Patent Application: US-60188303-A

Abstract:
methods for agrobacterium - mediated transformation and regeneration of dandelion plants are disclosed . the invention encompasses regenerated , fertile dandelion plants , transgenic seeds produced therefrom , and subsequent generations .

Description:
the present invention involves methods for the transformation and preparation of transgenic dandelion plants . an appropriate dna sequence is selected for introduction into the dandelion plant cells . as useful gene for introduction into dandelion plant cells , the kinds of genes do not have to be limited . however , as a preferred example , rubber - biosynthesis related genes ( rubber polymerase , cis - prenyltransferase , isopentenyl pyrophosphate synthase ); medical proteins ( albumin ) can be exemplified . the sequence typically contains a gene of interest , a promoter functional to direct transcription of the gene , and a selectable marker to facilitate identification of the transformed plant cells . examples of selectable markers include , but are not limited to , the neomycin phosphotransferase , hygromycin phosphotransferase , and epsps genes . expression of the selectable marker confers resistance to a selective agent . growth of plant cells on medium containing the selective agent allows phenotypic differentiation of the transgenic and non - transgenic plant cells . cells lacking the selectable marker are unable to grow in the presence of the selective agent . explants are obtained from either dandelion cultures grown in micropropagation media or from pot - grown dandelion leaves . the explants are placed onto preculture plates and placed under mixed white and red lights ( 1 : 1 ) prior to transformation . co - culturing of leaf explants and a liquid culture of agrobacterium tumefaciens bacteria harboring the dna plasmid is performed for approximately 15 - 30 minutes . the bacterial culture is removed , and the explants are briefly dried and stored in the dark or under low light conditions at approximately 22 . degree . c . for about two days to continue co - culturing with the agrobacterium tumefaciens . explants are moved to shoot induction medium for about seven days at approximately 22 . degree . c . the samples are kept under mixed white and red lights ( 1 : 1 ) during the incubation in shoot induction medium . the samples are transferred onto selection medium containing about 1 % maltose and appropriate selective agents , and cultured for about three weeks . subcultures are performed approximately every three weeks . transformed explants produce green shoots and green callus . explants containing green shoots and callus are selected for further processing . shoots are rooted on rooting medium for about three to four weeks . shoots are potted in soil to grow into dandelion plants . the following protocols are included to specify conditions , components , and methods involved in the preparation of transgenic dandelion plants . one skilled in the art will recognize that changes to the compositions , concentrations , times , and steps may be made without deviating from the scope and spirit of the invention . where alternative compositions or methods are available , they are indicated by different letters , e . g . media a , media b , method a , method b . dandelion seeds are surface - sterilized in approximately 70 % ( volume / volume , v / v ) ethanol for 30 sec in the bottle . the ethanol is removed and the seeds are soaked in approximately 1 % ( v / v ) bleach with gentle agitation for about 15 minutes . the bleach is poured off and the leaves are rinsed thoroughly about 3 - 4 times with sterilized distilled water . seeds are germinated and in vitro cultured in the sterile bottles containing seedling medium ( table 1 ). over one month - old seedlings are used as stock plants for leaf and root explants . stock plants are also grown in pots containing soil mixture ( peat : vermiculite : perite = 1 : 1 : 1 ) in the greenhouse . the leaves or roots are surface sterilized by rinsing in water in a sterile bottle . the leaves or roots are then briefly immersed in approximately 70 % ( v / v ) ethanol for 30 sec in the bottle . the ethanol is removed and the leaves are soaked in approximately 1 % ( v / v ) bleach with gentle agitation for about 15 minutes . the bleach is poured off and the leaves are rinsed thoroughly about 3 - 4 times with sterilized distilled water . the leaves and roots of stock plants are placed in a petri plate with droplets of sterile water . explants of leaves ( ca . 0 . 5 cm in diameter ) and of roots ( ca . 0 . 5 cm in length ) were excised from dandelion plants . approximately 15 to 20 explants per plate are positioned with the adaxial surface down onto pre - incubation medium ( table 2 ). the plates are incubated under mixed white and red lights for about 6 days at about 22 . degree . c . agrobacterium is cultured overnight from a frozen stock in 50 ml media containing rifampicin , and kanamycin ( denoted lb - kr , table 3 ) until the optical density at 660 nanometers reaches to about 0 . 8 . the overnight culture is centrifuged , and the pellet is suspended in 50 ml induction solution ( table 4 ). the suspended culture is incubated for 1 h at 28 ° c . this agrobacterium culture is used as the inoculum . after the six - day pre - incubation period , the leaf or root explants are then incubated in the tube with the agrobacterium suspension for about 15 - 30 minutes . enough agrobacterium suspension is added to just cover explants . the tissue is blotted on a sterile whatman filter paper ( whatman is a registered trademark of whatman international , ltd ., hillsboro , oreg .) and placed on co - cultivation plates containing pre - incubation medium ( table 2 ). the plates are then incubated in the dark for about 2 days at approximately 22 . degree . c . after the approximately 2 day co - culture period , the explants are washed with washing solution ( table 5 ), briefly dried , and transferred to shoot induction medium ( table 6 ). the explants are incubated on these plates for 7 days at approximately 22 - 25 . degree . c . after 7 days , the leaves are transferred to selection medium ( table 7 ). the explants are cultured at about 22 . degree . c . and in mixed white and red lights ( 1 : 1 ). the explants are subcultured to fresh medium every two weeks . shoots are rooted on root induction medium ( table 8 ). the rooting step may take 4 - 5 weeks . regenerated plants are potted into 3 - inch pots containing a mixture of peat , vermiculite and perite ( 1 : 1 : 1 ). the containers are covered with plastic wrap for 3 days . subsequently , several holes were made on the cover of plastic wrap to allow for airflow for 3 more days . the wrap is partially opened after 6 days and plants stay under half - opened cover for an additional 10 to 15 days . plants are then transplanted into 6 - inch pots containing a mixture of peat , vermiculite and perite ( 1 : 1 : 1 ). greenhouse temperatures range from about 20 - 25 . degree . c . the following examples are included to demonstrate preferred embodiments of the invention . it should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention , and thus can be considered to constitute preferred modes for its practice . however , those of skill in the art should , in light of the present disclosure , appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention . table 9 displays percentage of leaf explants displaying adventitious shoot formation after four weeks of culture on seedling media ( table 1 ) containing 6 - benzyladenine or kinetin in combination with α - naphthalene acetic acid at different concentrations . parentheses indicate the number of regenerated shoots per explant . combinations of 1 or 2 mg / l 6 - benzyladenine and 0 . 1 mg / l α - naphthalene acetic acid were the most suitable for shoot regeneration of dandelion , based on the percentage of explants displaying shoot formation , the number of regenerated shoots per leaf explant , and regenerated leaf morphology ( table 9 ). in contrast , a combination of either 0 . 05 mg / l α - naphthalene acetic acid and 1 . 0 mg / l kinetin or 0 . 1 mg / l α - naphthalene acetic acid and 2 . 0 mg / l kinetin was also suitable for dandelion shoot regeneration ( table 9 ). leaf explants derived from dandelion plants were cultured in shoot induction media containing no maltose ( w / v ) or 1 % maltose ( w / v ) to compare the efficacy of maltose on shoot production . table 10 demonstrates that maltose improves shoot production in comparison to no maltose . leaf or root explants derived from dandelion plants were cultured in shoot induction media to evaluate the effects of explant sources on regeneration . the results indicate that roots are better source of explants than leaves ( table 11 ). dandelion leaf explants were co - cultivated with agrobacterium tumefaciens strain eha105 carrying a binary vector , pcambia1301 . the explants were transferred to shoot induction medium and incubated under mixed white and red lights for 7 days at approximately 22 . degree . c . afterwards , the explants were placed in selection medium containing 50 mg / l of hygromycin and selected for hygromycin resistant calli . table 12 shows that 7 day incubation in shoot induction medium before hygromycin selection improves the transformation efficiency by agrobacterium up to 1 1 %. to confirm that the gus gene is integrated in the dandelion genome in hygromycin - resistant plants , pcr was performed using genomic dna as a template and primers specific for both the gus gene and the 18s ribosomal protein as a control . a pcr product was observed with the 18s control , but not with gus primers in wild type plants ( fig1 ). pcr products with both gus and 18s primers were observed in hygromycin - resistant shoots , indicating that the gus gene is integrated into the dandelion genome ( fig1 ). to further determine whether gus is transcribed in hygromycin - resistant plants , rt - pcr was performed using primers specific for the gene . no pcr band was observed with wild - type dandelion rna ( fig2 a ). rna obtained from r 1 generation - transformed plants exhibited a 986 bp band ( fig2 a ), but except transgenic plant number 2 , implying the presence of false - positive transgenic plants among hygromycin - resistant plants . expression of the gus gene in transgenic plant no . 3 was low relative to that in other transgenic plants ( fig2 a ), suggesting incomplete transformation or unstable expression . to stabilize gus gene expression , we generated r 2 transgenic plants from the root tissues of r 1 plants . roots were a better source for adventitious shoot induction than leaves as previously observed by other groups . rt - pcr analysis revealed similar levels of gus expression in five independent r 2 transgenic lines ( fig2 b ), suggesting stabilized gene expression . roots were induced from r 2 - transgenic shoots . transgenic plants were transferred to soil pots and grown in the greenhouse . all of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure . while the compositions and methods of this invention have been described in terms of preferred embodiments , it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept , spirit and scope of the invention . more specifically , it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved . all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit , scope and concept of the invention . 6 , 274 , 791 * 8 / 2001 dhir et al . 800 / 278 6 , 483 , 013 * 11 / 2002 sonville et al . 800 / 278 booth , a . et al . 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