CN1329517C - Shenzhen No.5 flameray gerbera transgene technology - Google Patents
Shenzhen No.5 flameray gerbera transgene technology Download PDFInfo
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- CN1329517C CN1329517C CNB2005100374053A CN200510037405A CN1329517C CN 1329517 C CN1329517 C CN 1329517C CN B2005100374053 A CNB2005100374053 A CN B2005100374053A CN 200510037405 A CN200510037405 A CN 200510037405A CN 1329517 C CN1329517 C CN 1329517C
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Abstract
The present invention relates to a transgenic technique for Shenzhen five number Africa chrysanthemum. The present invention is characterized in that the present invention comprises three steps: leading meloidogynosis agrobacterium in plasmid; preparing and conserving LBA4404 seed solution; transforming the agrobacterium. The third step-transforming the agrobacterium comprises: activating the agrobacterium; culturing outer plants and the agrobacterium liquid; degerming; screening antibiotic; rejuvenating a quasi-transgenic plant; breeding. The method transforms a gene with the embedded CaMV35S. HPT and a gene with embedded CaMV35S. GUS into Shenzhen five number Africa chrysanthemum bud, and obtains the transgenic plant. The method provides a technique for transforming other object genes and obtaining new transgene Africa chrysanthemum strain (species). The method has the advantages of high stability, high regeneration and conversion efficiency.
Description
(1) technical field
The present invention relates to the transgenic technology in African chrysanthemum (Gerbera hybrida) Shenzhen No. 5 (S5), promptly obtain the method for transfer-gen plant by transgenic technology.
(2) background technology
African chrysanthemum another name African daisy for one of the world's six big cut-flowers, also is important pot flowers.Its garden-variety is many, the ornamental value height.All to insert the life-span long being devoted to obtain various patterns, flower type, the fragrance of a flower and bottle always for the breeding work person, has resistance, heat-resisting, cold-resistant new variety.Traditional breeding technology exists cross-breeding time length, hybridization to be difficult to introduce the good character that affinity is far planted, and often follows some bad shortcomings such as proterties introducing when introducing a certain or some good character.Utilize genetic engineering technique will be expected to realize African chrysanthemum pattern, flower shape, resistance, florescence, bottle are inserted proterties such as life-span, the fragrance of a flower and improved.
So far, the African chrysanthemum transgenosis has been carried out preliminary research work abroad, but also only limited to breadboard report one or two, and transformation efficiency is very low.For example, Elomaa etc. (1993) are explant with the petiole of red kind Terra Regina test-tube plantlet, utilize agrobacterium tumefaciens that foreign gene is imported explant, carrying out kantlex (Kan) then selects, last per 100 explants obtain 0.1-2 transgenic seedling (Biotechnology, 1993,11:508-511).Domestic African chrysanthemum transgenic research also just is in the stage of groping conversion condition, still fails to obtain transfer-gen plant [Ye Hua etc., 2003, Yunnan University's journal (natural science edition), 25 (supplementary issue): 128-130; Liu Hui, 2004, Hua Zhong Agriculture University's master thesis].No. 5 (S5) ligulate flowers in Shenzhen are orange, and interior wheel plate-like flower is black, and pattern is bright-coloured, liked by people.This kind plant regeneration frequency is very low at present.The infringement of various in addition viruses, microorganism and insect and itself thermotolerance, resistance to cold are not high, make the quality raising of African chrysanthemum and be widely used in market to be very limited.
(3) Fa Ming content
The object of the present invention is to provide the transgenic method of No. 5 African chrysanthemum in a kind of Shenzhen, this method has the high advantage of good stability, regeneration and transformation efficiency.To contain chimeric CaMV35S.HPT gene (hygromycin phosphotransferase gene) and the chimeric No. 5 African chrysanthemum simple buds in CaMV35S.GUS gene transformation Shenzhen, obtained transfer-gen plant, transformed other goal gene for producing, obtain transgenosis African chrysanthemum new lines (kind) technology is provided.
Method of the present invention comprises the steps:
Step 1, plasmid imports agrobacterium tumefaciens: method (" the plant genetic engineering philosophy and technique " that can write in 2002 with reference to Wang Guanlin and the Fang Hong skin of bamboo that directly imports Agrobacterium according to the intermediate carrier plasmid DNA of routine, Beijing: Science Press), pCAMBIA1301 (CAMBIA company, Australia) is imported in the agrobacterium tumefaciens lba4404; Coated plate is cultivated, and substratum adopts prescription to be the solid medium A of YEB+50～100mg/L kantlex+50～100mg/L Streptomycin sulphate, at 28 ± 1 ℃ of condition constant temperature culture 46～50h;
Step 2, the preparation of LBA4404 seed liquor and preservation: the single bacterium colony of the LBA4404 that contains the pCAMBIA1301 plasmid that the above-mentioned cultivation of picking obtains, be seeded to substratum B: promptly in the liquid nutrient medium of YEB+50～100mg/L kantlex+50～100mg/L Streptomycin sulphate, shaking culture 24～36h under 28 ± 1 ℃, 180～220r/min condition; The adding concentration of volume percent is 80% glycerine, and final glycerol concentration is 20% in glycerol stock liquid mixed solution; With glycerol stock liquid mixed solution, promptly LBA4404 seed liquor branch is filled in the eppendof pipe, place under-70～-80 ℃ of conditions preserve standby;
Step 3, the conversion of Agrobacterium: comprise the steps
(1) activation Agrobacterium: get step 2 gained LBA4404 seed liquor 5～8 μ l and be inoculated among the substratum B of 10～15ml step 2, two temperature and shaking culture condition are cultivated 23～25h set by step; The bacterium liquid of getting 1ml is transferred among 40～50ml fresh culture B, and two temperature and shaking culture condition continue to cultivate 10～14h set by step; The bacterium liquid of cultivating is changed in the eppendof pipe, 5000r/min, centrifugal 5min removes supernatant liquor; With the plant inducing culture is 1/2MS+3% sucrose, and the substratum of pH5.2 suspends and precipitates; On spectrophotometer, measure the OD of gained bacterium liquid
600Value, OD
600Value be 0.4～0.5 o'clock be activatory bacterium liquid, in this bacterium liquid, add Syringylethanone, make the Syringylethanone ultimate density be 20～30mg/L, standby;
(2) explant and bacterium liquid are cultivated altogether: will be step by step (1) standby bacterium liquid of being obtained, shaking culture 1～2h under 28 ± 1 ℃, 180～220r/min condition; Cut the simple bud of No. 5 African chrysanthemum tissue cultured seedling in Shenzhen, in bacterium liquid, soak 10～15min and be placed in the common substratum of prescription for MS+BA2～3mg/L+IAA0.2～0.5mg/L+ Syringylethanone 20～30mg/L+ sucrose 3%+ agar 1%, pH5.2～5.5, cultivate 3～4d altogether under 24 ± 1 ℃, dark condition;
(3) degerming is cultivated: will step by step (2) simple bud explant after cultivating with the aseptic water washing that contains the 500mg/L Cephradine, surface-moisture is removed in suction; Change MS+BA2～3mg/L+IAA0.2～0.5mg/L+ Cephradine 400～500mg/L+ sucrose 3%+ agar 0.8% over to, 3～4d is cultivated in the degerming that removes on the bacterium culture medium of pH5.6～5.8, and culture condition is 24 ± 1 ℃, 12h illumination/12h dark;
(4) antibiotic-screening: change the simple bud after the degerming cultivation over to MS+BA2～3mg/L+IAA0.2～0.5mg/L+ Cephradine 300～400mg/L+ Totomycin 10mg/L+ sucrose 3%+ agar 0.8%, cultivate on the selection substratum of pH5.6～5.8, per 7～10d rolling bottle once, behind the rolling bottle 3～4 times, the simple bud base portion induces indefinite bud;
(5) intend the rejuvenation and the multiplication culture of transfer-gen plant: will step by step (4) indefinite bud cutting-out of inducing, change MS+BA0.5～0.8mg/L+IAA0.3～0.5mg/L+ sucrose 3%+ agar 0.8% over to, in the indefinite bud rejuvenation and proliferated culture medium of pH5.6～5.8, carry out rejuvenation and propagation 10～15d; Again indefinite bud is changed over to and continue to cultivate 30～40d on the selection substratum identical with (4) step by step; At last that vitality is strong plan transgenosis indefinite bud goes on the root media of MS+IAA0.3～0.5mg/L+10mg/L Totomycin and cultivates, and 20～30d can obtain to have the complete plan transfer-gen plant of root, is used for Molecular Identification.
In this method step 1, the method that the intermediate carrier plasmid DNA directly imports Agrobacterium generally comprises steps such as the competent preparation of agrobacterium tumefaciens, matter DNA conversion and detection; Constant temperature culture is generally carried out in constant incubator.In step 2 and the step 3 they (2), shaking culture is generally carried out in the constant-temperature shaking culture case.In step 3 they (2), the preparation method of tissue cultured seedling belongs to prior art, generally comprises the step such as sterilization, inoculation, cultivation, seedling proliferation and succeeding transfer culture of holder explant and obtains the simple bud of healthy and strong seedling.In step 3 they (3), the general aseptic water washing 2～3 times that contains Cephradine with 30～50mL of simple bud explant removes surface-moisture with the thieving paper suction then.
The present invention has following advantage and effect
1,, shows that through GUS stability detection of expression and PCR, Southern hybridization detected result the transgenic seedling induction frequency can reach 7.43% by transgenosis.
2, adopting the simple bud of aseptic tissue cultured seedling in the transgenic method is explant, and the acquisition of experiment material has just had assurance like this, and the influence that not changed by season and external environmental condition.
3, adopting simple bud directly to induce this gene transformation approach of indefinite bud (simple bud propagation) can (be inoculated in common substratum from the simple bud explant and obtain to intend the transgenosis indefinite bud to obtaining resistant buds about 30～45d), can obtain complete plan transgenosis tissue cultured seedling through 20～30d again in the short period.
4, the research of adventitious shoot regeneration approach in each kind of African chrysanthemum is very ripe in this transgenic method, therefore can realize the foundation and the follow-up engineered research of other kind transformation system of African chrysanthemum.
(4) concrete embodiment
Below in conjunction with embodiment 1-3 the present invention is further detailed.
Embodiment 1:
Step 1, plasmid import agrobacterium tumefaciens: directly import the method for Agrobacterium according to the intermediate carrier plasmid DNA of routine, pCAMBIA1301 is imported in the agrobacterium tumefaciens lba4404; Coated plate is cultivated, and substratum adopts prescription to be the solid medium A of YEB+50mg/L kantlex+50mg/L Streptomycin sulphate, at 28 ± 1 ℃ of condition constant temperature culture 46h;
Step 2, the preparation of LBA4404 seed liquor and preservation: the single bacterium colony of the LBA4404 that contains the pCAMBIA1301 plasmid that the above-mentioned cultivation of picking obtains, be seeded to substratum B: promptly in the liquid nutrient medium of YEB+50mg/L kantlex+50mg/L Streptomycin sulphate, shaking culture 24h under 28 ± 1 ℃, 180r/min condition; Be that 80% glycerine mixes with cultured bacterium liquid with the concentration of volume percent that has prepared, make the final glycerol concentration in the glycerol stock liquid mixed solution reach 20%.Glycerol stock liquid mixed solution branch is filled in the eppendof pipe of 1.5ml, place under-70 ℃ of conditions preserve standby;
Step 3, the conversion of Agrobacterium:
(1) activation Agrobacterium: get step 2 gained LBA4404 seed liquor 5 μ l and be inoculated among the substratum B of 10ml step 2, two temperature and shaking culture condition are cultivated 23h set by step; The bacterium liquid of getting 1ml is transferred among the 40ml fresh culture B, and two temperature and shaking culture condition continue to cultivate 10h set by step; The bacterium liquid of cultivating is changed in the 10ml eppendof pipe, 5000r/min, centrifugal 5min removes supernatant liquor; With 10ml plant inducing culture is 1/2MS+3% sucrose, and the substratum of pH5.2 suspends and precipitates; Get the bacterium liquid 1ml of gained, on spectrophotometer, survey the OD of bacterium liquid
600Value, OD as a result
600Value is 0.4, adds Syringylethanone in this bacterium liquid, makes ultimate density reach 20mg/L, and is standby;
(2) explant and bacterium liquid are cultivated altogether: will be step by step (1) standby bacterium liquid of being obtained, shaking culture 1h under 28 ± 1 ℃, 220r/min condition; Cut the simple bud of No. 5 African chrysanthemum tissue cultured seedling in Shenzhen, in bacterium liquid, soak 10min and be placed in the common substratum of prescription for MS+BA2mg/L+IAA0.2mg/L+ Syringylethanone 20mg/L+ sucrose 3%+ agar 1%, pH5.5, cultivate 3d altogether under 24 ± 1 ℃, dark condition;
(3) degerming is cultivated: will step by step (2) simple bud explant after cultivating with the aseptic water washing that contains the 500mg/L Cephradine 3 times, remove surface-moisture with the thieving paper suction; Change MS+BA2mg/L+IAA0.2mg/L+ Cephradine 400mg/L+ sucrose 3%+ agar 0.8% over to, pH5.6 cultivates 3d except that degerming on the bacterium culture medium, and culture condition is 24 ± 1 ℃, 12h illumination/12h dark;
(4) antibiotic-screening: change the simple bud after the degerming cultivation over to MS+BA2mg/L+IAA0.2mg/L+ Cephradine 300mg/L+ Totomycin 10mg/L+ sucrose 3%+ agar 0.8%, cultivate on the selection substratum of pH5.6, every 7d rolling bottle once, behind the rolling bottle 3 times, the simple bud base portion induces indefinite bud;
(5) intend the rejuvenation and the multiplication culture of transfer-gen plant: will step by step (4) indefinite bud cutting-out of inducing, change MS+BA0.5mg/L+IAA0.3mg/L+ sucrose 3%+ agar 0.8% over to, in the indefinite bud rejuvenation and proliferated culture medium of pH5.8, carry out rejuvenation and propagation 10d; Again indefinite bud is changed over to and continue to cultivate 30d on the selection substratum identical with (4) step by step; At last that vitality is strong plan transgenosis indefinite bud goes on the root media of MS+IAA0.3mg/L+10mg/L Totomycin and cultivates, and 20d can obtain to have the complete plan transfer-gen plant of root, is used for Molecular Identification.
Detected result: GUS transient expression rate is 20% in the simple bud explant, and resistance indefinite bud occurrence frequency reaches 20%, finally can obtain transgenosis indefinite bud frequency and reach 7.43%.
Embodiment 2: other is with embodiment 1, and different is:
Step 1: culture medium A is the solid medium of YEB+100mg/L kantlex+100mg/L Streptomycin sulphate, and incubation time is 50h;
Step 2: substratum B is the liquid nutrient medium of YEB+100mg/L kantlex+100mg/L Streptomycin sulphate, 28 ± 1 ℃ of culture condition, 220r/min, incubation time 36h; LBA4404 seed liquor branch is filled in the 1.5ml eppendof pipe, place under-80 ℃ of conditions preserve standby.
Step 3:
(1) activation Agrobacterium: get step 2 gained LBA4404 seed liquor 8 μ 1 and be inoculated among the substratum B of 15ml step 2, two temperature and shaking culture condition are cultivated 25h set by step; The bacterium liquid of getting 1ml is transferred among the 50ml fresh culture B, and two temperature and shaking culture condition continue to cultivate 14h set by step; The bacterium liquid of cultivating is changed in the 10mleppendof pipe, 5000r/min, centrifugal 5min removes supernatant liquor; With 10ml plant inducing culture is 1/2MS+3% sucrose, and the substratum of pH5.2 suspends and precipitates; Get the bacterium liquid 1ml of gained, on spectrophotometer, survey the OD of bacterium liquid
600Value, OD as a result
600Value is 0.5, adds Syringylethanone in this bacterium liquid, makes ultimate density reach 30mg/L, and is standby;
(2) explant and bacterium liquid are cultivated altogether: will be step by step (1) standby bacterium liquid of being obtained, shaking culture 2h under 28 ± 1 ℃, 180r/min condition; Cut the simple bud of No. 5 African chrysanthemum tissue cultured seedling in Shenzhen, in bacterium liquid, soak 15min and be placed in the common substratum of prescription for MS+BA3mg/L+IAA0.5mg/L+ Syringylethanone 30mg/L+ sucrose 3%+ agar 1%, pH5.2, cultivate 4d altogether under 24 ± 1 ℃, dark condition;
(3) degerming is cultivated: will step by step (2) simple bud explant after cultivating with the aseptic water washing that contains the 500mg/L Cephradine 3 times, remove surface-moisture with the thieving paper suction; Change MS+BA3mg/L+IAA0.5mg/L+ Cephradine 500mg/L+ sucrose 3%+ agar 0.8% over to, pH5.8 cultivates 4d except that degerming on the bacterium culture medium, and culture condition is 24 ± 1 ℃, 12h illumination/12h dark;
(4) antibiotic-screening: change the simple bud after the degerming cultivation over to MS+BA3mg/L+IAA0.5mg/L+ Cephradine 400mg/L+ Totomycin 10mg/L+ sucrose 3%+ agar 0.8%, cultivate on the selection substratum of pH 5.8, every 10d rolling bottle once, behind the rolling bottle 4 times, the simple bud base portion induces indefinite bud;
(5) intend the rejuvenation and the multiplication culture of transfer-gen plant: will step by step (4) indefinite bud cutting-out of inducing, change MS+BA0.8mg/L+IAA0.5mg/L+ sucrose 3%+ agar 0.8% over to, in the indefinite bud rejuvenation and proliferated culture medium of pH5.8, carry out rejuvenation and propagation 15d; Again indefinite bud is changed over to and continue to cultivate 40d on the selection substratum identical with (4) step by step; At last that vitality is strong plan transgenosis indefinite bud goes on the root media of MS+IAA0.5mg/L+10mg/L Totomycin and cultivates, and 30d can obtain to have the complete plan transfer-gen plant of root, is used for Molecular Identification.
Detected result: GUS transient expression rate is 18% in the simple bud explant, and resistance indefinite bud occurrence frequency reaches 18%, finally can obtain transgenosis indefinite bud frequency and reach 6.86%.
Embodiment 3: other is with embodiment 1, and different is:
Step 1: culture medium A is the solid medium of YEB+100mg/L kantlex+100mg/L Streptomycin sulphate;
Step 2: substratum B is the liquid nutrient medium of YEB+100mg/L kantlex+100mg/L Streptomycin sulphate, incubation time 36h;
Step 3:(1) finish after, OD
600Value is 0.5; (2) the standby bacterium liquid shaking culture time is 2h in; (3) explant is that 3d is cultivated in degerming under 5.8 conditions at the degerming medium pH; (4) medium pH is 5.8 in; (5) indefinite bud rejuvenation and proliferated culture medium pH value are 5.6 in; The time of cultivating on root media is 30d.
Detected result: GUS transient expression rate is 20.3% in the simple bud explant, and resistance indefinite bud occurrence frequency reaches 20.3%, finally can obtain transgenosis indefinite bud frequency and reach 7.35%.
Claims (2)
1, the transgenic method of No. 5 African chrysanthemum in a kind of Shenzhen is characterized in that comprising the steps:
Step 1, plasmid import agrobacterium tumefaciens: directly import the method for Agrobacterium according to the intermediate carrier plasmid DNA of routine, pCAMBIA1301 is imported in the agrobacterium tumefaciens lba4404; Coated plate is cultivated, and substratum adopts prescription to be the solid medium A of YEB+50～100mg/L kantlex+50～100mg/L Streptomycin sulphate, at 28 ± 1 ℃ of condition constant temperature culture 46～50h;
Step 2, the preparation of LBA4404 seed liquor and preservation: the single bacterium colony of the LBA4404 that contains the pCAMBIA1301 plasmid that the above-mentioned cultivation of picking obtains, be seeded to substratum B: promptly in the liquid nutrient medium of YEB+50～100mg/L kantlex+50～100mg/L Streptomycin sulphate, shaking culture 24～36h under 28 ± 1 ℃, 180～220r/min condition; The adding concentration of volume percent is 80% glycerine, and final glycerol concentration is 20% in glycerol stock liquid mixed solution; With glycerol stock liquid mixed solution, promptly LBA4404 seed liquor branch is filled in the eppendof pipe, place under-70～-80 ℃ of conditions preserve standby;
Step 3, the conversion of Agrobacterium: comprise the steps
(1) activation Agrobacterium: get step 2 gained LBA4404 seed liquor 5～8 μ l and be inoculated among the substratum B of 10～15ml step 2, two temperature and shaking culture condition are cultivated 23～25h set by step; The bacterium liquid of getting 1ml is transferred among 40～50ml fresh culture B, and two temperature and shaking culture condition continue to cultivate 10～14h set by step; The bacterium liquid of cultivating is changed in the eppendof pipe, 5000r/min, centrifugal 5min removes supernatant liquor; With the plant inducing culture is 1/2MS+3% sucrose, and the substratum of pH5.2 suspends and precipitates; On spectrophotometer, measure the OD of gained bacterium liquid
600Value, OD
600Value be 0.4～0.5 o'clock be activatory bacterium liquid, in this bacterium liquid, add Syringylethanone, make the Syringylethanone ultimate density be 20～30mg/L, standby;
(2) explant and bacterium liquid are cultivated altogether: will be step by step (1) standby bacterium liquid of being obtained, shaking culture 1～2h under 28 ± 1 ℃, 180～220r/min condition; Cut the simple bud of No. 5 African chrysanthemum tissue cultured seedling in Shenzhen, in bacterium liquid, soak 10～15min and be placed in the common substratum of prescription for MS+BA 2～3mg/L+IAA 0.2～0.5mg/L+ Syringylethanone 20～30mg/L+ sucrose 3%+ agar 1%, pH 5.2～5.5, cultivate 3～4d altogether under 24 ± 1 ℃, dark condition;
(3) degerming is cultivated: will step by step (2) simple bud explant after cultivating with the aseptic water washing that contains the 500mg/L Cephradine, surface-moisture is removed in suction; Change MS+BA 2～3mg/L+IAA0.2～0.5mg/L+ Cephradine 400～500mg/L+ sucrose 3%+ agar 0.8% over to, 3～4d is cultivated in the degerming that removes on the bacterium culture medium of pH5.6～5.8, and culture condition is 24 ± 1 ℃, 12h illumination/12h dark;
(4) antibiotic-screening: change the simple bud after the degerming cultivation over to MS+BA 2～3mg/L+IAA0.2～0.5mg/L+ Cephradine 300～400mg/L+ Totomycin 10mg/L+ sucrose 3%+ agar 0.8%, cultivate on the selection substratum of pH5.6～5.8, per 7～10d rolling bottle once, behind the rolling bottle 3～4 times, the simple bud base portion induces indefinite bud;
(5) intend the rejuvenation and the multiplication culture of transfer-gen plant: will step by step (4) indefinite bud cutting-out of inducing, change MS+BA 0.5～0.8mg/L+IAA 0.3～0.5mg/L+ sucrose 3%+ agar 0.8% over to, in the indefinite bud rejuvenation and proliferated culture medium of pH5.6～5.8, carry out rejuvenation and propagation 10～15d; Again indefinite bud is changed over to and continue to cultivate 30～40d on the selection substratum identical with (4) step by step; At last that vitality is strong plan transgenosis indefinite bud goes on the root media of MS+IAA 0.3～0.5mg/L+10mg/L Totomycin and cultivates, and 20～30d obtains to have the complete plan transfer-gen plant of root.
2, the method for claim 1 is characterized in that: in the step 1, constant temperature culture is carried out in constant incubator; In step 2 and the step 3 they (2), shaking culture is carried out in the constant-temperature shaking culture case; In step 3 they (3), the simple bud explant is inhaled with thieving paper then and is removed surface-moisture with the aseptic water washing 2～3 times that 30～50mL contains Cephradine.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1127015A (en) * | 1993-05-20 | 1996-07-17 | 国际花卉开发有限公司 | Transgenic flowering plants |
| CN1413255A (en) * | 1999-12-21 | 2003-04-23 | 太阳基因两合公司 | Production of transgenic plants of tagetes species |
| WO2004020637A1 (en) * | 2002-08-30 | 2004-03-11 | International Flower Developments Pty. Ltd. | Flavonoid 3',5'hydroxylase gene sequences and uses therefor |
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2005
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1127015A (en) * | 1993-05-20 | 1996-07-17 | 国际花卉开发有限公司 | Transgenic flowering plants |
| CN1413255A (en) * | 1999-12-21 | 2003-04-23 | 太阳基因两合公司 | Production of transgenic plants of tagetes species |
| WO2004020637A1 (en) * | 2002-08-30 | 2004-03-11 | International Flower Developments Pty. Ltd. | Flavonoid 3',5'hydroxylase gene sequences and uses therefor |
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