Patent Application: US-15309993-A

Abstract:
a method of producing a genetically transformed fluid - producing plant comprises : i ) inserting into the plant tissue a gene or gene fragment controlling the expression of a target product , and ii ) regenerating a plant from said tissue , the genetically transformed plant being capable of expressing the target product in the fluid that it produces . there are also provided clones of genetically transformed fluid - producing plants which contain in their cells a chromosomal insert such that the target product is expressed in the fluids that the plants produce . there is further described a method of producing a protein or other target product which comprises : i ) harvesting the fluid from a genetically transformed fluid - producing tree or plant , or a clone thereof , and ii ) recovering the target protein or other product from said fluid . most preferably , the plants are rubber plants and the genes are foreign genes that code for pharmaceutically valuable protein products which can be harvested in the latex produced by the plants .

Description:
this invention will now be illustrated further by the following examples . example 1 relates to the genetic transformation of hevea brasiliensis using the particle gun method , while examples 2 and 3 show the use of the agrobacterium vector system and the imbibition technique , respectively . the enzyme glucuronidase is used as an example of the target protein , but the same procedures will generally apply for other products with the gene controlling the production of glucuronidase being substituted by the appropriate gene for the target protein . the following abbreviations are quoted in the examples : embryogenic hevea anther callus was initiated from individual anthers of the staminal column . the callus was initiated and maintained at 25 ° c . on mb medium ( chen et al ., 1984 ) and was used for transformation after four weeks on this medium . all the tissue culture procedures essentially followed the protocol described in chen et al . ( 1984 ). the following plasmids were used for transformation : pb1221 . 1 ( jefferson , 1987 ) containing the β - glucuronidase ( gus ) gene , pmon9793 ( gasser , monsanto company , unpublished ) containing the gus and nptii genes , pde10 ( rhodes , norwich , uk ) containing the cat and nptii genes and php23 ( paszkowski and saul , 1988 ) containing nptii . these genes were under the control of the strong camv 35s promoter . recombinant plasmids were grown in e . coli , isolated by alkali lysis and purified by caesium chloride / ethidium bromide density gradient centrifugation ( sambrook , fritsch and maniatis , 1989 ). plasmid dna was quantified by its absorbance at 260 nm and by gel electrophoresis . plasmid dna was precipitated onto tungsten particles as described previously ( gordon - kamm et al ., 1990 ). the precipitation mixture included 130 mg tungsten particles , 25 μg plasmid dna , 1 . 1m cacl 2 · 2h 2 o and 8 . 7 mm spermidine in a total volume of 575 μl . after addition of components in the above order , the mixture was vortexed at 4 ° c . for 10 minutes , centrifuged at 500 × g for 5 minutes , and then 550 μl of supernatant were discarded . the pellet was resuspended in the remaining 25 μl of supernatant and , from this , 1 μl of the tungsten suspension was loaded onto a microprojectile and accelerated toward the target with a biolistic particle gun ( spearline precision engineering ltd ., uk ). the anther calli were placed in the centre of a whatman no 1 filter paper disc ( 7 cm ) which was then positioned in the centre of a plastic petri dish ( 9 cm diameter ) containing mb medium prior to bombardment . the callus cultures were positioned 5 cm below the microprojectile stopping plate , and a 100 - μm mesh stainless steel screen was placed halfway between the stopping plate and the tissue to aid the dispersion of the tungsten particles . each plate was bombarded once and the calli were then incubated for a short while in a growth chamber at 25 ° c . in the dark . after post - bombardment incubation at 25 ° c . in the dark , calli were gently removed from the filter paper and placed on a fresh plate containing mb medium and further incubated for 10 days at 25 ° c . in the dark . bombarded calli and controls were then transferred to mb selection medium containing 100 μg kanamycin sulphate ml - 1 . antibiotic resistant colonies were isolated four weeks after bombardment and transferred to differentiation medium containing 100 μg kanamycin sulphate ml - 1 and incubated at 25 ° c . in the light ( 250 μe m - 2 s - 1 ). this stimulated the development of shoots and roots ; however , even without antibiotic selection only a small percentage ( 3 %) of the somatic embryos differentiate into plantlets ( chen et al ., 1981 b ). nptii : nptii activity in transformed tissue was determined using an elisa kit ( 5 prime - 3 prime , inc ., obtained through cp laboratories , uk ). approximately 400 μg of extracted protein were used per assay . gus : histochemical analysis of β - glucuronidase activity in transformed anther callus , embryoids and roots of hevea was performed using the substrate 5 - bromo - 4 - chloro - 3 - indolyl glucuronide ( x - gluc ) according to the protocol of jefferson ( 1987 ). tissue was examined for blue stained cells 18 - 36 hours after the addition of x - gluc . photomicrographs were taken using a nikon szm - u 1 : 10 zoom binocular microscope with kodacolor tungsten 160t film . fluorometric assay of gus activity was performed using the substrate 4 - methyl - umbeliferyl - beta - d - glucuronide ( jefferson et al ., 1987 ). cat : cat activity was determined as described by gorman et al ., 1982 . positive controls were run in parallel with bombarded tissue samples using 1 unit of bacterial cat enzyme ( sigma ). negative controls included samples of the cat buffer and extracts from non - bombarded embryoids . dna isolation : genomic dna was isolated using the protease method as described by draper et al ,, ( 1988 ). fifty mg of lyophilised hevea tissue were ground to a fine powder using a pestle and mortar . the powdered tissue was then mixed thoroughly with 400 μl of protease buffer ( 100 mm tris - hcl ph8 . 5 , 100 mm nacl , 50 mm edta ph8 . 0 , 2 . 0 % sds , 0 . 1 mg proteinase k ml - 1 ) and incubated at 37 ° c . for 1 - 2 hours with occasional gentle inversion . the tissue / buffer homogenate was extracted with 80 μl of phenol and 80 μl of 24 : 1 ( v / v ) chloroform / isoamylalcohol . the aqueous phase was separated by centrifugation and the extraction procedure repeated . the collected aqueous phase after the second extraction was precipitated overnight at - 20 ° c . using 0 . 54 volumes of isopropanol . the following day , the precipitate was washed with 70 % ethanol and resuspended in 50 μl te buffer ( 10 mm tris - hcl ph 8 . 0 , 1 . 0 mm edta ph 8 . 0 ). for restriction of hevea genomic dna , 2 μl of 10 × bsa / spermidine ( 2 . 5 mg bsa ml - 1 , 40 mm spermidine ) were routinely included in the restriction buffer to overcome problems due to the presence of impurities . pcr : each pcr reaction was carried out in 50 μl containing 10 mm tris - hcl ph 8 . 8 , 50 mm kcl , 1 . 5 mm mgcl 2 , 50 μl mineral oil , 200 μm datp , 200 μm dttp , 200 μm dctp , 200 μm dgtp , taq polymerase ( 0 . 5 - 1 . 0 u ), dna ( 2 - 10 ng ) and oligonucleotide primers : 5 &# 39 ; ggtgggaaagcgcgtyacaag 3 &# 39 ; and 5 &# 39 ; gtitacgcgttgctyccgcca 3 &# 39 ; ( positions 400 - 420 and 1599 - 1579 respectively in the gus gene , jefferson et al ., 1986 ). 100 ng of each primer were used in the pcr reaction . taq polymerase was obtained from amersham international pic . the denaturation temperature was 92 ° c . ( duration 1 minute ), annealing temperature was 55 ° c . ( duration 1 . 5 minute ) and extension temperature was 72 ° c . ( duration 2 . 0 minute ). the reactions were programmed for 30 cycles , using a programmable thermal controller ( the hybaid thermal reactor ). dna was detected after running samples on 1 . 0 % agarose / ethidium bromide gels using tbe ( 0 . 9m tris - hcl , 25 mm edta , 0 . 9 mm h 3 bo 3 ) as running buffer . the biolistic protocol adopted followed the procedure established for the introduction of genes into barley embryos for studying transient gene expression ( m . g . k . jones , rothamsted , u . k .). for particle bombardment the anther callus used was derived from a highly embryogenic hevea clone ( clone gl1 ). upon transferring the bombarded callus onto selection medium , kanamycin resistant microcalli appeared to grow very slowly , however , these transformed calli appeared healthy and white among the dark and dying non - bombarded calli ( fig1 ). the percentage of antibiotic selected calli and embryoids showing blue coloration upon adding x - gluc reached 90 % in callus and 80 % in embryoids after bombardment using plasmid pmon9793 . using pb1221 . 2 , but without kanamycin selection , the percentage of blue coloration reached 60 % in callus and 70 % in the embryoids ( table 2 ). the higher transfer efficiency exhibited in callus and embryoids bombarded using plasmid pmon9793 is probably due to kanamycin selection . table 2______________________________________percentage of callus and embryoids expressing gus activity______________________________________plasmids 10transformants 10no of calli 9no &# 39 ; s of calli expressing gus 8percentage 90pmon9793 10anther callus 10embryoids 6pbi221 . 2 7anther callus 60embryoids 70______________________________________ the histochemical assay for gus often demonstrated a heterogeneous pattern of enzyme activity in callus maintained without kanamycin selection , but exhibited a homogeneous pattern in cells after kanamycin selection . none of the control ( non - bombarded ) anther callus , embryoids or roots were stained blue when using x - gluc . the results of these experiments are summarized in photomicrographs shown in fig1 . the results obtained from histochemical staining for gus activity were confirmed using a fluorometric assay ( fig2 ). gus fluorometric activity was evident in 90 % of the transformed embryoids analysed and , overall , there was a 4 - fold increase in gus activity in transformed embryoids compared with non - bombarded control values . npt ii levels were quantified in transformed calli using the elisa technique ( fig3 ). overall , npt ii protein levels were approximately four - fold above background control values and ranged from 28 to 32 ng npt ii per mg total protein . in addition to the use of gus as a reporter gene , we also used a cat gene to monitor gene delivery into hevea . the results for these experiments are summarised in the autoradiograph shown in fig4 . it is clear that plasmids containing the cat gene can be delivered by bombardment and are efficiently expressed in hevea callus and embryoid tissue . only a very low level of cat activity was observed in the non - bombarded control tissues ( fig4 lane 3 ). a direct check for the presence of transferred reporter genes in hevea callus was performed using the pcr technique . the method of hamill et al . ( 1991 ) was used to amplify the internal sequence of gus . after 30 cycles of amplification a single band was visible on agarose / ethidium bromide gels using as little as 0 . 8 ng template dna ( fig5 ). this amplified band was identical in size to the gus gene . finally , we managed to regenerate two hevea plantlets ( axenically grown ) after particle bombardment ( fig6 ). the plantlets appeared quite normal and the roots were stained blue after treatment with x - gluc . the presence of the gus gene in one of these plantlets was confirmed using the same pcr protocol performed on dna derived from callus . as shown in fig7 successful amplification of the gus gene was achieved with as little as 4 . 0 ng of hevea leaf dna . we could not amplify the gus gene in any of the control ( non - bombarded ) callus , embryoids or plantlets using the same pcr conditions as described above . these results show that microprojectiles / microparticles can deliver foreign dna into the cells of hevea brasiliensis , as determined by reporter gene assays , the recovery of kanamycin - resistant transformants and the use of the pcr technique . the dna carried into the cell apparently desorbs from the surface of the microprojectile and is transported by some passive or active mechanism into the nucleus where its genes may be expressed . male flowers of selected hevea cultivars were disinfected with chlorox ( 5 . 25 % a . i . sodium hypochlorite ) for five minutes followed by several washes with sterile distilled water . the anthers were excised from the staminal column and inoculated on mb medium ( 20 anthers / petri plate ). the cultures are incubated at 25 ° c . in the dark . four week old hevea anther callus was infected with agrobacterium tumefaciens that bears the genes for the enzymes glucoronidase ( gus ) and neomycin phosphotransferase . the latter confers resistance to the antibiotic kanamycin . the callus was immersed in an overnight culture of agrobacterium tumefaciens suspension , ph 5 . 8 , for one minute , diluted with liquid mb medium containing 7 % sucrose but without hormone supplementation , to give a final estimated bacterial population of 3 . 7 × 10 8 cells / ml . estimation of population density was carried out by measuring absorbance at 600 nm of the overnight culture . the excess bacterial suspension was blotted from the callus using sterile whatman no . 1 filter paper , and the latter was transferred to the petri plate for a co - cultivation period of two days . the callus was transferred to mb medium containing the antibiotics cefotaxime ( 250 μg / ml ) and ticar ( 500 μg / ml ) to kill off the agrobacterium . after one week , the callus was placed on selective plates ( mb , km 100 μg / ml ) but still maintaining cefotaxime and ticar . the concentrations of cefotaxime and ticar were gradually reduced in subsequent sub - cultures at weekly intervals . the callus culture was plated on mb medium and incubated for a period of one week and then transferred to fresh medium ( mb ) every seven days . after three weeks , the culture was transferred to selective differentiation medium . this phase lasts approximately 2 months , with transfers carried out to fresh medium after 30 days . cultures were again incubated in the dark at 25 ° c . both the initiation medium ( mb ) and the differentiation medium contained kanamycin sulphate ( 100 μg / ml ) to select for the transformed cells . after a period of about 60 days in the differentiation medium , the developed embryoids were transferred to the developmental medium for the formation of shoot and roots . the plantlet was planted out in the soil and can then be nurtured to maturity . hevea brasiliensis plantlets have been successfully regenerated after insertion of the gus gene by agrobacterium mediation . the plantlets appeared normal and samples of the leaves were stained blue after treatment with x - gluc , indicating expression of the gus gene . samples of leaves from in vitro - cultured hevea plants that were not transformed were unstained when similarly treated with x - gluc . the results are shown in fig8 a and 8b . the procedure of example 2 was essentially followed except that , instead of the agrobacterium vector system , the genetic transformation was performed by imbibition as follows : four week old hevea anther callus was dehydrated by placing the tissues in a 37 ° c . incubator for 30 minutes . the callus was imbibed for 30 minutes in a dna solution containing the gene for glucoronidase ( 100 μg of pbi221 . 1 dna in sterile water ). the culture was transferred to mb medium and subsequently to a differentiation medium . unlike example 2 , however , the media did not include kanamycin sulphate . fig1 . gus activity in hevea tissue after treatment with the chromogenic substrate x - gluc . ( a ) control callus . ( b ) cells from a 3 week old bombarded anther callus . ( c ) control embryoid . ( d ) embryoids after particle bombardment . all photographs are magnified × 25 . fig2 . a bar chart showing gus activity in bombarded hevea embryoids ( t . e .) compared with control values ( u . t . e .). the results are presented as mean ± s . d . ; n = 20 embryoids . fig3 . npt ii protein levels in bombarded hevea callus compared with control values . the results are presented for 3 different plasmids ( see methodology ) harbouring the npt ii gene . the values are presented as mean ± s . d . ; n - x replicates . fig4 . autoradiograph showing cat activity in hevea tissue previously bombarded with plasmid pde10 . the positions of [ 14 c ] chloramphenicol ( cap ), [ 14 c ] 1 - acetylchloramphenicol ( 1 - cap ), and [ 14 c ] 3 - acetylchloramphenicol ( 3 - cap ) are indicated . lane 1 : commercial cat ( sigma ) used as positive control . lane 2 : negative control ( containing only buffer ). lane 3 : control ( non - bombarded ) anther callus . lanes 4 - 6 : different transformed anther calli three weeks after selection on kanamycin sulphate . lane 7 : embryoid tissue regenerated from bombarded callus in the presence of kanamycin sulphate . fig5 . amplification of the gus gene from dna isolated from x week old antibiotic selected callus of hevea . lane 1 : 1 kb ladder . lane 2 : 500 ng dna . lane 3 : 100 ng dna . lane 4 : 20 ng dna . lane 5 : 4 ng dna . lane 6 : 0 . 8 ng dna . fig6 a . hevea plantlet grown from the particle bombarded tissue shown in fig1 . gus activity after treatment with the chromogenic substrate x - gluc . fig6 b . control root . fig6 c . roots regenerated from transformed embryoids . all photographs are magnified × 25 . fig7 . detection of the gus gene by pcr of genomic dna isolated from a regenerated hevea brasiliensis plantlet . lane 1 : 1 kb ladder . lane 2 : 4 . 0 ng dna from a control ( non - bombarded ) plant . lane 3 : 4 . 0 ng dna from a transformed callus . lane 4 : 4 . 0 ng dna from a transformed embryoid . lane 5 : 4 . 0 ng dna isolated from a single leaf of a transformed hevea plantlet . fig8 a . surfaces of leaf samples incubated in x - gluc . the untransformed sample ( top ) is unstained ; 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