CN101044840A - Method for fast breeding western azalea and culture medium therefor - Google Patents
Method for fast breeding western azalea and culture medium therefor Download PDFInfo
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- CN101044840A CN101044840A CN 200610050993 CN200610050993A CN101044840A CN 101044840 A CN101044840 A CN 101044840A CN 200610050993 CN200610050993 CN 200610050993 CN 200610050993 A CN200610050993 A CN 200610050993A CN 101044840 A CN101044840 A CN 101044840A
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Abstract
A tissue culture method for the fast reproduction of Western azalea and making its test-tube plantlet to bloom includes such steps as preparing the explant, creating an aseptic culture system, reproduction, culturing the strong test-tube plantlet, rooting culture, and inducing the test-tube plantlet to bloom. Its culture medium is also disclosed.
Description
Technical field
The present invention relates to tissue-culturing quick-propagation and test tube flowering method and the used nutrient component of a kind of Western cuckoo, belong to flowers culture technique field.
Background technology
The West cuckoo (being commonly called as western cuckoo) (Rhododendron hybridum west) just is listed in one of the world ten big potted flowers in early days, very is in great demand in International Flower market.Its florescence reaches four or five ten days, views and admires than common azalea is more anti-.Except that being used for potted plant viewing and admiring, also can be used for the garden decoration and form multiple uses such as spending hedge.In recent years, along with the raising of people's living standard, demand is increasing, and traditional propagation technique cultivation cycle is long, and output is little, but also is subjected to the influence of season and cottage propagation maternal plant, is difficult to satisfy the needs in market.
Summary of the invention
The objective of the invention is to, a kind of Western cuckoo quick breeding by group culture and the method for test tube flowering and used medium are provided.Utilize the present invention to make the cultivation cycle of Western cuckoo short, be not subjected to the influence in season, improved the output of Western cuckoo.
Technical scheme of the present invention.The method of the West cuckoo quick breeding by group culture and test tube flowering adopts tender stem apex and stem section to form the method for quickly breeding of the bud of growing thickly, and on the basis of breeding fast its test-tube plantlet is carried out bud and induces, and it comprises the following steps:
The preparation of a, explant
Get the 1-1.5cm long stem apex and the stem section that contain the tender stem of New Development that 1-3 axillalry bud save, put under the running water, stir 2-3 time gently with glass bar during this period towards 1-2h; Through 70% alcohol rinsing 30-60s, sterile water dashes once, changes 0.1% mercury chloride (HgCl under aseptic condition
2) solution disinfection 6-8 minute, sterile water is towards 4-5 time; Remove the part of blackening on stem apex and the stem section, it is standby to put into the sterile petri dish that filter paper is housed;
The foundation and the propagation of b, aseptic culture system
On superclean bench, with stem apex and stem section oblique cutting in the explant induction medium, the explant induction medium is Read, MS organic principle thing, sucrose 20～30g/L, ZT (zeatin) 1.0～2.5mg/L, NAA (methyl) 0.05～0.1mg/L, contain agar 9～11g/L in the explant induction medium, pH5.0～5.4; 25 ± 1 ℃ of temperature, the dark 12h of illumination 12h/, intensity of illumination 1500～2000lx;
The bud that derives is put into the adventitious buds proliferation medium carry out enrichment culture, the culture of multiplicative stage was cultivated 25～30 days; The adventitious buds proliferation medium is Read, MS organic principle thing, sucrose 20～30g/L, ZT (zeatin) 1.0～2.5mg/L, NAA (methyl) 0.05～0.1mg/L, contains agar 9～11g/L in the adventitious buds proliferation medium, pH5.0～5.4; 25 ± 1 ℃ of temperature, the dark 12h of illumination 12h/, intensity of illumination 1500～2000lx;
The strong sprout of c, test-tube plantlet and culture of rootage
The bud of growing thickly after the propagation is severed, be inoculated in the strong seedling culture base, cultivated 25～35 days; The strong seedling culture base is Read, MS organic principle thing, 6-BA (6-benzyladenine) 0.5～1.0mg/L, NAA (methyl) 0.05～0.1mg/L; Contain sucrose 20～30g/L in the strong seedling culture base, agar 9～11g/L, pH5.0～5.4; 25 ± 1 ℃ of temperature, the dark 8h of illumination 16h/, intensity of illumination 1500～2000lx;
Selection is grown preferably, and seedling is seeded in the root media, make 25～35 days culture of rootage, root media is Read, MS organic principle thing, NAA (methyl) 0.5～2.0mg/L, IBA (indolebutyric acid) 0.5～1.0mg/L, ZT (zeatin) 0.1～0.2mg/L; Contain sucrose 20～30g/L in the root media, agar 9～11g/L, pH5.0～5.4; 25 ± 1 ℃ of temperature, the dark 8h of illumination 16h/, intensity of illumination 1500～2000lx;
D, test-tube plantlet flower induction medium
To grow preferably, test-tube plantlet is inoculated in the test-tube plantlet flower induction medium, test-tube plantlet flower induction medium is 1/2Read, MS organic principle thing, IBA (indolebutyric acid) 1.0～2.0mg/L, 0.2% active carbon, the test-tube plantlet top induces bud after 3 months, cultivates to bloom after 10 days; Contain sucrose 20～30g/L in the test-tube plantlet flower induction medium, agar 9～11g/L, pH5.0～5.4; 25 ± 1 ℃ of temperature, the dark 8h of illumination 16h/, intensity of illumination 1500～3000lx.
The medium that above-mentioned Western cuckoo quick breeding by group culture and test tube flowering are used, it comprises the various medium with following raw material preparation:
The explant induction medium:
Read
MS organic principle thing
Sucrose 20～30g/L
ZT (zeatin) 1.0～2.5mg/L
NAA (methyl) 0.05～0.1mg/L
The adventitious buds proliferation medium:
Read
MS organic principle thing
Sucrose 20～30g/L
ZT (zeatin) 1.0～2.5mg/L
NAA (methyl) 0.05～0.1mg/L
The strong seedling culture base:
Read
MS organic principle thing
6-BA (6-benzyladenine) 0.5～1.0mg/L
NAA (methyl) 0.05～0.1mg/L
Root media:
Read
MS organic principle thing
NAA (methyl) 0.5～2.0mg/L
IBA (indolebutyric acid) 0.5～1.0mg/L
ZT (zeatin) 0.1～0.2mg/L
Test-tube plantlet flower induction medium:
1/2Read
MS organic principle thing
IBA (indolebutyric acid) 1.0～2.0mg/L or NAA (methyl) 2.0～5.0mg/L or IBA (indolebutyric acid) 1.0～2.0mg/L add NAA (methyl) 1.0～2.0mg/L
0.2% active carbon.
The medium that above-mentioned Western cuckoo quick breeding by group culture and test tube flowering are used is the various medium with following raw material preparation better:
The explant induction medium:
Read
MS organic principle thing
Sucrose 30g/L
ZT (zeatin) 2.0mg/L
NAA (methyl) 0.05mg/L
The adventitious buds proliferation medium:
Read
MS organic principle thing
Sucrose 30g/L
ZT (zeatin) 2.0mg/L
NAA (methyl) 0.05mg/L
The strong seedling culture base:
Read
MS organic principle thing
6-BA (6-benzyladenine) 1.0mg/L
NAA (methyl) 0.05mg/L
Root media:
Read
MS organic principle thing
NAA (methyl) 2.0mg/L
IBA (indolebutyric acid) 1.0mg/L
ZT (zeatin) 0.2mg/L
Test-tube plantlet flower induction medium:
1/2Read
MS organic principle thing
IBA (indolebutyric acid) 2.0mg/L
0.2% active carbon.
The present invention has the advantages that cultivation cycle is short, the branch branch growth is good, be not subjected to seasonal effect.Common western cuckoo needs about 3 months product flowers, and test-tube plantlet of the present invention only needed to bloom in 2 months.
For the best approach of the quick breeding by group culture that obtains Western cuckoo, the applicant has done a series of tests:
One, grow thickly the inducing of bud
The stem apex of peeling off out is inoculated on the bud inducing culture, and statistics sees Table 1 after 40 days.The result shows, the explant best inductivity of growing is the highest on Read+ZT (2.0mg/L)+NAA (0.05mg/L) (unit under with).The explant growth induces indefinite bud rapidly and at base portion after 35 days, and inductivity is 90%.About the long 3cm of explant stem this moment, base portion has a small amount of yellow green callus.Though the generation that 6-BA also can induced bud, inductivity is low, and the downright bad phenomenon of stem also appears in the part explant.KT (kinetin) can not induce basically and sprout.So select the optimum medium that Read+ZT (2.0)+NAA (0.05) induces for Western cuckoo bud.The explant that choosing will insert is the earliest cut off, and is inoculated in the root media and takes root.Bud and the callus of removing apical dominance are changed in the medium of front in the lump, and after 2 weeks, the increase of base portion callus induces more bud simultaneously, and then forms the bud of growing thickly, and mostly is the 6-10 offspring.After cultivating for 5 weeks, with the high about 3cm of stem, the slightly about 0.5cm of stem, the seedling preferably of growing cut off and change in the medium that bud induces.
Different medium of table 2 and of the influence of different hormone combinations to inducing clumping bud
| Handle | Medium | Inductivity |
| 1 2 3 4 5 6 7 8 9 | Read+BA(0.5)+NAA(0.05) Read+BA(1.0)+NAA(0.05) Read+BA(2.0)+NAA(0.05) Read+KT(0.5)+NAA(0.05) Read+KT(1.0)+NAA(0.05) Read+KT(2.0)+NAA(0.05) Read+ZT(0.5)+NAA(0.05) Read+ZT(1.0)+NAA(0.05) Read+ZT(2.0)+NAA(0.05) | 0 13.3％ 23.3％ 0 0 6.66％ 83.33％ 86.66％ 90％ |
Two, bud is induced combination
To grow preferably that seedling cuts off from callus, and be inoculated into to contain on the 0.2% active carbon 1/2Read medium, IBA (indolebutyric acid), NAA (methyl), the IAA (heteroauxin) of additional variable concentrations induce the generation of its bud, see Table 2.With the Read medium that does not add active carbon, do not reduce by half is contrast.
The influence that the different medium of table 2 and different hormone combinations are induced bud
| Numbering | Medium and hormone combination | The upgrowth situation of bud | The bud inductivity |
| A B C D E F G | 1/2Read+IBA (1.0)+0.2% active carbon 1/2Read+IBA (2.0)+0.2% active carbon 1/2Read+NAA (2.0)+IBA (2.0)+0.2% active carbon 1/2Read+NAA (5.0)+0.2% active carbon 1/2Read+NAA (8.0)+0.2% active carbon 1/2Read+IAA (0.2)+0.2% active carbon Read+NAA (2.0)+IBA (1.0)+ZT (0.2) | +++ ++++ +++ +++ - ++ ++ | 16.6％ 33.3％ 33.3％ 33.3％ 0 25％ 5.5％ |
"-" expression does not induce bud, "+, ++, +++, ++ ++ " upgrowth situation of expression bud
From top experiment as can be known, induce in the experiment at bud, though numbering " B ", " C ", " D " medium are the same to the inductivity of bud, from the upgrowth situation and the flowering time of bud, " B " medium optimum is induced bud.Explant on " B " medium is better than the explant upgrowth situation on other medium, and bud is bigger than the bud of other medium, and flowering time is also done sth. in advance 1 month than bud on other medium." A " " C " " D " " F " is though also can induce the formation of bud, and yellow leaf phenomenon appears in explant in these several medium, and with rhizoid, be that many blades all send out roots, and the basic unrooted of explant, even if the explant base portion that has has produced root, also lacking very." G " though medium is not high to the inductivity of bud, and its base portion easily goes out root, root is many, and is sturdy.Be suitable for inducing to the explant root.With the bud anatomic observation, development of floral organs is all normal.Therefore, medium 1/2Read+IBA (2.0)+0.2% active carbon is for inducing the bud optimum medium.
Embodiment
Embodiments of the invention.Adopt tender stem apex and stem section to form the method for quickly breeding of the bud of growing thickly, on the basis of breeding fast its test-tube plantlet is carried out bud and induce, it comprises the following steps:
The preparation of a, explant
Get the stem apex and the stem section of the tender stem of New Development that contains 1-3 axillalry bud joint of 1-1.5 centimeter length, put under the running water, stir 2-3 time gently with glass bar during this period, make its surperficial dust and bacterium wash towards 1-2 hour; Through 70% alcohol rinsing 30-60 second, sterile water dashes once, changes 0.1%HgCl under aseptic condition
2(mercury chloride) solution disinfection 6-8 minute, sterile water is towards 4-5 time; Remove the part of blackening on stem apex and the stem section, it is standby to put into the sterile petri dish that filter paper is housed.
The foundation and the propagation of b, aseptic culture system
On superclean bench, with stem apex and stem section oblique cutting in the explant induction medium, the explant induction medium is Read, MS organic principle thing, sucrose 30g/L, ZT (zeatin) 2.0mg/L, NAA (methyl) 0.05mg/L, contain agar 11g/L in the explant induction medium, pH5～6; 25 ± 1 ℃ of temperature, the dark 12h of illumination 12h/, intensity of illumination 1500～2000lx;
The bud that derives is put into the adventitious buds proliferation medium carry out enrichment culture, the culture of multiplicative stage was cultivated 30 days; The adventitious buds proliferation medium is identical with the explant induction medium, for Read, MS organic principle thing, sucrose 30g/L, ZT (zeatin) 2.0mg/L, NAA (methyl) 0.05mg/L, contains agar 11g/L in the explant induction medium, pH5～6; 25 ± 1 ℃ of temperature, the dark 12h of illumination 12h/, intensity of illumination 1500～2000lx.
The culture medium preparation method: joining the 1L medium is example
The macroelement, trace element, the molysite that at first add the needed Read of 1L medium add required MS organic principle thing in the 1L medium again, and each material and content see the following form.
The constituent table (mg/L) of table 3 Read medium
| Macroelement | Content | Moderate-element | Content | Trace element | Content | Other | Content |
| NH 4NO 3 (NH 4) 2SO 4 KNO 3 CaCl 2·2H 2O MgSO 4·7H 2O KH 2PO 4 | 400 132 202 440 370 408 | Na 2·EDTA FeSO 4·7H 2O H 3BO 4 MnSO 4·H 2O ZnSO 4·H 2O | 37.8 27.8 6.2 16.9 8.6 | NaMOO 4·2H 2O CuSO 4·5H 2O CoCl 2·6H 2O | 0.25 0.025 0.025 | VB 1Inositol | 0.4 100 |
The constituent table (mg/L) of table 4 MS medium
| Macroelement | Content | Trace element | Content | Molysite | Content | Organic matter | Content |
| NH 4NO 3KNO 3CaCl 2·2H 2O MgSO 4·7H 2O KH 2PO 4 | 1650 1900 440 370 170 | KI H 3BO 4 MnSO 4·4H 2O ZnSO 4·7H 2O NaMoO 4·2H 2O CuSO 4·5H 2O CoCl 2·6H 2O | 0.83 6.2 22.3 8.6 0.25 0.025 0.025 | Na 2·EDTA FeSO 4·7H 2O | 37.8 27.8 | Inositol glycine Cobastab 1Cobastab 6Nicotinic acid (nicotinic acid) | 100 2.0 0.1 0.5 0.5 |
The strong sprout of c, test-tube plantlet and culture of rootage
The bud of growing thickly after the propagation is severed, be inoculated in the strong seedling culture base, cultivated 30 days, seedling length is 3-4 centimetre, about 0.5 centimetre of diameter; The strong seedling culture base is Read, MS organic principle thing, 6-BA (6-benzyladenine) 1.0mg/L, NAA (methyl) 0.05mg/L; Contain sucrose 30g/L in the strong seedling culture base, agar 11g/L, pH5.4; 25 ± 1 ℃ of temperature, the dark 8h of illumination 16h/, intensity of illumination 1500～2000lx.
Selection grows preferably that seedling is seeded in the root media, makes 30 days culture of rootage, and root media is Read, MS organic principle thing, NAA (methyl) 2.0mg/L, IBA (indolebutyric acid) 1.0mg/L, ZT (zeatin) 0.2mg/L; Contain sucrose 30g/L in the root media, agar 11g/L, pH5.4; 25 ± 1 ℃ of temperature, the dark 8h of illumination 16h/, intensity of illumination 1500～2000lx.
D, test-tube plantlet flower induction medium
To grow preferably, test-tube plantlet is inoculated in the test-tube plantlet flower induction medium, test-tube plantlet flower induction medium is 1/2Read, MS organic principle thing, IBA (indolebutyric acid) 2.0mg/L, 0.2% active carbon, the test-tube plantlet top induces bud after 3 months, cultivates to bloom after 10 days; Contain agar 11g/L, pH5.4 in the test-tube plantlet flower induction medium; 25 ± 1 ℃ of temperature, the dark 8h of illumination 16h/, intensity of illumination 1500～3000lx.
Claims (3)
1, the method for Western cuckoo quick breeding by group culture and test tube flowering is characterized in that, adopts tender stem apex and stem section to form the method for quickly breeding of the bud of growing thickly, and on the basis of breeding fast its test-tube plantlet is carried out bud and induces, and it comprises the following steps:
The preparation of a, explant
Get the 1-1.5cm long stem apex and the stem section that contain the tender stem of New Development that 1-3 axillalry bud save, put under the running water, stir 2-3 time gently with glass bar during this period towards 1-2h; Through 70% alcohol rinsing 30-60s, sterile water dashes once under aseptic condition, changes 0.1% mercuric chloride solution sterilization 6-8 minute over to, and sterile water is towards 4-5 time; Remove the part of blackening on stem apex and the stem section, it is standby to put into the sterile petri dish that filter paper is housed;
The foundation and the propagation of b, aseptic culture system
On superclean bench, with stem apex and stem section oblique cutting in the explant induction medium, the explant induction medium is Read, MS organic principle thing, sucrose 20～30g/L, zeatin 1.0～2.5mg/L, methyl 0.05～0.1mg/L, contain agar 9～11g/L in the explant induction medium, pH5.0～5.4; 25 ± 1 ℃ of temperature, the dark 12h of illumination 12h/, intensity of illumination 1500～2000lx;
The bud that derives is put into the adventitious buds proliferation medium carry out enrichment culture, the culture of multiplicative stage was cultivated 25～30 days; The adventitious buds proliferation medium is Read, MS organic principle thing, sucrose 20～30g/L, zeatin 1.0～2.5mg/L, methyl 0.05～0.1mg/L, contains agar 9～11g/L in the adventitious buds proliferation medium, pH5.0～5.4; 25 ± 1 ℃ of temperature, the dark 12h of illumination 12h/, intensity of illumination 1500～2000lx;
The strong sprout of c, test-tube plantlet and culture of rootage
The bud of growing thickly after the propagation is severed, be inoculated in the strong seedling culture base, cultivated 25～35 days; The strong seedling culture base is Read, MS organic principle thing, 6-benzyladenine 0.5～1.0mg/L, methyl 0.05～0.1mg/L; Contain sucrose 20～30g/L in the strong seedling culture base, agar 9～11g/L, pH5.0～5.4; 25 ± 1 ℃ of temperature, the dark 8h of illumination 16h/, intensity of illumination 1500～2000lx;
Selection grows preferably that seedling is seeded in the root media, makes 25～35 days culture of rootage, and root media is Read, MS organic principle thing, methyl 0.5～2.0mg/L, indolebutyric acid 0.5～1.0mg/L, zeatin 0.1～0.2mg/L; Contain sucrose 20～30g/L in the root media, agar 9～11g/L, pH5.0～5.4; 25 ± 1 ℃ of temperature, the dark 8h of illumination 16h/, intensity of illumination 1500～2000lx;
D, test-tube plantlet flower induction medium
To grow preferably, test-tube plantlet is inoculated in the test-tube plantlet flower induction medium, test-tube plantlet flower induction medium is 1/2Read, MS organic principle thing, indolebutyric acid 1.0～2.0mg/L, 0.2% active carbon, the test-tube plantlet top induces bud after 3 months, cultivates to bloom after 10 days; Contain sucrose 20～30g/L in the test-tube plantlet flower induction medium, agar 9～11g/L, pH5.0～5.4; 25 ± 1 ℃ of temperature, the dark 8h of illumination 16h/, intensity of illumination 1500～3000lx.
2, the medium used of Western cuckoo quick breeding by group culture and test tube flowering is characterized in that, it comprises the various medium with following raw material preparation:
The explant induction medium:
Read
MS organic principle thing
Sucrose 20～30g/L
Zeatin 1.0～2.5mg/L
Methyl 0.05～0.1mg/L
The adventitious buds proliferation medium:
Read
MS organic principle thing
Sucrose 20～30g/L
Zeatin 1.0～2.5mg/L
Methyl 0.05～0.1mg/L
The strong seedling culture base:
Read
MS organic principle thing
6-benzyladenine 0.5～1.0mg/L
Methyl 0.05～0.1mg/L
Root media:
Read
MS organic principle thing
Methyl 0.5～2.0mg/L
Indolebutyric acid 0.5～1.0mg/L
Zeatin 0.1～0.2mg/L
Test-tube plantlet flower induction medium:
1/2Read
MS organic principle thing
Indolebutyric acid 1.0～2.0mg/L or methyl 2.0～5.0mg/L or
Indolebutyric acid 1.0～2.0mg/L adds methyl 1.0～2.0mg/L
0.2% active carbon.
3, the medium used of Western cuckoo quick breeding by group culture according to claim 2 and test tube flowering is characterized in that, it comprises the various medium with following raw material preparation: the explant induction medium:
Read
MS organic principle thing
Sucrose 30g/L
Zeatin 2.0mg/L
Methyl 0.05mg/L
The adventitious buds proliferation medium:
Read
MS organic principle thing
Sucrose 30g/L
Zeatin 2.0mg/L
Methyl 0.05mg/L
The strong seedling culture base:
Read
MS organic principle thing
6-benzyladenine 1.0mg/L
Methyl 0.05mg/L
Root media:
Read
MS organic principle thing
Methyl 2.0mg/L
Indolebutyric acid 1.0mg/L
Zeatin 0.2mg/L
Test-tube plantlet flower induction medium:
1/2Read
MS organic principle thing
Indolebutyric acid 2.0mg/L
0.2% active carbon.
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| CN102440190A (en) * | 2011-09-28 | 2012-05-09 | 天津农学院 | Method for effectively flowering and culturing impatiens balsamina through test tube |
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| CN102342246A (en) * | 2010-07-30 | 2012-02-08 | 贵州省生物研究所 | Rhododendron decorum tissue-culture quick propagation method |
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| CN103371102B (en) * | 2013-07-30 | 2015-04-22 | 杭州植物园 | Rapid propagation method for rhododendron spiciferum Franch tissue cultur |
| CN103548677A (en) * | 2013-10-21 | 2014-02-05 | 中国林业科学研究院林业研究所 | Multiplication method of salix matsudana stems |
| CN103548677B (en) * | 2013-10-21 | 2015-04-22 | 中国林业科学研究院林业研究所 | Multiplication method of salix matsudana stems |
| CN104885932A (en) * | 2014-12-11 | 2015-09-09 | 华南农业大学 | Tissue culture and rapid propagation method for rhododendron moulmainense |
| CN108207629A (en) * | 2018-01-08 | 2018-06-29 | 井冈山大学 | A kind of Rhododendron jinggangshanicum Tam method for tissue culture |
| CN112970583A (en) * | 2021-01-29 | 2021-06-18 | 浙江万里学院 | Establishment method and application of Rhododendron erythropolis regeneration technology system |
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