Patent Application: US-60002500-A

Abstract:
the present invention is concerned with methods of producing transgenic plants , in particular poppy plants , by way of transfecting and / or regenerating plant material under specified culture conditions which prevent , reduce the rate of or delay the rise in ph of the culture medium .

Description:
the method comprises in part the use of conventional methods of plant transformation and regeneration of transgenic plants but in addition includes steps which improve the conventional methods by stabilising the ph of the medium by way of either preventing or delaying the rapid rise in ph of the culture medium or the plant material . in particularly advantageous variants of the methods of the present invention the ph is stabilised within the range of ph 5 . 5 – 6 . 5 during both transformation and regeneration of transgenic plants . in most plant species studied , the introduction of recombinant dna and the culture of seedling explants on standard tissue culture media is not accompanied by a large rise in ph , however , this hitherto unexpected phenomena has been identified in poppy cultures . it has been observed by the applicants that there is an unexpected and rapid rise in the ph of tissue culture media used to support the growth of papaver somniferum tissues or cells . this phenomenon may also apply to other plants and this can be simply ascertained by those skilled in the art . this very rapid and substantial rise , for example , from ph 5 . 6 to ph & gt ; 6 . 4 in the immediate area around a type ii callus in b5o medium within 30 minutes , rising ultimately to ph 8 . 7 , has been identified as a major cause of poor growth and the difficulty in producing transgenic poppies . the preferred medium for transformation and culturing of transgenic plants is 1 gd ( also referred to as the callusing medium ) buffered with mes . however , any method or medium modification which for preference results in the medium ph remaining within the range of ph 5 . 5 – 6 . 5 is suitable for the method described herein , including , addition of mes buffer ( eg 10 mm ), addition of bis - tris buffer ( eg 10 mm ), addition of ada ( eg 10 mm ), modifying the ammonium and nitrate ion amounts and ratio in the medium ( such as for example no 3 − / nh 4 of 1 : 3 , total n of 30 mm ). in addition to helping control the medium ph during culture , the buffering agents may produce direct or indirect benefits to the process such as improving agrobacterium - mediated gene transfer , type ii callus formation or somatic embryo formation and development . a number of authors have indicated the significance of ph on t - dna transfer by agrobacterium ( holford et al , 1992 ; fenning et al 1996 a . b ; li and komatsuda . 1995 ). the preferred exogenous genetic material used in transformation is the binary vector tab101 containing 35s 5 ′: pat :: 35s 3 ′ ( see fig1 ). another preferred exogenous genetic material is the binary vector bsf16 ( see fig2 .) a further preferred vector is ppop5 ( see fig3 ) which has two genes in the t - dna : the pat gene conferring basta ( or ppt ) resistance ; the papaver somniferum p450 reductase gene which enables the cytochrome p450 reductase enzyme to accumulate to higher levels in the transgenic tissues ; this enzyme donates electrons to reconstitute the cytochrome p450 complex which can be rate limiting for a number of cytochrome p450 - dependent enzymes involved in morphine biosynthesis ; the transgene is therefore expected to increase the biosynthesis of morphinan alkaloids . the pat gene serves two purposes , as a selectable marker in vitro and as the herbicide resistance gene in the transgenic plant . as a selectable gene , it enables selection of transgenic cells in the culture using basta herbicide or the active ingredient , glufosinate ammonium or phosphinothricin ( also known as ppt ). those skilled in the art will know that alternative selectable genes could be employed such as those conferring hygromycin resistance , kanamycin resistance or spectinomycin resistance . it will also be known to those skilled in the art that it is possible to introduce exogenous genetic material coding for more than one desirable property . for instance , the pbsf16 vector has three genes in the t - dna : the bar gene conferring basta ( or ppt ) resistance ; the sunflower albumin gene , sf8g , which enables a novel sunflower seed albumin to accumulate in the seeds of the transgenic plant ; the gus reporter gene , which encodes β - glucuronidase and enables the detection of transgenic tissues . the genotypes of papaver somniferum used were c 046 - 3 - 5 . c 058 , c 060 , c 048 - 6 - 14 - 64 and d 233 ( norman ) obtained from tasmanian alkaloids . seeds are surface sterilised by washing for 30 – 60 seconds in 70 % ethanol then in 1 %( w / v ) sodium hypochlorite solution plus 1 – 2 drops of autoclaved tween 20 or triton x for 20 minutes with agitation . seeds are rinsed three to four times in sterile distilled water or until no smell of bleach remains and placed on 90 × 25 mm petri dishes containing b5o medium ( see below ). dishes are sealed with micropore tape and are usually stored at 4 ° c . for 24 to 48 hours . seeds are germinated at 24 ° c . in a 16 hour light - 8 hour dark cycle . hypocotyls are excised from seedlings after 7 – 8 days of culture and are cut into 3 – 6 mm explants ( usually 1 – 3 explants per seedling ) and used in transformation experiments . b5o medium consists of b5 macronutrients , micronutrients , iron salts and vitamins ( gamborg et al . 1968 ), 20 g / l sucrose using 0 . 8 % sigma agar as the gelling agent . ph is adjusted with 1m koh to ph 5 . 6 . callusing medium ( also referred to as 19d ) is identical to b5o except that it includes 1 mg / l 2 , 4 dichloro phenoxy acetic acid ( 2 , 4 - d ). 19d may be buffered with the appropriate buffering agent selected from mes , bis - tris , ada or modified no 3 / nh 4 + ionic ratios . medium # 7 is 19d but modified in no 3 − / nh 4 + content to achieve a ratio of 1 : 3 with a total n of 30 mm . suitable antibiotics , such as timentin , are added to all media after autoclaving and cooling to 55 – 65 ° c . explant and type i callus cultures are grown in petri dishes sealed with micropore tape at 24 ° c . type ii callus and somatic embryos are cultured at 18 – 21 ° c . the disarmed agrobacterium tumefaciens strains aglo and agl1 ( lazo et al ., 1991 ) are used in transformation experiments . dna constructs are based on the binary vector ppzp201 ( hajdukiewicz et al , 1994 ), e . g . ptab101 ( see fig1 ) with 35s 5 ′: pat : 35s 3 ′. agrobacterium strains are maintained in glycerol at − 80 ° c . or on lb agar plates plus appropriate selection at 4 ° c . fresh cultures are grown overnight at 28 ° c . in 10 ml mg broth ( garfinkle and nester , 1980 ) without antibiotics . this agrobacterium suspension is diluted to approximately 5 × 10 8 cells ml − 1 ( od600 = 0 . 25 ) for use in transformation experiments . hypocotyls are excised from seedlings and immediately inoculated by immersion in liquid agrobacterium culture for 10 − 15 minutes . explants are then transferred directly to 19d , with or without buffering agent , or medium # 7 . after four to five days co - cultivation explants are washed in sterile distilled water , until the water is clear of agrobacterium , blotted on sterile filter paper and transferred to 1 gd , with or without buffering agent , or medium # 7 containing 150 mg / l timentin plus 10 mg / l ppt ( phosphinothricin , the active ingredient of basta herbicide ). timentin is included to control agrobacterium overgrowth and it will be clear to those skilled in the field that suitable alternative antibiotics or agents may also be used . explants are transferred to fresh 1 gd , with or without buffering agent , or medium # 7 , at three weekly intervals . they initially produce friable brownish type i callus and may subsequently form small regions of very white , compact embryogenic callus ( type ii ) by about 7 – 8 weeks culture . type ii callus is transferred to b5o containing 150 mg / l timentin plus 10 mg / l ppt and cultures are transferred to fresh medium every three weeks . meristemoid / embryo development usually occurs after one or two periods on b5o medium and are seen from about 14 – 16 weeks total culture time . plantlet development from embryos is slow and may require a further 3 months in tissue culture before shoot and root growth is sufficient to ensure successful transplantation to soil . if the initial ph of the medium is 5 . 8 and buffering agent is omitted , the ph of poppy cultures rapidly rises to ph 8 . 0 or higher when the callus mass reaches about 1 cm diameter . fresh agar - solidified b5 - based medium adjusted to ph 5 . 6 rose to ph & gt ; 6 . 4 in the immediate area around type ii callus within 30 mins . the inclusion of chlorophenyl red in the medium was sometimes used to observe these localised increases in ph ; the medium turns purple at ph6 . 4 . the whole plate was ph & gt ; 7 within 24 h . at the end of the culture period ph values were measured at 8 . 7 . this rapid rise in ph results in very poor growth which is not compensated for by frequent changes of medium . the rapid rise was significantly delayed even by 2 . 5 mm mes , but 10 mm mes is preferred to adequately buffer the medium and support improved growth over the 3 week subculture period . as shown in table 1 , the use of mes buffer , especially when used throughout the entire transformation and culture process , resulted in a substantial increase in recovery of transgenic plants . an experiment was set up to investigate any possible effects of total nitrogen levels , and the ratio of no 3 − : nh 4 − . this experiment was prompted by literature implying the involvement of n interconversions in medium as a driving force for ph changes ( galvez and clark , 1991 ; nidez , 1994 ; schmitz and lörz , 1990 ; smith and krikorian , 1990 ). mes was not added to any of the media . after twelve weeks of culture , which included two transfers to fresh media , type i and type ii calli were weighed . results are presented in fig4 a and 4b . there are obviously a number of media treatments that appear superior to our standard callusing medium 19d ( medium # 12 in fig4 a and 4b ), especially in terms of type ii ( embryogenic ) callus production . the failure of the standard medium to produce any type ii callus in this experiment is attributable to the absence of mes . the medium # 7 was chosen for further studies and as an alternative way to control ph changes in the medium . the following experiments have focussed on the control of ph increases . the inbred cultivar c 048 - 6 - 14 - 64 was used throughout and the agrobacterium used carried the ppop5 binary vector with pat gene for selection ( ppt as the selection agent ). with agrobacterium co - cultivation and ppt selection , the unbuffered medium ( 19d ) did permit unacceptable rises in ph ( fig5 c ) and accumulation of black or brown pigments even though the amounts of tissue involved were very small . the ph was controlled by the addition of 10 mm of the buffers mes , ada or bis / tris and the pigmentation was less severe . unacceptable increases in medium ph were also controlled by a buffering strategy based on an alteration of the nitrate to ammonium ratio in medium # 7 which has no 3 − : nh 4 + of 1 : 3 molar . the rise in ph was most extreme when explants were under ppt selection but had not been treated with agrobacterium ( fig5 a ). after the first three weeks these cultures showed no growth as expected . without ppt selection and without agrobacterium cocultivation ( fig5 b ) the upward pressures on ph were not evident until the third culture period when the amount of tissue had increased . adequate control of ph under the conditions used was achieved with 10 mm mes ( fig6 a , b , c ). 50 mm mes in the absence of agrobacterium and no ppt permitted healthy unpigmented growth . however , in the presence of agrobacterium and with ppt selection , 20 and 50 mm mes were less effective gave no growth . pooled data over a number of experiments , using two poppy cultivars and a number of binary plasmids are shown in table 1 . these experiments are all under ppt selection . we have confirmed the transgenic status of 23 poppy plants in soil , firstly by pat assay ( eg fig7 ). these plants represent at least five independent transformation events . two of the events are in the cultivar c 058 and three are in norman . all plants in the glasshouse have flowered and seed has been collected . seed ( t 1 ) from one line of c 058 and 4 lines of norman have been sterilised and plated onto medium with and without 10 μg / ml ppt to check the viability of seed , stability of pat gene expression and segregation of the transgene . we have shown that the t 1 seed is viable and most progeny have inherited ppt resistance ( table 3 ). seedlings were also germinated and grown without ppt selection and the segregation of pat enzyme activity has been determined to date on one line ( table 4 ): we have further confirmed the transgenic status of 9 plants representing at least 5 independent events by southern blot analysis . some lines appear to have only a single copy of the pat gene , whereas other lines show multiple inserts ( table 5 ). we have further confirmed the transgenic status of plants and their progeny by demonstrating the accumulation of sunflower seed albumin in the seeds of transgenics produced using the pbsf16 binary vector . this was demonstrated using antibodies specific for the sunflower albumin with western blots of proteins prepared from control and transgenic seed ( see fig8 ). the transgenic status of plants derived from pbsf16 was further demonstrated by incubating various tissues with a substrate for β - glucuronidase , enzyme encoded by the gus gene . in the presence of the substrate , x - glucuronide , leaves , stems , capsules and seeds all stained intensely blue , while the same tissues from non - transgenic controls did not develop any blue colour . although the invention has been described with reference to specific embodiments , modifications that are within the knowledge of those skilled in the art are also contemplated as being within the scope of the present invention . fenning t m . tymens s s . brasier c m , gartland j s , gartland k m a , ahuja m r , boerjan w , and neale d b . 1996 . a strategy for the genetic manipulation of english elm . in “ somatic cell genetics and molecular genetics of trees ”, pp . 105 – 112 . kluwer academic publishers . dordrecht , netherlands . fenning t m , tymens s s , gartland j s , brasier c m , and gartland k m a . 1996 . transformation and regeneration of english elm using wild - type agrobacterium tumefaciens . plant science ( limerick ) 116 : p 37 – 46 . galvez , l ., and r . b . clark . 1991 . nitrate and ammonium uptake and solution ph changes for al - 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