Patent Application: US-46882699-A

Abstract:
a method of genetic improvement of coffee plants , using technique of molecular bleeding , is disclosed . the method provides a transformant of coffee plants produced from embryogenic calli , using agrobacterium method .

Description:
leaf explants of coffee ( coffea canephora ) were prepared from leaves of greenhouse - grown trees , according to the method previously described ( hatanaka et al . 1991 ). the leaf explants were cultured on woody plant agar ( 0 . 9 %) media ( wpm ) which consisted of mccown &# 39 ; s woody plant salt mixture ( lloyd and mccown , 1981 ), gamborg &# 39 ; s b 5 ( gamborg et al . 1968 ) vitamins , 3 % sucrose and 5 μm n 6 -[ 2 - isopentenyl ]- adenosine ( 2 - ip ). the medium was adjusted to ph 5 . 7 before autoclaving at 120 ° c . for 15 min . the culture room was maintained at 25 ° c . with 16 - h light illumination of 24 μmol m − 2 s − 1 ( white fluorescent tubes ). after 4 months of the above culture , embryogenic calli induced from the leaf explants were transferred to callus proliferation medium ( cm ) which consisted of ms salts ( murashige and skoog , 1962 ), 0 . 25 % gellan gum , b 5 vitamin , 3 % sucrose and 10 μm 2 , 4 - dichlorophenoxyacetic acid ( 2 , 4 - d ). the cm medium was also adjusted to ph 5 . 7 before autoclaving at 120 ° c . for 15 min . agrobacterium tumefaciens eha101 harboring pig121 - hm containing β - glucuronidase ( gus )-, hygromycin phosphotransferase ( hpt )- and neomycin phosphotransferase ii ( npt ii ) genes in the t - dna region of the plasmid was used for the transformation . freshly subcultured embryogenic calli ( 3 days after culture ) were co - cultivated in bacterial suspension ( absorbance of 0 . 6 at 600 nm ) for 30 min at 25 ° c . in wpm liquid medium containing 5 μm 2 - ip and 50 mg / l acetosyringone , then these calli were transferred to wpm agar medium containing 50 mg / l acetosyringone , 3 % sucrose and 5 μm 2 - ip at 25 ° c . in the dark for four days . to eliminate bacteria , the calli were washed 5 times with sterilized water , followed by water containing 300 mg / l cefotaxime once . thereafter the embryogenic calli were cultured on wpm agar medium containing 300 mg / l cefotaxime , 5 mg / l hygromycin , and 5 μm 2 - ip , and subcultured on the same medium at 2 week intervals . after 2 months of culture , embryogenic calli were transferred to fresh medium with an increased concentration of hygromycin ( 50 mg / l ). after 2 months of culture , each line of embryogenic callus was maintained by transferring to fresh wpm agar medium containing 5 μm 2 - ip and 100 mg / l hygromycin . after selection at the concentration of 100 mg / l hygromycin , survived embryogenic calli were transferred to wpm medium containing 5 μm 2 - ip in 10 × 2 cm plastic petri dishes . partially germinated embryos ( about 1 - 2 cm in length ) were transferred to phytohormone - free wpm agar medium containing both 100 mg / l hygromycin and 100 mg / l kanamycin for final selection of transgenic plantlets . after selection , they were cultured on wpm agar medium without growth regulators to support continued growth in 300 ml culture bottles . plantlets with both shoots and roots were transferred to plastic pots containing soil and peat moss ( 1 : 1 v / v ) in a greenhouse . histochemical assays of gus were performed for hygromycin - resistant embryogenic calli , somatic embryos , and leaves and roots of plantlets , according to the method of van boxtel et al . ( 1995 ). for staining , the materials were incubated in 5 - bromo - 4 - chloro - 3 - indolyl - β - d - glucuronide ( x - gluc ) solution with a composition modified to 50 mm na 2 hpo 4 , 10 mm na 2 edta , 0 . 3 % triton x - 100 , 0 . 5 mm k 3 fe ( cn ) 6 , 0 . 5 mm k 4 fe ( cn ) 6 , and antioxidants ( 0 . 5 % caffeine , 1 % pvp and 1 % sodium ascorbate ). after 16 hours at 37 ° c ., these explants were immersed in 99 . 5 % ethanol for chlorophyll bleaching and observed under a dissecting microscope . friable and yellow ( fig1 ) embryogenic calli were obtained from leaf explants of coffee ( coffea canephora ) after 4 months of culture on wpm agar medium with 5 μm 2 - ip . embryogenic calli were co - cultivated with agrobacterium tumefaciens eha101 , a super - virulent line for rice transformation ( hiei et al . 1994 ; yokoi et al . 1996 ), in wpm medium containing 50 mg / l 2 acetosyringone and 5 μm 2 - ip for 30 min . after washing in sterilized water , embryogenic calli were transferred to cm solid medium containing 50 mg / l acetosyringone in the dark for 4 days . it has been reported that acetosyringone treatment is highly effective for increasing the transformation efficiency ( james et al . 1993 ). to eliminate remnant bacteria , embryogenic calli were transferred to wpm medium containing 300 mg / l cefotaxime , 5 mg / l hygromycin and 5 μm 2 - ip . after 2 months of culture , about 90 % of the calli ( 267 out of 298 calli ) survived on wpm medium with 5 mg / l hygromycin , and 96 calli ( 36 . 0 %) demonstrated gus positive blue spots after immersion in x - gluc solution ( fig1 ). thereafter , the survived calli were transferred to the same medium containing 50 mg / l hygromycin . after 2 months of culture , 81 ( 43 . 3 %) out of 187 calli that survived on medium with 50 mg / l hygromycin had blue spots by x - gluc reaction ( fig1 ). these 187 calli were transferred to wpm medium containing 100 mg / l hygromycin and 131 calli ( 70 . 1 %) continued to proliferate even after 2 months of incubation . when the hygromycin resistant embryogenic calli were reacted with x - gluc solution , 90 calli ( 68 . 7 %) showed a strong gus positive reaction ( fig2 arrows , fig1 ). however , embryogenic calli without co - cultivation did not show any gus activity ( fig2 arrowhead ). after selection of survived embryogenic calli in the presence of 100 mg / l hygromycin , embryogenic calli were transferred to wpm medium with 5 μm 2 - ip . numerous somatic embryos were formed from the putative transgenic calli after 2 months of culture ( fig3 ). the x - gluc reaction revealed that somatic embryos ( fig4 arrows ) were formed from hygromycin resistant embryogenic calli to be positive . somatic embryos from non - transformed embryogenic calli ( fig4 arrowhead ) were stained negatively except for intrinsic reaction with pale blue color . it had been reported that intrinsic gus - like activity was observed in immature and mature somatic embryos of coffee ( van boxtel et al . 1995 ). somatic embryos germinated and regenerated to small plantlets with shoots and roots after transferring to wpm medium lacking growth regulators . to check finally the transgenic plantlets , the small plantlets ( 1 - 2 cm in length ) were transferred to wpm medium containing both 100 mg / l hygromycin and 100 mg / l kanamycin . in this medium , non - transformed plantlets did not grow at all and rapidly browned ( fig5 arrowheads ), whereas transformed plantlets grew very well ( fig5 arrows ). especially , the roots thrived without showing any growth suppression and browning . eighty seven % of plantlets survived on this medium . after transfer to a medium without growth regulators in petri dishes ( fig6 ) or 300 ml culture bottles , these plantlets grew to about 7 cm in height with about 6 - 10 leaves and formed well - developed roots after 3 - 5 months of culture . transgenic plantlets were transferred to a mixture of autoclaved soil in a greenhouse . most of the plants survived without wilting and the loss of their green color ( fig9 ). the leaves ( fig7 arrow ) and roots ( fig8 arrow ) of the putative transgenic plantlets demonstrated a deep blue color on reaction with x - gluc . explants from non - transformed plantlets ( fig7 - 8 , arrowheads ) did not react with x - gluc . while leaf tissues in the transformed case were not always stained by x - gluc , the roots always showed a strong gus positive reaction . furthermore , surgical wounding on leaf surfaces increased their positivity ( fig7 ), suggesting a blocking effect of the well - developed cuticle of the coffee leaf . dna extraction from leaves of coffee plantlets having positive gus activity was carried out according to the described procedure ( kikuchi et al . 1998 ) using the modified ( addition of 3 % 2 - mercaptoethanol in solution 1 ) benzyl chloride method ( isoplant kit , wako co .). the primers [ seq id nos . : 1 - 4 ] used for amplifying the gus gene were 5 ′- aattgatcagcgttggtgg - 3 ′ and 5 ′- acgcgtggttacagtcttgc - 3 ′ and those for the hpt gene were 5 ′- gcgtgacctattgcatctcc - 3 ′ and 5 ′- ttctacacagccatcggtcc - 3 ′. the reaction mixture for pcr was incubated in a dna thermal cycler ( perkin elmer cetus , 9700 ) under the following conditions : 96 ° c . for 5 min , followed by 30 cycles of 94 ° c . for 30 sec , 58 ° c . for 30 sec , and 72 ° c . for 2 min with a final 5 min extension at 72 ° c . examination of the leaves of gus positive transgenic plantlets ( t ) by pcr revealed amplified fragments coinciding with the gus ( 515 bp band in fig1 ) and hpr ( 713 bp band in fig1 ) genes . in non - transformed plantlets ( n ), neither gus nor hpr genes were detectable . leaf explants of coffee ( coffea arabica ) were prepared from leaves of greenhouse - grown trees , according to the method described previously ( hatanaka et al . 1991 ). leaf explants were cultured on murashige and skoog agar ( 0 . 9 %) medium ( murashige and skoog , 1962 ) containing gamborg &# 39 ; s b 5 ( gamborg et al . 1968 ) vitamins , 3 % sucrose and 10 μm n 6 -[ 2 - isopentenyl ]- adenosine ( 2 - ip ). the medium was adjusted to ph 5 . 7 before autoclaving at 120 ° c . for 15 min . the culture room was maintained at 25 ° c . with 16 - h light illumination of 24 μmol m − 2 s − 1 ( white fluorescent tubes ). after selection of embryogenic callus , these calli were serially subcultured by two - week intervals onto ms medium supplemented with 0 . 9 % agar , b 5 vitamin , 3 % sucrose and 10 μm 2 - ip to induce friably embryogenic callus . agrobacterium tumefaciens eha101 harboring psmbuba containing bar and hygromycin phosphotransferase ( hpt ) genes in the t - dna region of the plasmid was used for the transformation . freshly subcultured embryogenic calli ( 3 days after culture ) were co - cultivated in bacterial suspension ( absorbance of 0 . 6 at 600 nm ) for 30 min at 25 ° c . in ms liquid medium containing 10 μm 2 - ip and 10 mg / l acetosyringone , then these calli were transferred to ms agar medium containing 10 mg / l acetosyringone , 3 % sucrose and 10 μm 2 - ip at 25 ° c . in the dark for four days . to eliminate bacteria , the calli were washed for 5 times with sterilized water , followed by water containing 300 mg / l cefotaxime once . thereafter the embryogenic calli were cultured on ms agar medium containing 300 mg / l cefotaxime and 10 μm 2 - ip , and subcultured on the same medium at 2 week intervals . after 2 months of culture , embryogenic calli were transferred to fresh ms medium containing hygromycin ( 25 mg / l ) for one month . thereafter , each line of embryogenic callus was maintained by transferring to fresh ms agar medium containing 10 μm 2 - ip and 50 mg / l hygromycin by three weeks of culture cycle . to induce somatic embryos , embryogenic callus maintained on ms medium with 10 μm 2 - ip was transferred to ms medium with 3 μm 2 - ip . somatic embryos developed spontaneously from embryogenic callus . after selection of somatic embryos , they were cultured on ½ ms agar medium with 10 μm ga 3 to support germination . after 3 weeks of culture , small plantlets were transferred to ½ ms agar medium in 300 ml erlenmeyer flasks to support the further growth . hygromycin - resistant embryogenic calli , somatic embryos , and small plantlets survived on medium containing 50 mg / l hygromycin were transferred to ½ ms medium containing 2 mg / l bialaphos . after one month of culture , survival rate was examined . dna extraction from small coffee plantlets resistant to hygromycine was carried out according to a described procedure ( kikuchi et al . 1998 ) using a modified ( addition of 3 % 2 - mercaptoethanol in solution 1 ) benzyl chloride method ( isoplant kit , wako co .). the primers [ seq id nos . : 5 - 6 and 3 - 4 , respectively ] used for amplifying the bar gene were 5 ′- atgagcccagaacgacgcccg - 3 ′ ( forward ) and 5 ′- gctcttgaagccctgtgcctcc - 3 ′ ( reverse ), and those for the bpt gene were 5 ′- gcgtgacctattgcatctcc - 3 ′ ( forward ) and 5 ′- ttctacacagccatcggtcc - 3 ′ ( reverse ). the reaction mixture for pcr was incubated in a dna thermal cycler ( perkin elmer cetus , 9700 ) under the following conditions : 96 ° c . for 5 min , followed by 30 cycles of 94 ° c . for 30 sec , 58 ° c . for 30 sec , and 72 ° c . for 2 min with a final 5 min extension at 72 ° c . yellow ( fig1 ) embryogenic calli were obtained from excised margins of leaf explants of coffee ( coffea arabica ) after 4 months of culture on ms agar medium with 10 μm 2 - ip . these calli were selected and maintained on that medium by 3 weeks of subculture cycle ( fig1 ). embryogenic calli were co - cultivated with agrobacterium tumefaciens eha101 in ms liquid medium containing 10 mg / l acetosyringone and 10 μm 2 - ip and transferred to ms solid medium containing 10 mg / l acetosyringone and 10 μm 2 - ip in the dark for 4 days . to eliminate remnant bacteria , the co - cultivated embryogenic calli were transferred to ms medium containing 300 mg / l cefotaxime and 10 μm 2 - ip . after 2 months of culture , these calli were transferred to the same medium containing 25 mg / l hygromycin . after 2 months of culture , survived embryogenic calli were transferred to ms medium containing 50 mg / l hygromycin . after selection of survived embryogenic calli ( fig1 ) in the presence of 50 mg / l hygromycin , these calli were transferred to ms agar medium containing 2 mg / l bialaphos . in 33 % of embryogenic callus , proliferation and colour was not influenced by the bialaphos treatment . whereas , in non - transformed callus , colour of callus rapidly turn to brown and did not proliferlated further after 2 weeks of culture . to induce somatic embryos from embryogenic callus , embryogenic calli were transferred to ms medium containing 3 μm 2 - ip . prolonged culture of embryogenic callus stimulated somatic embryo formation from embryogenic cells . over one month of subculture cycle was efficient for somatic embryo induction from callus . numerous somatic embryos were formed from the putative transgenic calli after 2 months of culture ( fig1 ). when somatic embryos were transferred to ½ ms medium containing 10 μm ga 3 , germination frequency was strongly enhanced . all ( 100 %) the embryos turn to green after 3 weeks of culture on ga 3 containing medium ( fig1 ). whereas , only 37 % of somatic embryos were in green colour after 3 weeks of culture on ga 3 - free medium and germination speed of somatic embryos was very slow . somatic embryo and small plantlets survived on 50 mg / l hygromycin also tolerant to the 2 mg / l bialaphos . eighty three percent of somatic embryos and 92 % of small plantlets were grew normally in 2 mg / l bilaphos without change of colour and growth ability ( fig1 ). while , in non - transformed somatic embryos and plantlets , most of them were browned and eventually dead after one to two months of culture ( fig1 ). these survived plantlets were transferred to ½ strength ms medium in 300 ml culture bottles for further growth ( fig2 ). examination of transgenic small plantlets ( t ) by genomic pcr revealed amplified fragments coinciding with the bar ( 362 bp band in fig2 ) and hpt ( 713 bp band in fig2 ) genes . in non - transformed plantlets ( n ), neither bar nor hpt genes were detectable ( fig2 , fig2 ). barton cr , adams tl , zarowitz ma ( 1991 ) stable transformation of foreign dna into coffea arabica plants . in : 14 ème colloq sci int café , asic , paris , pp 460 - 464 gamborg ol , miller ra , ojima k ( 1968 ) plant cell cultures . 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