Patent Application: US-719404-A

Abstract:
the present invention is directed to a method of acacia mangium regeneration through organogenesis and a method of genetic transformation of acacia mangium . the method of regeneration comprises inducing callus from different parts of seedlings and vegetatively micropropagated plantlets as explants ; adventitious bud induction followed by pinnate leaf and bud elongation and eventually , elongated shoots were induced to root . based on the regeneration system , a marker gene gus under cauliflower mosaic virus promoter was introduced to acacia mangium via agrobacterium infection . gus staining in the regenerated plants and southern blot hybridization prove the incorporation of the foreign gene into the host genome and expression of the foreign gene .

Description:
the present invention is directed to methods for genetically transforming tissue of trees , especially acacia mangium , and regenerating whole plants via organogenesis from the transformed tissue . the present invention is further detailed in the following examples , which are offered by way of illustration and are not intended to limit the invention in any manner . standard techniques well known in the art or the techniques specifically described below are utilized . con - sigma centration cat . name molecular formula ( mg / l ) no . macronutrients ammonium nitrate nh 4 no 3 1 , 650 a - 3795 potassium nitrate kno 3 1 , 900 p - 8291 calcium chloride cacl 2 • 2h 2 o 440 c - 2536 dihydrate magnesium sulfate mgso 4 • 7h 2 o 370 — 7774 heptahydrate potassium phosphate kh 2 po 4 170 p - 8416 monobasic , anhydrous ferrous sulfate feso 4 • 7h 2 o 27 . 8 f - 8263 heptahydrate ethylenediamine - c 10 h 14 n 2 o 8 na 2 • 2h 2 o 37 . 3 e - 6635 tetraacetic ( na 2 edta ) acid ( edta ) micronutrients potassium iodide ki 0 . 83 p - 8166 boric acid h 3 bo 3 6 . 2 b - 9645 manganese sulfate mnso 4 • h 2 o 16 . 9 — 7899 monohydrate zinc sulfate znso 4 • 7h 2 o 8 . 6 z - 1001 molybdic acid na 2 moo 4 • 2h 2 o 0 . 25 — 1651 ( sodium salt : dihydrate ) cupric sulfate cuso 4 • 5h 2 o 0 . 025 c - 3036 ( pentahydrate ) cobalt chloride cocl 2 • 6h 2 o 0 . 025 c - 2911 ( hexahydrate ) organic reagents myo - inositol c 6 h 12 o 6 100 i - 3011 nicotinic acid c 6 h 5 no 2 0 . 5 — 0765 glycine c 2 h 5 no 2 2 . 0 g - 6143 thiamine c 12 h 17 cln 4 os • hcl 0 . 1 t - 3902 ( vitamin b1 ) pyridoxine c 8 h 11 no 3 • hcl 0 . 5 p - 9755 ( vitamin b6 ) hydrochloride sigma cat . name molecular formula no . l - ascorbic acid ( vitamin c ) c 6 h 8 o 6 a - 2174 casein enzymatic hydrolysate ( ch ) c - 7290 l - glutamine ( gln ) c 5 h 10 n 2 o 3 g - 9273 l - asparagine monohydrate ( asn ) c 4 h 8 n 2 o 3 • h 2 o a - 4284 l - proline ( pro ) c 5 h 9 no 2 p - 4655 sucrose s - 5390 sigma cat . name no . indole - 3 - acetic acid ( iaa ) i - 2886 α - naphthaleneacetic acid ( naa ) — 0640 1 - phenyl - 3 -( 1 , 2 , 3 - thiadiazol - 5 - yl ) urea ( thidiazuron , tdz ) p - 6186 6 - benzylaminopurine ( 6 - ba ) b - 3408 kinetin ( kt ) k - 0753 gibberellic acid ( ga 3 ) g - 7645 sigma cat . name no . type m a - 4800 purified a - 7921 phytagel p - 8169 e . antibiotics timentin ( t ) beecham pharmaceuticals ( pte ) ltd kanamycin ( k ) sigma genetins ( g418 sulfate ) clontech 8056 - 2 am - 5 ms + 2 , 4 - d 2 . 0 mg / l + kt 3 . 0 mg / l am - 6 ms + 2 , 4 - d 2 . 0 mg / l + kt 0 . 5 mg / l am - 7 ms + naa 2 . 0 mg / l + kt 3 . 0 mg / l am - 8 ms + naa 2 . 0 mg / l + kt 0 . 5 mg / l am - 14 ms + 2 , 4 - d 0 . 5 mg / l + 6 - ba 3 . 0 mg / l am - 15 ms + 2 , 4 - d 1 . 0 mg / l + 6 - ba 3 . 0 mg / l am - 16 ms + 2 , 4 - d 2 . 0 mg / l + 6 - ba 3 . 0 mg / l am - 17 ms + 2 , 4 - d 0 . 5 mg / l + 6 - ba 0 . 5 mg / l am - 18 ms + 2 , 4 - d 1 . 0 mg / l + 6 - ba 0 . 5 mg / l am - 19 ms + 2 , 4 - d 2 . 0 mg / l + 6 - ba 0 . 5 mg / l am - 20 ms + 2 , 4 - d 0 . 5 mg / l + 6 - ba 1 . 0 mg / l am - 21 ms + 2 , 4 - d 1 . 0 mg / l + 6 - ba 1 . 0 mg / l am - 22 ms + 2 , 4 - d 2 . 0 mg / l + 6 - ba 1 . 0 mg / l am - 27 ms + naa 0 . 5 mg / l + 6 - ba 3 . 0 mg / l am - 28 ms + naa 1 . 0 mg / l + 6 - ba 3 . 0 mg / l am - 29 ms + naa 2 . 0 mg / l + 6 - ba 3 . 0 mg / l am - 30 ms + naa 0 . 5 mg / l + 6 - ba 0 . 5 mg / l am - 31 ms + naa 1 . 0 mg / l + 6 - ba 0 . 5 mg / l am - 32 ms + naa 2 . 0 mg / l + 6 - ba 0 . 5 mg / l am - 33 ms + naa 0 . 5 mg / l + 6 - ba 1 . 0 mg / l am - 34 ms + naa 1 . 0 mg / l + 6 - ba 1 . 0 mg / l am - 35 ms + naa 2 . 0 mg / l + 6 - ba 1 . 0 mg / l am - 231 ms + 2 , 4 - d 5 . 0 mg / l + kt 0 . 5 mg / l am - 233 ms + 2 , 4 - d 0 . 5 mg / l + kt 1 . 0 mg / l am - 234 ms + 2 , 4 - d 1 . 0 mg / l + kt 1 . 0 mg / l a . am - 265 : ms basic medium with tdz 1 . 0 mg / l , iaa 0 . 25 mg / l , ch 100 mg / l , vc 100 mg / l , gln 150 mg / l , asn 150 mg / l and pro 150 mg / l , ph 5 . 8 after autoclaving , phytagel 0 . 275 or 0 . 30 %, or agar 0 . 8 % ( a - 4800 , sigma ), sucrose 30 g / l . b . am - 261 : ms basic medium with tdz 1 . 0 mg / l , iaa 0 . 5 mg / l , ch 100 mg / l , vc 100 mg / l , gln 150 mg / l , asn 150 mg / l , pro 150 mg / l , ph 5 . 8 after autoclave , phytagel 0 . 275 or 0 . 30 %, or agar 0 . 8 % ( a - 4800 , sigma ), sucrose 30 g / l . c . am - 304 : ms basic medium with tdz 2 . 0 mg / l , iaa 0 . 25 mg / l , ch 100 mg / l , vc 100 mg / l , gln 150 mg / l , asn 150 mg / l and pro 150 mg / l , ph 5 . 8 after autoclave , phytagel 0 . 275 or 0 . 30 %, or agar 0 . 8 % ( a - 4800 , sigma ), sucrose 30 g / l . d . am - 308 : ms basic medium with tdz 1 . 0 mg / l , iaa 2 . 0 mg / l , ch 100 mg / l , vc 100 mg / l , gln 150 mg / l , asn 150 mg / l and pro 150 mg / l , ph 5 . 8 after autoclaving , phytagel 0 . 275 or 0 . 30 %, or agar 0 . 8 % ( a - 4800 , sigma ), sucrose 30 g / l . a . am - 337 : ms basic medium with tdz 0 . 01 mg / l , ch 100 mg / l , vc 100 mg / l , gln 150 mg / l , asn 150 mg / l and pro 150 mg / l , ph 5 . 8 after autoclaving , phytagel 0 . 275 or 0 . 30 %, or agar 0 . 8 % ( a - 4800 , sigma ), sucrose 30 g / l . b . am - 4 1 : ms basic medium supplemented with 6 - ba 2 mg / l , ch 100 mg / l , vc 100 mg / l , gln 150 mg / l , asn 150 mg / l and pro 150 mg / l , ph 5 . 8 after autoclaving , phytagel 0 . 275 or 0 . 30 %, or agar 0 . 8 %, sucrose 30 g / l . a . modified am - 8 : ms basic medium with naa 2 . 0 mg / l , kt 0 . 5 mg / l , ch100 mg / l , vc 100 mg / l , gln 150 mg / l , asn 150 mg / l and pro 150 mg / l , ph 5 . 8 after autoclaving , phytagel 0 . 30 %, sucrose 30 g / l . b . am - 357 : ½ ms basic medium with naa 2 . 0 mg / l , kt 0 . 5 mg / l , ch 100 mg / l , vc 100mg / l , gln 150 mg / l , asn 150 mg / l and pro 150 mg / l , ph 5 . 8 after autoclaving , phytagel 0 . 30 %, sucrose 30 g / l . c . am - 451 : ½ ms basic medium with naa 2 . 0 mg / l , kt0 . 1 mg / l , ch 100 mg / l , vc 100 mg / l , gln 150 mg / l , asn 150 mg / l and pro 150 mg / l , ph 5 . 8 after autoclaving , phytagel 0 . 35 %, sucrose 20 g / l . stain the tissue overnight at 37 ° c . in gus staining solution . gus staining solution is described by jefferson ( 1987 ). it is x - gluc — 1 mm , sodium phosphate ( ph 7 . 0 )— 100 mm , edta — 10 mm , and triton x - 100 — 0 . 1 %. gus staining showed a positive blue reaction in adventitious buds and stem and leaf ( fig8 , 9 , and 10 ). a method for performing southern blots is described in sambrook et al . ( 1989 ). the method is : 2 - 5 g fresh sample was frozen in liquid n 2 . this was ground with a mortar and pestle in liquid nitrogen to a fine powder . the powder was transferred to a centrifuge tube ( 50 ml ). 15 ml of extraction buffer was added , 2 ml 10 % sds was added and mixed thoroughly . this was incubated at 65 ° c . for 15 minutes . 5 ml 5 m kac was added and shaken vigorously . the mixture was incubated in ice for 20 minutes and then spun at 25 , 000 × g for 20 minutes . the supernatant was filtered through microcloth into a new tube . the dna was precipitated with ½ volume of isopropanol , mixed and incubated at − 20 ° c . for 30 minutes . the dna was pelleted at 25 , 000 × g for 30 minutes , the supernatant was poured off , and the tube was inverted and air dried for 30 minutes . the pellet was dissolved with 0 . 7 ml of 1 × te ( ph 8 . 0 ) and transferred to an eppendorf tube . this was spun 10 minutes . the supernatant was transferred to a new tube , 7 μl rnase ( 10 mg / ml ) was added and left at room temperature for 10 minutes , then 75 μl 3 m naac ( ph 5 . 3 ) and 500 μl of isopropanol were added . the solution was mixed and the dna was pelleted in a microcentrifuge at full speed for 5 minutes . the pellet was washed with 70 % ethanol , air dried and dissolved with 100 μl 1 × te ( ph 8 . 0 ). 100 mm tris - hcl , ph 8 50 ml 50 mm edta , ph 8 50 ml 500 mm nacl 50 ml 10 mm β - me 0 . 6 ml ddh 2 o to 500 ml reaction system i : dna sample 100 μl ( 20 μg ), 10 × hindiii buffer 40 μl , hindiii 8 μl ( 80 units ), add sterile double distilled h 2 o 252 μl to total volume 400 μl . the reaction was incubated at 37 ° c . for overnight . 40 μl of 3 m naac ( ph 5 . 3 ) and ⅔ volume of 100 % ethanol were added to the reaction system and this was incubated at − 20 ° c . for 30 minutes . this was spun at full speed at 4 ° c . for 20 minutes . the supernatant was poured off and the tube was air dried for 30 minutes then the pellet was dissolved in 30 μl sterile double distilled water . electrophoresis was performed on a 0 . 8 % agarose gel in 1 × tbe at 28v for overnight . this step was performed as described in sambrook et al . ( 1989 ). a prehybridization solution of 6 × ssc , 5 × denhardt &# 39 ; s reagent , 0 . 5 % sds , 100 μg / ml denatured , fragmented salmon sperm dna ( stratagene products ) and 50 % formamide was prepared . 50 × denhardt &# 39 ; s is : 5 g of ficoll , 5 g of polyvinylpyrrolidone , 5g of bovine serum albumin and ddh 2 o to 500 ml , filtered and stored at − 20 ° c . after fixing dna to the membrane , the membrane was placed into a hybridization tube containing suitable prehybridization solution using 0 . 2 ml prehybridization solution for each square centimeter of nylon membrane . the membrane was incubated at 42 ° c . for 6 hours . during the prehybridization , labeled probe was prepared using a boehringer mannheim - high primer dna labeling kit . 50 ng of nptii were added then brought to a volume of 8 μl with h 2 o . the dna was denatured in a 100 ° c . heat block for 10 minutes , chilled quickly in ice , and pulse spun . on ice the denatured dna was mixed with : 4 μl high prime reaction mixture , 3 μl of datp , dgtp , dttp mixture , and 5 μl of α - 32 p dctp , 3000 ci / mmol ( biolab ). this was incubated at 37 ° c . for 10 minutes . the reaction was stopped by adding 20 μl of 50 mm edta ( ph 8 . 0 ). the labeled probe was purified by running through a small sephadex g50 column prepared on a small pasteur pipette . the eluent was monitored with a counter and the first peak was collected . the probe was added into the hybridization tube , then incubated at 42 ° c . for 10 - 24 hours . the hybridization solution was poured off and the membrane was submerged in 2 × ssc , 0 . 5 % sds at room temperature for 10 minutes . the membrane was transferred into 2 × ssc , 0 . 1 % sds at room temperature for 15 minutes . the solution was replaced with 0 . 5 × ssc , 0 . 1 % sds and the membrane was incubated at room temperature for 15 minutes . the solution was replaced with 0 . 1 × ssc , 0 . 1 % sds and incubation continued at 55 ° c . for 30 - 55 minutes . the membrane was transferred into 0 . 1 × ssc at room temperature for 3 - 5 minutes , then air dried on 3mm whatman paper for 30 minutes . the membrane was exposed to x - ray film ( kodak ) to obtain an autoradiographic image at − 80 ° c . for one day or more . using nptii fragment as a probe , southern blotting showed that the nptii gene had integrated in adventitious buds ( fig1 ). the results demonstrate that this protocol of acacia mangium transformation is very successful . mature seeds ( black coat ) were pre - treated with 98 % h 2 so 4 for 2 - 3 minutes and washed with tap water several times . treated seeds were sterilized with 70 % ethanol for 2 - 3 minutes and washed times with sterile ddh 2 o . seeds were then immersed in 0 . 1 % hgcl 2 for 6 minutes and washed 5 times with sterile ddh 2 o , again sterilized with bleach 30 % ( market product ) for 6 minutes , then washed 5 times with sterile ddh 2 o . sterilized seeds were soaked in sterile ddh 2 o overnight for isolating zygotes for embryo culture . ms basic medium ( murashige and skoog , 1962 ), ph 5 . 8 , sucrose 30 g / l , phytagel 0 . 25 % or agar 0 . 7 % ( sigma , a - 4800 ), with or without activated charcoal , was used to culture mature embryo . isolated mature zygotic embryos were cultured on ms using a photoperiod of 12 / 12 hours or 16 / 8 hours ( l / d ) or complete dark , at 25 - 28 ° c . germinated hypocotyls or leaves or petioles or stems were used as explants to induce callus formation . different media were used to induce callus , including medium nos . am - 5 , 6 , 7 , 8 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 231 , 233 and 234 at 12 / 12 hours or 16 / 8 hours ( l / d ) or dark at 25 ° c . callus induced from young leaflet : am - l ; from young petiole : am - p ; from hypocotyl : am - h ; from young stem : am - s ; from bud : am - b ; from root : am - r . all above calli were used to induce adventitious buds , using a photoperiod of 16 / 8 hours , 1800 - 2000 lux , and 28 ° c . the results of callus induction on different media were different ( table . 1 ). generally callus induction was not difficult . all explants produced calli at the photoperiod of 12 / 12 hrs or 16 / 8 hrs ( l / d ) ( see , e . g ., fig1 a ; fig2 a ; fig3 a ), and 25 ° c . for 20 days . calli induced from leaflet , petiole , young stem and buds were cultured on am - 261 , am - 265 , am - 304 , and am - 308 , at a photoperiod of 16 / 8 hrs ( l / d ), at 28 ° c . one month later , there were some bud recoveries from callus ( fig1 b ; fig2 b ). bud recovery from friable callus first appeared as compact and smooth bud terminal . the ratio of bud recovery reaches 15 % on am - 265 . but on these media , induced buds could not easily form pinnate leaves and elongate . bud recoveries from callus were transferred into am - 337 or am - 41 with ga 3 2 . 5 mg / l , at a photoperiod of 16 / 8 hrs ( l / d ), 1800 - 2000 lux , and 28 ° c . after culture for one month , some of them could form pinnate leaves and elongate ( fig1 c , d , e ; fig2 c ; and fig3 b ). the ratio of pinnate leaf formation and bud elongation reaches 16 . 67 %. elongated buds were cultured in am - 357 or am - 451 for root formation . 20 days later , adventitious roots began to appear from basic stem of buds ( fig3 c ). after root formation , plantlets were transferred to ms basic medium without any plant growth regulators . plantlets grow normally and fast , and the root system grew well ( fig3 d ). phytagel on plantlets was washed away and the plantlets were transferred to pot ( peat soil : white sand 3 : 1 ) and grown in a growth chamber at a photoperiod of 16 / 8 hours and 25 ° c . one month later the plantlets were transferred to a green house ( fig3 e ). the auxiliary buds of a two - year old young tree ( fig5 a ) were cultured on ms basic medium with naa 0 . 1 mg / l , 6 - ba 3 . 0 mg / l , ch100 mg / l , vc 100 mg / l , pro 150 mg / l , asn 150 mg / l and gln 150 mg / l , phytagel 0 . 275 % or agar 0 . 8 % ( a - 4800 , sigma ), ph 5 . 8 after autoclaving at 121 ° c ., sucrose 30 g / l . sixty days later , some adventitious buds with phyllodes were obtained ( fig5 b ). induced adventitious buds with phyllodes were subcultured on am - 41 ( ms basic medium with 6 - ba 2 mg / l , ch 100 mg / l , vc 100 mg / l , pro 150 mg / l , asn 150 mg / l and gln 150 mg / l , phytagel 0 . 275 % or agar 0 . 8 % ( a - 4800 , sigma ), ph 5 . 8 after autoclaving at 121 ° c ., sucrose 30 g / l ). after sub - culturing adventitious buds with phyllodes twice in about two months , the rejuvenation of adventitious shoots with pinnate leaf was obtained ( fig5 c ). adventitious buds can be used as explants for transformation ( fig6 a ). agrobacterium strain lba4404 ( ooms et al ., 1981 ) harbouring binary vector plasmid pbi121 ( size of vector 13 . 0 kb , clontech , fig4 ) was used for this experiment . pb 121 was derived from pbi101 with an 800 - bp hindiii - bamhi fragment containing the cauliflower mosaic virus ( camv ) 35s promoter cloned upstream of the gus gene . from a permanent glycerol stock stored at − 70 ° c ., agrobacterium tumefaciens pbi12i / lba4404 was streaked onto a solid lb ( ph 7 . 0 ) medium plate containing streptomycin 100 mg / l ( str 100 ) and kanamycin 50 mg / l ( k 50 ). this was incubated 2 - 3 days at 28 ° c . fresh pbi121 / lba4404 was streaked onto solid lb medium ( ph 7 . 0 ) with streptomycin 100 mg / l ( str 100 ) and kanamycin 50 mg / l ( k 50 ) for overnight or 24 hours at 28 ° c . in the dark . several colonies of pbi121 / lba4404 were picked and pbi121 / lba4404 was inoculated in liquid 50 ml lb medium with streptomycin 100 mg / l ( str 100 ) and kanamycin 50 mg / l ( k 50 ), and cultured at 28 ° c . under dark , 250 rpm for 10 hours until od 600 = 0 . 70 - 1 . 10 . this was spun down at 3500 rpm for 30 minutes or 5000 rpm for 10 minutes , and resuspended in 4 - 5 volumes of yep medium ( ph 7 . 0 ) with streptomycin 100 mg / l ( str 100 ) and kanamycin 50 mg / l ( k 50 ), od 600 = 0 . 10 - 0 . 20 , incubated at 28 ° c . in the dark , 250 rpm for 8 - 10 hours , od 600 = 0 . 70 - 1 . 20 . this at 3500 rpm for 30 minutes and resuspended in the same volume of sterile nac 10 . 9 %. this was spun down at 3500 rpm for 30 minutes and resuspended in 2 - 3 . 5 volumes of induction medium , od 600 = 0 . 2 - 0 . 3 , and incubated at 28 ° c . in the dark , 120 rpm for 8 - 15 hours , od 600 = 0 . 70 - 1 . 20 for infecting explants or callus or a cell suspension . stem pieces were cultured on am - 265 for 3 days using a photoperiod of 16 / 8 hrs ( l / d ), 1800 - 2000 lux , at 28 ° c . after preculture , stem pieces were soaked in 0 . 5 m mannitol for 20 - 25 minutes and then transferred to activated pbi121 / lba4404 suspension for 15 minutes . infected young stem pieces were washed once and dried on sterile whatman paper before being cultured on am - 265 with 100 μm acetosyringone at ph 5 . 2 at 22 ° c . in the dark for 3 days . after co - culturing for 3 days , stem pieces were washed with sterile ddh 2 o for 10 times and dried on sterile whatman paper . then stem pieces were cultured on am - 265 containing timentin 250 mg / l and g418 12 mg / l , phytagel 0 . 275 %, using a photoperiod of 16 / 8 hrs ( l / d ) and 28 ° c . for selecting transformed callus or transformed adventitious buds . subsequently , stem pieces were cultured on the above media with g418 12 mg / l for 25 days , g418 20 mg / l for 60 days , g418 30 mg / l for 25 days , then on g418 12 mg / l . after continuous selecting for 4 months on antibiotics , 33 . 75 % of stem pieces formed adventitious buds , and 2 . 5 mg / l to 5 mg / l ga 3 was added into the same media to promote adventitious bud elongation ( fig7 ). after selection for five months , timentin was no longer used in the medium and some adventitious buds were used for gus staining . gus staining showed positive blue colour reaction in adventitious buds ( fig8 , fig9 , and fig1 ). adventitious buds were transferred to am - 41 with ga 3 2 . 5 mg / l to promote pinnate leaf formation . transformed adventitious buds were transferred into am - 357 or am - 451 with or without g418 ( 10 mg / l ) using a photoperiod of 16 / 8 hrs ( l / d ) at 28 ° c . while the invention has been disclosed in this patent application by reference to the details of preferred embodiments of the invention , it is to be understood that the disclosure is intended in an illustrative rather than in a limiting sense , as it is contemplated that modifications will readily occur to those skilled in the art , within the spirit of the invention and the scope of the appended claims . ahmad d h ( 1991 ). “ micropropagation of acacia mangium from aseptically germinated seedlings ” journal of tropical forest science 3 ( 3 ): 204 - 208 . anwar c ( 1992 ). “ the growth of shorea seedlings on soil media of several age levels of acacia mangium stands ” buletin penelitian hutan 0 ( 544 ): 9 - 16 . awang k ( 1994 ). “ growth of three multipurpose tree species on tin tailing in malaysia ” journal of tropical forest science 7 ( 1 ): 106 - 112 . basu p k , ganguly d k , and mandal g s ( 1987 ). “ introduction of exotics in south - 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