tool is flourishing; Spring, summer, three season of autumn the dark green leaf color look, harmonious comfortable.Be good sight leaf ground quilt and color lump seeds, appreciation effect is eye-catching.Be suitable for the East China and use, the outdoor cropping seeds of this zone ornamental in winter are fewer, winter hardy tree species beautiful in colour still less, the red autumnal leaves heavenly bamboo is desirable new selection, the market development potential is huge.Conventional cuttage seedling raising method, the breeding cycle is long, reproduction rate is extremely low, because plant type compactness, stipes cripetura, branches and leaves are intensive, available cuttage stem section is very limited, propagation technique becomes the huge obstacle of heavenly bamboo extensive use.At present, domestic report to heavenly bamboo Regenerated Plantlets aspect seldom, the training of red autumnal leaves heavenly bamboo group has no report, has limited so to a certain extent the spread of red autumnal leaves heavenly bamboos.
Summary of the invention
The present invention is directed to problems of the prior art, the object of the invention is to design the technical scheme of the method for tissue culture that a kind of red autumnal leaves heavenly bamboo is provided, this cultural method survival rate is high, reproduction speed is fast.
The method for tissue culture of described red autumnal leaves heavenly bamboo is characterized in that may further comprise the steps:
1) from red autumnal leaves heavenly bamboo seedling, choose health, fancy points shows typical plant as material; In the morning of fine day, the clip shoot is removed winglike compound leaf, cleans for subsequent use;
2) on superclean bench, the branch that cleans up is also washed with alcoholic solution, mercuric chloride solution soaking disinfection successively;
3) strip out the interior stem apex of branch, downcut to be placed in the culturing room in the first end parts access medium and cultivate;
4) cultivate a period of time after, the stem apex differentiation and proliferation forms some indefinite buds, indefinite bud is transferred cultivate in the proliferated culture medium;
5) after cultivation a period of time, with the tender stem cutting-out of indefinite bud growth formation, after root media was cultivated, the healthy and strong group of formation was trained the seedling of taking root.
The method for tissue culture of described red autumnal leaves heavenly bamboo is characterized in that in the step 1): gather and spray 700 times of-900 times of carbendazim every the whole strain of 7-10d by the previous moon of newborn branch; Branch after the collection is wiped out winglike compound leaf, and running water is rinsed well, and liquid detergent solution soaks 5-10min, and is for subsequent use with running water flushing 0.3-0.6h again.
The method for tissue culture of described red autumnal leaves heavenly bamboo, it is characterized in that step 2) in: alcohol concentration is 70%-80%, soak time is 20-30s, outwell behind the alcohol aseptic water washing 4-5 time, then add the 0.05%-0.2% mercuric chloride solution, soak 8-10 min, constantly shake therebetween, outwell mercuric chloride solution aseptic water washing 7-8 time.
The method for tissue culture of described red autumnal leaves heavenly bamboo is characterized in that in the step 3): strip stem apex with sterile razor blade after the branch sterilization, downcutting tip length is the part of 3-6mm, in the inductive differentiation medium that access prepares; The prescription of inductive differentiation medium is to add 0.5-1.5mgL in the MS medium
-16-benzyl aminopurine, 0.05-0.15 mgL
-1Indolebutyric acid, 2-5% sucrose and 0.5-0.8% agar are made, and are that 5.8-6.0 ends to medium pH.
The method for tissue culture of described red autumnal leaves heavenly bamboo is characterized in that in the step 4): cultivation temperature is 25 ± 2 ℃ in the culturing room, light application time 11-13h/d, light intensity 1500-2000Lx; Stem apex access medium 60d-65d, stem apex is differentiated to form sprouting, and grows young shoot, and the stem section of being longer than 0.4-0.6 cm children shoot and cutting into 0.5-1.0cm is transferred into proliferated culture medium, and proliferation culture medium formula is to add 0.5-1.5 mgL in the MS medium
-16-benzyl aminopurine, 0.1-0.2 mgL
-1Indolebutyric acid, 2-5% sucrose and 0.5-0.8% agar are made, and are that 5.8-6.0 ends to medium pH.
The method for tissue culture of described red autumnal leaves heavenly bamboo, it is characterized in that in the step 5): the stem section is cultivated 60d-65d at proliferated culture medium, and stem section propagation forms indefinite bud, and pumping goes out tender stem, the tender stem that the length that stem section differential growth is gone out surpasses 2-4 cm downcuts, the access root media; The prescription of root media is to add 0.1-0.5 mgL in the 1/2MS medium
-1Indolebutyric acid, 0.05-0.15 mgL
-1The sucrose of methyl α-naphthyl acetate, 1-3%, the agar of 0.5-0.8% and 0.1-0.3 gL
-1Active carbon is made, and is that 6.3-6.7 ends to medium pH; Behind the 60-75d, form the healthy and strong seedling of taking root after cultivating.
Described MS medium, liquid detergent can be by directly buying on the market.Described BA is 6-benzyl aminopurine, and IBA is indolebutyric acid, and NAA is methyl α-naphthyl acetate.
The method for tissue culture of above-mentioned red autumnal leaves heavenly bamboo, survival rate is high, reproduction speed is fast, and growth coefficient reaches 7.3, and rooting rate reaches 87%, and the red autumnal leaves heavenly bamboo that obtains is best in quality, has wide market prospects.
The percentage composition that relates in the present specification unless otherwise indicated all refers to weight percentage.
Embodiment
Now in conjunction with embodiments of the invention, the invention will be further described.
Embodiment 1:
1) from red autumnal leaves heavenly bamboo seedling, choose health, fancy points shows typical plant as material, gather and spray 800 times of carbendazim every the whole strain of 8d by the previous moon of newborn branch, clip shoot in the morning at annual 3-5 fine day in the month, remove winglike compound leaf, wash surface dirt with running water, then soak 7min with liquid detergent solution, for subsequent use with running water flushing 0.5h again;
2) on superclean bench, the branch that cleans up is soaked 25s with 75% alcoholic solution, outwell behind the alcohol with aseptic water washing 5 times, add again 0.1% mercuric chloride solution and soak 9min, constantly shake therebetween, then outwell mercuric chloride solution for subsequent use with aseptic water washing 8 times;
3) blade with sterilization strips out stem apex on the branch, and cutting length is 5mm stem apex tip, places in the culturing room in the inductive differentiation medium that access prepares and cultivates; Culture medium prescription is to add 1.0 mgL in the MS medium
-1BA, 0.1 mgL
-1IBA, 3% sucrose and 0.7% agar are made, and are 6.0 to end to medium pH;
4) cultivation temperature is 25 ℃ in the culturing room, light application time 12h/d, light intensity 1500Lx; Stem apex is cultivated 60d at inductive differentiation medium, is differentiated to form some sproutings, and pumping children shoot, greater than the young shoot cutting-out of 0.5cm, is divided into the stem section of 0.5cm with wherein, and switching enters in the proliferated culture medium to cultivate; Proliferation culture medium formula is to add 1.5 mgL in the MS medium
-1BA, 0.1 mgL
-1IBA, 3% sucrose and 0.7% agar are made, and are 6.0 to end to medium pH;
5) the stem section is cultivated 60d at proliferated culture medium, and stem section propagation forms indefinite bud, and pumping goes out tender stem, and the tender stem that the length that stem section differential growth is gone out surpasses 3cm downcuts the access root media; The prescription of root media is to add 0.25 mgL in the 1/2MS medium
-1IBA, 0.1 mgL
-1NAA, 2% sucrose, 0.7% agar and 0.2 gL
-1Active carbon is made, and is 6.5 to end to medium pH; Behind the 65d, form the healthy and strong seedling of taking root after continuing to cultivate.
Embodiment 2:
Spray 700 times of carbendazim every the whole strain of 7d in the step 1), liquid detergent solution soaks 5min, running water flushing 0.3h; Step 2) alcohol concentration is 70% in, and soak time is 30s, outwells behind the alcohol aseptic water washing 4 times, then adds 0.05% mercuric chloride solution, and soak time is 10 min, constantly shakes therebetween, outwells mercuric chloride solution with aseptic water washing 7 times; Downcutting tip length in the step 3) is the part of 3mm, and culture medium prescription is the MS medium, adds 0.5mgL
-1BA, 0.15mgL
-1IBA, 2% sucrose and 0.8% agar are made, and are 5.8 to end to medium pH; Cultivation temperature is 27 ℃ in the step 4), light application time 11h/d, light intensity 2000Lx; Greater than the young shoot cutting-out of 0.6cm, be divided into the stem section of 0.6cm with wherein; Proliferation culture medium formula is to add 0.5 mgL in the MS medium
-1BA, 0.15 mgL
-1IBA, 4% sucrose and 0.8% agar are made, and are 5.8 to end to medium pH; The stem section is cultivated 65d at proliferated culture medium in the step 5), and the tender stem that the length that stem section differential growth is gone out surpasses 2 cm downcuts, and the prescription of root media is to add 0.1 mgL in the 1/2MS medium
-1IBA, 0.05 mgL
-1NAA, 3% sucrose, 0.6% agar and 0.3 gL
-1Active carbon is made, and is 6.3 to end to medium pH; Behind the 60d, form the healthy and strong seedling of taking root after continuing to cultivate; Other step is with embodiment 1.
Embodiment 3: spray 900 times of carbendazim every the whole strain of 9d in the step 1), liquid detergent solution soaks 10min, running water flushing 0.6h; Step 2) alcohol concentration is 80% in, and soak time is 20s, then adds 0.15% mercuric chloride solution, and soak time is 9 min, constantly shakes therebetween, outwells mercuric chloride solution aseptic water washing 7 times; Downcutting tip length in the step 3) is the part of 4mm, and culture medium prescription is to add 1.5 mgL in the MS medium
-1BA, 0.05 mgL
-1IBA, 5% sucrose and 0.5% agar are made, and are 5.9 to end to medium pH; Cultivation temperature is 26 ℃ in the step 4), light application time 11h/d, light intensity 1600Lx; Greater than the young shoot cutting-out of 0.6cm, be divided into the stem section of 1.0cm with wherein; Proliferation culture medium formula is to add 1.5mgL in the MS medium
-1BA, 0.05 mgL
-1IBA, 5% sucrose and 0.6% agar are made, and are 5.9 to end to medium pH; The stem section is cultivated 62d at proliferated culture medium in the step 5), and the tender stem that the length that stem section differential growth is gone out surpasses 4 cm downcuts, and the prescription of root media is to add 0.30 mgL in the 1/2MS medium
-1IBA, 0.15 mgL
-1NAA, 3% sucrose, 0.8% agar and 0.1 gL
-1Active carbon is made, and is 6.7 to end to medium pH; Behind the 70d, form the healthy and strong seedling of taking root after cultivating; Other step is with embodiment 1.
Embodiment 4: liquid detergent solution soaks 9min in the step 1), running water flushing 0.4h; Step 2) alcohol concentration is 78% in, and soak time is 23s; Culture medium prescription is to add 1.2 mgL in the MS medium in the step 3)
-1BA, 0.08mgL
-1IBA, 4% sucrose and 0.6% agar are made; Cultivation temperature is 24 ℃ in the step 4), light application time 13h/d, light intensity 1700Lx; Proliferation culture medium formula is to add 1.2 mgL in the MS medium
-1BA, 0.09 mgL
-1IBA, 3% sucrose and 0.5% agar are made; The prescription of root media is to add 0.5 mgL in the 1/2MS medium in the step 5)
-1IBA, 0.12 mgL
-1NAA, 3% sucrose, 0.6% agar and 0.25 gL
-1Active carbon is made, and is 6.6 to end to medium pH; Behind the 75d, form the healthy and strong seedling of taking root after cultivating; Other step is with embodiment 1.
Embodiment 5: spray 650 times of carbendazim every the whole strain of 10d in the step 1), liquid detergent solution soaks 6min; Step 2) mercuric chloride solution concentration is 0.2% in, and soak time is 8 min, outwells mercuric chloride solution aseptic water washing 8 times; Cultivation temperature is 23 ℃ in the step 4), light application time 12h/d, light intensity 2000Lx; Other step is with embodiment 1.
The red autumnal leaves heavenly bamboo of adopting above-described embodiment 1-5 method for tissue culture the to obtain seedling of taking root, survival rate is high, reproduction speed is fast, and growth coefficient reaches 7.3, and rooting rate reaches 87%.
The invention will be further described below in conjunction with corresponding test data.
Test 1. arranges hormone combination tests different in the proliferated culture medium, 4 gradient 0.5 mgL of BA concentration
-1, 1.0 mgL
-1, 1.5 mgL
-1, 2.0 mgL
-1, IBA2 concentration gradient 0.1 mgL
-1, 0.2mgL
-1, and set up contrast, see Table 1.
Different hormone combination result of the test table in table 1 proliferated culture medium
| Process | BA(mg/L) | IBA(mg/L) | Average bud number (individual) | Average plant height (cm) | The vitrifying situation | Leaf color |
| 1 | 0．5 | 0．1 | 0 | 3．5 | Nothing | ++ |
| 2 | 0．5 | 0．2 | 0 | 3．4 | Nothing | +++ |
| 3 | 1．0 | 0．1 | 2．3 | 4．5 | Nothing | ++ |
| 4 | 1．0 | 0．2 | 2．1 | 4．4 | Nothing | +++ |
| 5 | 1．5 | 0．1 | 3．3 | 4．6 | Nothing | ++ |
| 6 | 1．5 | 0．2 | 3．1 | 4．5 | Nothing | ++ |
| 7 | 2．0 | 0．1 | 5．5 | 2．8 | Vitrifying | + |
| 8 | 2．0 | 0．2 | 4．9 | 2．7 | Vitrifying | + |
| Contrast | 0.0 | 0.0 | 0 | 2．1 | Nothing | +++ |
Result of the test shows: BA concentration is 1.5 mgL
-1The time plant average height be up to 4.6 cm, the number that on average sprouts is 3.3; Be improved though be higher than this concentration number that on average sprouts, the plant average height descends and in various degree vitrification phenomenon occurs, and petiole is extremely short, the flavescence of leaf look; When BA concentration is 2.0 mgL
-1The time, the callus spherical water stain shape that is white in color, the callus surface is covered with the bud point.Obtain best propagation prescription according to experimental result, growth coefficient is 7.3.
Test 2. arranges the test of IBA variable concentrations in the root media, and the IBA concentration gradient is: 0,0.25,0.5 mgL
-1, rooting rate is 30-87.33%, concentration 0.25m gL
-1The time obtained the highest rooting rate 87.33%.
Claims (5)
1. the method for tissue culture of red autumnal leaves heavenly bamboo is characterized in that may further comprise the steps:
1) from red autumnal leaves heavenly bamboo seedling, choose health, fancy points shows typical plant as material; In the morning of fine day, the clip shoot is removed winglike compound leaf, cleans for subsequent use;
2) on superclean bench, the branch that cleans up is also washed with alcoholic solution, mercuric chloride solution soaking disinfection successively;
3) strip out the interior stem apex of branch, downcut to be placed in the culturing room in the first end parts access inductive differentiation medium and cultivate; The prescription of inductive differentiation medium is to add 0.5-1.5mgL in the MS medium
-16-benzyl aminopurine, 0.05-0.15 mgL
-1Indolebutyric acid, 2-5% sucrose and 0.5-0.8% agar are made, and medium pH is 5.8-6.0;
4) stem apex access medium 60d-65d, the stem apex differentiation and proliferation forms some sproutings, and pumping children shoot, greater than the young shoot cutting-out of 0.5cm, is divided into the stem section with wherein, and switching enters in the proliferated culture medium to cultivate; Proliferation culture medium formula is to add 0.5-1.5 mgL in the MS medium
-16-benzyl aminopurine, 0.1-0.2 mgL
-1Indolebutyric acid, 2-5% sucrose and 0.5-0.8% agar are made, and medium pH is 5.8-6.0;
5) the stem section is cultivated 60d-65d at proliferated culture medium, and stem section propagation forms indefinite bud, and pumping goes out tender stem, and the tender stem that the length that stem section differential growth is gone out surpasses 2cm downcuts, and the access root media after root media is cultivated, forms the healthy and strong group training seedling of taking root; The prescription of root media is to add 0.1-0.5 mgL in the 1/2MS medium
-1Indolebutyric acid, 0.05-0.15 mgL
-1The sucrose of methyl α-naphthyl acetate, 1-3%, the agar of 0.5-0.8% and 0.1-0.3 gL
-1Active carbon is made, and medium pH is 6.3-6.7.
2. the method for tissue culture of red autumnal leaves heavenly bamboo as claimed in claim 1 is characterized in that in the step 1): previous month of clip shoot is sprayed 700 times of-900 times of carbendazim every the whole strain of 7-10d; Branch behind the clip is wiped out winglike compound leaf, and running water is rinsed well, and liquid detergent solution soaks 5-10min, and is for subsequent use with running water flushing 0.3-0.6h again.
3. the method for tissue culture of red autumnal leaves heavenly bamboo as claimed in claim 1, it is characterized in that step 2) in: alcohol concentration is 70%-80%, soak time is 20-30s, outwell behind the alcohol aseptic water washing 4-5 time, then add the 0.05%-0.2% mercuric chloride solution, soak 8-10 min, constantly shake therebetween, outwell mercuric chloride solution aseptic water washing 7-8 time.
4. the method for tissue culture of red autumnal leaves heavenly bamboo as claimed in claim 1 is characterized in that: strips stem apex with sterile razor blade after the branch sterilization, downcutting tip length is the part of 3-6mm, in the inductive differentiation medium that access prepares.
5. the method for tissue culture of red autumnal leaves heavenly bamboo as claimed in claim 1, it is characterized in that: cultivation temperature is 25 ± 2 ℃ in the culturing room, light application time 11-13h/d, light intensity 1500-2000Lx.
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| CN103314853B (en) * | 2013-06-28 | 2014-08-06 | 江苏农林职业技术学院 | Tissue culture method for flame nandina |
| CN103843665B (en) * | 2014-03-25 | 2016-08-17 | 江苏农林职业技术学院 | A kind of Nandina domestica'Fire power' tissue culture and rapid propagation method based on LED light source |
| CN105284610B (en) * | 2015-09-28 | 2017-08-25 | 江苏农林职业技术学院 | The regeneration and cultivation method of Nandina domestica'Fire power' |
| CN107333656A (en) * | 2017-08-29 | 2017-11-10 | 江苏丰收大地种业发展有限公司 | A kind of Nandina domestica'Fire power' fast propagating culture medium series and tissue culture method |
| CN109287484A (en) * | 2018-11-02 | 2019-02-01 | 杭州市园林绿化股份有限公司 | Dwarf form bigleaf hydrangea method for tissue culture |
| CN112931199A (en) * | 2020-12-31 | 2021-06-11 | 河北北方学院 | College regeneration culture medium and culture method for potato northern 002 |
| CN115152627B (en) * | 2022-06-15 | 2023-02-28 | 浙江省园林植物与花卉研究所(浙江省萧山棉麻研究所) | Tissue culture and rapid propagation method for Nandina domestica Brisson |
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Effective date of registration: 20230522 Address after: Floor 19-20, Building A, Oceanwide International Center, No. 2 Xiangzhang Road, Qianjiang New City, Jianggan District, Hangzhou City, Zhejiang Province, 310007 Patentee after: ZHEJIANG SENHE SEED Co.,Ltd. Patentee after: ZHEJIANG SENHE ENVIRONMENTAL ENGINEERING Co.,Ltd. Address before: 310007 room 1703, 1 pan Hai International Center, Jianggan District, Hangzhou, Zhejiang. Patentee before: ZHEJIANG SENHE HUALI TECHNOLOGY Co.,Ltd. Patentee before: ZHEJIANG SENHE SEED Co.,Ltd. Patentee before: ZHEJIANG SENHE ENVIRONMENTAL ENGINEERING Co.,Ltd. |