Patent Application: US-44693395-A

Abstract:
this invention relates to a method for the production of stable , genetically transformed , zea mays plants that comprise an exogenous dna expressing phosphinothricin acetyl transferase to impart resistance to phosphinothricin in said plants . the method further comprises transforming said zea mays plants with a second gene and further still wherein said second gene imparts insect resistance , such as the expression of a bacillus thuringiensis toxin gene . the method for achieving stable transformation includes tissue culture methods and media , methods for the bombardment of recipient cells with said dna &# 39 ; s , and methods of regenerating fertile plants from transformed cells . the invention also pertains to a method of breeding said transgenic zea mays plants with a either a non - transgenic plant or a transgenic plant , and the obtention of fertile transgenic plants and seed therefrom .

Description:
for the first time , fertile transgenic maize plants have been produced , opening the door to new vistas of crop improvement based on in vitro genetic transformation . the inventors have succeeded where others have failed by combining and modifying numerous steps in the overall process leading from somatic cell to transgenic plant . although the methods disclosed herein are part of a unified process , for illustrative purposes they may be subdivided into : culturing cells to be recipients for exogenous dna ; cryopreserving recipient cells ; constructing vectors to deliver the dna to cells ; delivering dna to cells ; assaying for successful transformations ; using selective agents if necessary to isolate stable transformants ; regenerating plants from transformants ; assaying those plants for gene expression and for identification of the exogenous dna sequences ; determining whether the transgenic plants are fertile ; and producing offspring of the transgenic plants . the invention also relates to transformed maize cells , transgenic plants and pollen produced by said plants . tissue culture requires media and controlled environments . &# 34 ; media &# 34 ; refers to the numerous nutrient mixtures that are used to grow cells in vitro , that is , outside of the intact living organism . the medium is usually a suspension of various categories of ingredients ( salts , amino acids , hormones , sugars , buffers ) that are required for growth of most cell types . however , each specific cell type requires a specific range of ingredient proportions for growth , and an even more specific range of formulas for optimum growth . rate of cell growth will also vary among cultures initiated with the array of media that permit growth of that cell type . nutrient media is prepared as a liquid , but this may be solidified by adding the liquid to materials capable of providing a solid support . agar is most commonly used for this purpose . bactoagar and gelgro are specific types of solid support that are suitable for growth of plant cells in tissue culture . some cell types will grow and divide either in liquid suspension or on solid media . as disclosed herein , maize cells will grow in suspension , but regeneration of plants requires transfer from liquid to solid media at some point in development . the type and extent of differentiation of cells in culture will be affected not only by the type of media used and by the environment , for example , ph , but also by whether media is solid or liquid . table 1 illustrates the composition of various media useful for creation of recipient cells and for plant regeneration . it is believed by the inventors that the ability to prepare and cryopreserve suspension cultures of maize cells is an important aspect of the present invention , in that it provides a means for reproducibly and successfully preparing cells for transformation . the studies described below set forth techniques which have been successfully applied by the inventors to generate transformable and regenerable suspension cultures of maize cells . a variety of different types of media have been developed by the inventors and employed in carrying out various aspects of the invention , including in particular , the development of suspension cultures . the following table , table 1 , sets forth the composition of the media preferred by the inventors for carrying out these aspects of the invention . table 1______________________________________illustrative embodiments of tissue culturemedia which are used for type ii callusdevelopment , development of suspensioncultures and regeneration of plant cells ( specifically maize cells ) mediumid . optimal othernumber ms * n6 sucrose ph components ** ______________________________________ 52 + - 2 % 6 . 0 0 . 25 mg thiamine 1 mg 2 , 4 - d 10 . sup .- 7 m aba bactoagar101 + - 3 % 6 . 0 100 mg myo - inositol v bactoagar142 + - 6 % 6 . 0 5 mg bap v 0 . 186 mg naa 0 . 175 mg iaa 0 . 403 mg 2 - ip 200 mg myo - inositol bactoagar163 + - 3 % 6 . 0 3 . 3 mg dicamba v 100 mg myo - inositol bactoagar171 + - 3 % 6 . 0 0 . 25 mg 2 , 4 - d v 10 mg bap 100 mg myo - inositol bactoagar173 + - 6 % 6 . 0 5 mg bap v 0 . 186 mg naa 0 . 175 mg iaa 0 . 403 mg 2 - ip 10 . sup .- 5 m aba 200 mg myo - inositol bactoagar177 + - 3 % 6 . 0 0 . 25 mg 2 , 4 - d v 10 mg bap 10 . sup .- 5 m aba 100 mg myo - inositol bactoagar201 - + 2 % 5 . 8 25 mm proline v 1 mg 2 , 4 - d 100 ing casein hydrolysate gelgro ® 205 - + 2 % 5 . 8 25 mm proline v 0 . 5 mg 2 , 4 - d 100 mg casein hydrolysate gelgro ® 227 - + 2 % 5 . 8 25 mm proline v 13 . 2 mg dicamba 100 mg casein hydrolysate gelgro ® 401 + - 3 % 6 . 0 0 . 25 mg thiamine 1 mg 2 , 4 - d 2 mg naa 200 mg casein hydrolysate 500 mg k sulfate 100 mg myo - inositol 400 mg k phosphate ( monobasic ) 402 + - 3 % 6 . 0 0 . 25 mg thiamine 25 mm proline 1 mg 2 , 4 - d 200 mg casein hydrolysate 500 mg k sulfate 400 mg k phosphate ( monobasic ) 100 mg myo - inositol409 + - 3 % 6 . 0 0 . 25 mg thiamine 25 mm proline 10 mg dicamba 200 mg casein hydrolysate 500 mg k sulfate 400 mg k phosphate ( monobasic ) 100 mg myo - inositol501 - - 2 % 5 . 7 clark &# 39 ; s *** gelgro ® ______________________________________ * basic ms medium described in reference 30 . the medium described in ref . 30 is typically modified by decreasing the nh . sub . 4 no . sub . 3 from 1 . 64 g / l to 1 . 55 g / l , and omittmg the pyridoxine hcl , nicotinic acid , myoinositol and glycine . + = present ; - = absent ; v = vitamins ** naa = napthol acetic acid ; iaa = indole acetic acid ; 2ip = 2 , isopentyl adenine ; 2 , 4d = 2 , 4dichlorophenoxyacetic acid ; bap = 6benzyl aminopurine ; aba = abscisic acid *** basic medium described in reference 6 initiation of the suspension culture gii ( a188 × b73 ) 716 ( designated sc716 ) for use in transformation this example describes the development of a maize suspension culture , designated sc716 , which was employed in various of the transformation studies described hereinbelow . the type ii tissue used to initiate the cell suspension was derived from immature embryos of a188 × b73 plated onto n6 - based medium with 1 mg / ml 2 , 4 - d ( 201 ; see table 1 ). a type ii callus was initiated by visual selection of fast growing , friable embryogenic cells . the suspension was initiated within 6 months after callus initiation . tissue chosen from the callus to initiate the suspension consisted of very undifferentiated type ii callus , the characteristics of this undifferentiated tissue are the earliest stages of embryo development along with the soft , friable , undifferentiated tissue underlying it . approximately one gram of tissue was added to 20 mls of liquid medium . in this example , the liquid medium was medium 402 to which different slow - release hormone capsule treatments were added ( see example 12 below ). these capsule treatments included 2 , 4 - d , naa , 2 , 4 - d plus naa , and 2 naa capsules . one flask was initiated for each of the different 402 media plus hormone combinations . every 7 days each culture was subcultured into fresh medium by transferring a small portion of the cellular suspension to a new flask . this involved swirling the original flask to suspend the cells ( which tend to settle to the bottom of the culture vessel ), tilting the flask on its side and allowing the denser cells and cell aggregates to settle slightly . one ml of packed cells was then drawn off from this pool of settled cells together with 4 mls of conditioned medium . a sterile ten ml , wide tip , pipet was used for this transfer ( falcon 7304 ). any very large aggregates of cells which would not pass easily through the pipet tip were excluded . if a hormone capsule was present , it was also transferred to the new flask . after approximately 7 weeks , the loose embryogenic cell aggregates began to predominate and fragment in each of the cultures , reaching a state referred to as &# 34 ; dispersed .&# 34 ; the treatment which yielded the highest proportion of embryogenic clusters was the 402 medium plus a naa capsule . after the cultures became dispersed and were growing at a fast rate , doubling approximately every two to three days as determined by increase in packed cell volume , a one ml packed cell inoculum from each culture was transferred into 401 medium using a ten ml narrow tip pipet ( falcon 7551 ). these transfers were performed about every 31 / 2 days . an inoculum from the 402 plus 2 , 4 - d plus naa capsules culture was also used to initiate a culture in 409 medium ( 402 minus 2 , 4 - d and plus 10 mg / l dicamba ) either with or without 1 ml coconut water ( gibco 670 - 8130 ag ). the most dispersed cultures were cryopreserved after 2 weeks , 2 months or 5 months . the culture grown on 409 with coconut water was brought out of cryopreservation eight months later and thawed , cultured for two weeks on solid 201 culture medium using bms as a feeder layer ( 38 ) and transferred to media 409 without coconut water . the culture was maintained by subculturing twice weekly , using 409 media , by the method described above . initiation of the suspension culture ( a188 × b73 ) 82 ( designated sc82 ) for use in transformation this example describes the development of another cell line employed in various of the transformation studies set forth below , termed sc82 . in the development of sc82 , inoculum for suspension culture initiation was visually selected from a type ii callus that was derived from immature embryos plated on a n6 - based medium containing 13 . 2 mg / l dicamba ( 227 ) ( table 1 ). the suspension culture was initiated within 3 months of initiation of the type ii callus . small amounts ( 50 - 100 mg ) of callus distinguishable by visual inspection because of its highly proembryonic morphology , were isolated from more mature or organized structures and inoculated into a 50 ml flask containing 5 mls of filter - sterilized conditioned medium from the various gii ( a188 × b73 ) 716 suspension cultures ( 402 medium with four types of capsule treatments and 409 medium ). after one week , this 5 ml culture was sieved through a 710 micron mesh and used to inoculate 20 mls of corresponding fresh and filter - sterilized conditioned medium from the established gii ( a188 × b73 ) 716 cultures in 150 ml flasks . after one week or more of growth , two mls of packed cells were subcultured to fresh media by the method described above . the suspension culture maintained on 409 by this method was then cryopreserved within 3 months . the original cell line , which was maintained on 409 ( not a reinoculated cryopreserved culture ) was used in experiments 1 and 2 months later which resulted in stable transformation and selection ( see table 2 below ). the cryopreserved culture was used for experiment 6 ( see table 2 below ). studies following the fate of radioactively labelled plant hormones ( 2 , 4 - d and naa ) showed that within two days corn cells absorb most of the auxins present in suspension culture media . this problem of hormone depletion can be overcome by spiking the cultures with a small amount of auxin every other day . however , spiking cultures is very time consuming when done on a large scale and also increases the risk of contamination as the culture vessels must be opened frequently . slow release plant hormone capsules were developed to overcome these problems . in summary , these capsules comprise a plant hormone , usually in a crystalline state , encapsulated in a silicone matrix surrounded by a silicone limiting membrane . the rate of hormone release is controlled by the size of the diffusible area and the thickness of the membrane . they have the advantages of 1 ) supplying hormones at an acceptable and predictable rate ( e . g ., 20 - 100 μg / 20 ml culture media / day , 2 ) they are of a convenient size ( e . g ., 0 . 5 - 1 . 5 cm in length ) for use in liquid or solid culture medium , 3 ) they are very durable and easily sterilized by autoclaving , and 4 ) they can be stored dry until needed . the present formulation involves the controlled release of a plant hormone or selective agent for a plant tissue culture from an inner matrix containing crystals of the desired agent through an outer diffusion limiting membrane . a preferred embodiment of the formulation is to mix 30 % dry crystals of the desired agent with 70 % ( w / w ) room temperature vulcanizing ( rtv ) silicone which is then injected into silicone tubing having an appropriate diameter and wall thickness for the desired release rate of the desired agent . ( the preferred agents for employing in connection with the slow release capsules are 2 , 4 - d and naa , and the preferred dimensions are 0 . 062 &# 34 ; id × 0 . 125 &# 34 ; od ). the rtv silicone is then polymerized at room temperature or at a higher temperature to accelerate the vulcanization process . following vulcanization of the inner matrix , the tubing is cut to desired lengths and the ends sealed with rtv silicone . the preferred lengths for use in connection with the present invention are about 0 . 5 cm . after the end seals have polymerized , the resulting capsules can either be stored , as is , or autoclaved for 15 minutes on a fast exhaust cycle and stored indefinitely in a sterile form . prior to use the capsules may be equilibrated to establish a stable diffusion gradient across the membrane , or used directly without equilibration . another formulation for a much lower release rate is to enclose crystals of a desired substance suspended in a liquid such as water or silicone oil in a relatively nonpermeable tubing such as nylon - 11 . the release rate from this reservoir can then be regulated by drilling various size holes in the tubing and gluing a silicone window over the hole with silicone medical adhesive . once again the capsules can be sterilized by autoclaving and stored dry until use . an exemplary technique employed by the inventors for preparing slow release hormone capsules is as follows : 1 . two grams of dow corning mdx - 4 - 4210 medical grade elastomer and 0 . 2 grams of dow corning mdx - 4 - 4210 curing agent were weighed into a 10 ml syringe , the bottom of which was capped with a plastic cap . 2 . six - hundred mg of 2 , 4 - d ( or naa ), from which lumps have been removed by sieving through a 411μ stainless steel sieve , was added to the same syringe and thoroughly mixed with the elastomer and curing agent . 3 . the 10 ml syringe and its contents were then degassed for 1 / 2 hr in a vacuum centrifuge to remove bubbles . 4 . dow corning silastic medical grade silicone tubing ( 0 . 062 &# 34 ; id × 0 . 125 &# 34 ; od ) of medium durometer ( 50 shore a ) was preswelled 10 to 30 minutes by soaking in acetone . 5 . the plastic cap was removed from the end of the 10 ml syringe and the degassed silicone - 2 , 4 - d mixture was extruded into the preswollen tubing from which excess acetone had been removed by blowing a stream of air briefly through it . 6 . both ends of the filled tubing were then clamped shut and the tubing heated at 50 degrees ( the boiling point of acetone = 56 . 5 degrees ) overnight to accelerate the polymerization . 8 . the ends of the tubing sections were sealed with dow corning type a medical adhesive and allowed to dry for 24 hr . 9 . the finished capsules are autoclaved dry for 15 - 20 min and stored dry until use . 10 . before use the capsules may be preequilibrated for 48 hr by shaking in 25 ml of sterile 1 to 10 mm khco 31 or added to cultures without equilibration . cryopreservation is important not only because it allows one to maintain and preserve a cell culture for future use , but it also is believed by the inventors that this may be a means for enriching for recipient cells . cell suspensions were cryopreserved using modifications of methods previously reported ( 15 , 49 ). the cryopreservation protocol comprised adding a pre - cooled ( 0 ° c .) concentrated cryoprotectant mixture dropwise over a period of one hour while stirring the cell suspension , which was also maintained at 0 ° c . during this period . the volume of added cryoprotectant was equal to the initial volume of the cell suspension ( 1 : 1 addition ), and the final concentration of cryoprotectant additives was 10 % dimethyl sulfoxide , 10 % polyethylene glycol ( 6000 mw ), 0 . 23 m proline and 0 . 23 m glucose . the mixture was allowed to equilibrate at 0 ° c . for 30 minutes , during which time the cell suspension / cryoprotectant mixture was divided into 1 . 5 ml aliquot ( 0 . 5 ml packed cell volume ) in 2 ml polyethylene cryo - vials . the tubes were cooled at 0 . 5 ° c ./ minute to - 8 ° c . and held at this temperature for ice nucleation . once extracellular ice formation had been visually confirmed , the tubes were cooled at 0 . 5 ° c ./ minute from - 8 to - 35 ° c . they were held at this temperature for 45 minutes ( to insure uniform freeze - induced dehydration throughout the cell clusters ). at this point , the cells had lost the majority of their osmotic volume ( i . e . there is little free water left in the cells ), and they could be safely plunged into liquid nitrogen for storage . the paucity of free water remaining in the cells in conjunction with the rapid cooling rates from - 35 to - 196 ° c . prevented large organized ice crystals from forming in the cells . the cells are stored in liquid nitrogen , which effectively immobilizes the cells and slows metabolic processes to the point where long - term storage should not be detrimental . thawing of the extracellular solution was accomplished by removing the cryo - tube from liquid nitrogen and swirling it in sterile 42 ° c . water for approximately 2 minutes . the tube was removed from the heat immediately after the last ice crystals had melted to prevent heating the tissue . the cell suspension ( still in the cryoprotectant mixture ) was pipetted onto a filter , resting on a layer of agarose - immobilized bms cells ( the feeder layer which provided a nurse effect during recovery ). dilution of the cryoprotectant occurred slowly as the solutes diffused away through the filter and nutrients diffused upward to the recovering cells . once subsequent growth of the thawed cells was noted , the growing tissue was transferred to fresh culture medium . the cell clusters were transferred back into liquid suspension medium as soon as sufficient cell mass had been regained ( usually within 1 to 2 weeks ). after the culture was reestablished in liquid ( within 1 to 2 additional weeks ), it was used for transformation experiments . when necessary , previously cryopreserved cultures may be frozen again for storage . as mentioned previously , there are several methods to construct the dna segments carrying dna into a host cell that are well known to those skilled in the art . the general construct of the vectors used herein are plasmids comprising a promoter , other regulatory regions , structural genes , and a 3 &# 39 ; end . dna segments encoding the bar gene were constructed into a plasmid , termed pdpg165 , which was used to introduce the bialaphos resistance gene into recipient cells ( see fig1 a and c ). the bar gene was cloned from streptomyces hygroscopicus ( 53 ) and exists as a 559 - bp sma i fragment in plasmid pij4101 . the sequence of the coding region of this gene is identical to that published ( 45 ). to create plasmid pdpg165 , the sma i fragment from pij4104 was ligated into a puc19 - based vector containing the cauliflower mosaic virus ( camv ) 35s promoter ( derived from pbi221 . 1 . provided by r . jefferson , plant breeding institute , cambridge , england ), a polylinker , and the transcript 7 ( tr7 ) 3 &# 39 ; end from agrobacterium tumefaciens ( 3 &# 39 ; end provided by d . stalker , calgene , inc ., davis , calif .). an additional vector encoding gus , pdpg208 , ( fig1 b and d ) was used in these experiments . it was constructed using a 2 . 1 kb bamhi / ecori fragment from pagus1 ( provided by j . skuzeski , university of utah , salt lake city , utah ) containing the coding sequence for gus and the nos 3 &# 39 ;- end from agrobacterium tumefaciens . in pagus1 the 5 &# 39 ;- noncoding and 5 &# 39 ;- coding sequences for gus were modified to incorporate the kozak consensus sequence ( 24 ) and to introduce a new hindiii restriction site 6 bp into the coding region of the gene ( see fig1 e ). the 2 . 1 kb bamhi / ecori fragment from pagus1 was ligated into a 3 . 6 kb bamhi / ecori fragment of a puc19 - based vector pcev1 ( provided by calgene , inc ., davis , calif .). the 3 . 6 kb fragment from pcev1 contains puc19 and a 430 bp 35s promoter from cauliflower mosaic virus adjacent to the first intron from maize adh1 . in terms of a member of the r gene complex for use in connection with the present invention , the most preferred vectors contain the 35s promoter from cauliflower mosaic virus , the first intron from maize adh1 , the kozak consensus sequence , sn : bol3 cdna , and the transcript 7 3 &# 39 ; end from agrobacterium tumefaciens . one such vector prepared by the inventors is termed pdpg237 . to prepare pdpg237 ( see fig1 f ), the cdna clone of sn : bol3 was obtained from s . dellaporta ( yale university , usa ). a genomic clone of sn was isolated from genomic dna of sn : bol3 which had been digested to completion with hindiii , ligated to lambda arms and packaged in vitro . plaques hybridizing to two regions of cloned r alleles , r - nj and r - sc ( 97 ) were analyzed by restriction digest . a 2 kb sst - hincii fragment from the psn7 . 0 was used to screen a cdna library established in lambda from rna of light - irradiated scutellar nodes of sn : bol3 . the sequence and a restriction map of the cdna clone was established . the cdna clone was inserted into the same plant expression vector described for pdpg165 , the bar expression vector ( see above ), and contains the 35s cauliflower mosaic virus promoter , a polylinker and the transcript 7 3 &# 39 ; end from agrobacterium tumefaciens . this plasmid , ppdg232 , was made by inserting the cdna clone into the polylinker region ; a restriction map of pdpg232 is shown in fig1 g . the preferred vector , pdpg237 , was made by removing the cdna clone and tr7 3 &# 39 ; end from pdpg232 , with avai and ecori and ligating it with a bamhi / ecori fragment from pdpg208 . the ligation was done in the presence of a bamhi linker as follows : ( seq id no : 2 and seq id no : 3 ) the final construct of pdpg237 contained a cauliflower mosaic virus 35s promoter , the first intron of adh1 , kozak consensus sequence , the bamhi linker , cdna of sn : bol3 , and the tr7 3 &# 39 ; end and is shown in fig1 f . additional vectors have been prepared using standard genetic engineering techniques . for example , a vector , designated pdpg128 , has been constructed to include the neo coding sequence ( neomycin phosphotransferase ( aph ( 3 &# 39 ;)- ii )). plasmid pdpg128 contains the 35s promoter from camv , the neomycin phosphotransferase gene from tn5 ( 66 ) and the tr7 terminator from agrobacterium tumefaciens . another vector , pdpg154 , incorporates the crystal toxin gene and was also prepared by standard techniques . plasmid pdpg154 contains the 35s promoter , the entire coding region of the crystal toxin protein of bacillus thuringiensisvar . kurstaki hd 263 , and the tr7 promoter . various tandem vectors have also been prepared . for example , a bar / aroa tandem vector was constructed by ligating a blunt - ended 3 . 2 kb dna fragment containing a mutant epsp synthase aroa expression unit ( 93 ) to ndei - cut pdpg165 that had been blunted and dephosphorylated ( ndei introduces a unique restriction cut approximately 200 bp downstream of the tr7 3 &# 39 ;- end of the bar expression unit ). transformants having aroa in both orientations relative to bar were identified . a preferred dna delivery system that does not require protoplast isolation or introduction of agrobacterium dna is microprojectile bombardment ( 8 , 23 ). there are several potential cellular targets for microprojectile bombardment to produce fertile transgenic plants : pollen , microspores , meristems , and cultured embryogenic cells are but a few examples . germline transformation in maize has not been previously reported by bombardment of any of these types . one of the newly emerging techniques for the introduction of exogenous dna constructs into plant cells involves the use of microprojectile bombardment . the details of this technique and its use to introduce exogenous dna into various plant cells are discussed in klein , 1989 , wang , et al ., 1988 and christou , et al ., 1988 ( 22 , 50 , 8 ). one method of determining the efficiency of dna delivery into the cells via microprojectile bombardment employs detection of transient expression of the enzyme β - glucuronidase ( gus ) in bombarded cells . for this method , plant cells are bombarded with a dna construct which directs the synthesis of the gus enzyme . apparati are available which perform microprojectile bombardment . a commercially available source is an apparatus made by biolistics , inc . ( now dupont ), but other microprojectile or acceleration methods are within the scope of this invention . of course , other &# 34 ; gene guns &# 34 ; may be used to introduce dna into cells . several modifications of the microprojectile bombardment method were made by the inventors . for example , stainless steel mesh screens were introduced below the stop plate of the bombardment apparatus , i . e ., between the gun and the cells . furthermore , modifications to existing techniques were developed by the inventors for precipitating dna onto the microprojectiles . for bombardment , friable , embryogenic type - ii callus ( 1 ) was initiated from immature embryos essentially as set forth above in examples 1 and 2 . the callus was initiated and maintained on n6 medium ( 5 ) containing 2 mg / l glycine , 2 . 9 g / l l - proline , 100 mg / l casein hydrolysate , 13 . 2 mg / l dicamba or 1 mg / l 2 , 4 - d , 20 g / l sucrose , ph 5 . 8 , solidified with 2 g / l gelgro ( icn biochemicals ). suspension cultures initiated from these callus cultures were used for bombardment . in the case of sc82 , suspension culture sc82 was initiated from type - ii callus maintained in culture for 3 months . sc82 cells ( see example 1 ) were grown in liquid medium for approximately 4 months prior to bombardment ( see table 2 , experiments # 1 and # 2 ). sc82 cells were also cryopreserved 5 months after suspension culture initiation , stored frozen for 5 months , thawed and used for bombardment ( experiment # 6 ). in the case of suspension culture sc716 ( see example 2 ), it was initiated from type - ii callus maintained 5 months in culture . sc716 cells were cultured in liquid medium for 5 months , cryopreserved for 8 months , thawed , and used two months later in bombardment experiments # 4 and # 5 . sc94 was initiated from 10 month old type - ii callus ; and cultured in liquid medium for 5 months prior to bombardment ( experiment # 3 ). prior to bombardment , recently subcultured suspension culture cells were sieved through 1000 μm stainless steel mesh . from the fraction of cell clusters passing through the sieve , approximately 0 . 5 ml packed cell volume ( pcv ) was pipetted onto 5 cm filters ( whatman # 4 ) and vacuum - filtered in a buchner funnel . the filters were transferred to petri dishes containing three 7 cm filters ( whatman # 4 ) moistened with 2 . 5 ml suspension culture medium . the dish containing the filters with the immobilized cell suspensions was positioned 6 cm below the lexan plate used to stop the nylon macroprojectile . with respect to the dna , when more than a single plasmid was used , plasmid dna was precipitated in an equimolar ratio onto tungsten particles ( average diameter approximately 1 . 2 μm , gte sylvania ) using a modification of the protocol described by klein , et al . ( 1987 ). in the modified procedure , tungsten was incubated in ethanol at 65 degrees c . for 12 hours prior to being used for precipitation . the precipitation mixture included 1 . 25 mg tungsten particles , 25 μg plasmid dna , 1 . 1 m cacl 2 and 8 . 7 mm spermidine in a total volume of 575 μl . after adding the components in the above order , the mixture was vortexed at 4 ° c . for 10 min , centrifuged ( 500 × g ) for 5 min and 550 μl of supernatant was decanted . from the remaining 25 μl of suspension , 1 μl aliquots were pipetted onto the macroprojectile for bombardment . each plate of suspension cells was bombarded twice at a vacuum of 28 inches hg . in bombarding the embryogenic suspensions of a188 × b73 and a188 × b84 , 100 μm or 1000 μm stainless steel screens were placed about 2 . 5 cm below the stop plate in order to increase the number of foci while decreasing their size and also to ameliorate injury to the bombarded tissue . after bombardment , the suspension cells and the supporting filter were transferred onto solid medium or the cells were scraped from the filter and resuspended in liquid culture medium . cells from embryogenic suspension cultures of maize were bombarded with the bar - containing plasmid pdpg165 alone or in combination with a plasmid encoding gus , pdpg208 ( fig1 ). in experiments in which a gus plasmid was included , two of the filters containing bombarded cells were histochemically stained 48h post - bombardment . the total number of foci ( clusters of cells ) per filter transiently expressing gus was at least 1000 . in two separate studies designed to quantitate transiently expressing cells ( using an sc82 ( a188 × b73 ) suspension culture ), the mean number and standard deviation of gus - staining foci per filter was 1472 +/- 211 and 2930 +/-( n = 3 and 4 , respectively ). the number of cells in individual foci that expressed gus averaged 2 - 3 ( range 1 - 10 ). although histochemical staining can be used to detect cells transformed with the gene encoding gus , those cells will no longer grow and divide after staining . for detecting stable transformants and growing them further , e . g ., into plants , selective systems compatible with viability are required . it is believed that dna is introduced into only a small percentage of cells in any one experiment . in order to provide a more efficient system for identification of those cells receiving dna and integrating it into their genomes , therefore , one may desire to employ a means for selecting those cells that are stably transformed . one exemplary embodiment of such a method is to introduce into the host cell , a marker gene which confers resistance to some agent , e . g . an antibiotic or herbicide . the potentially transformed cells are then exposed to the agent . in the population of surviving cells are those cells wherein generally the resistance - conferring gene has been integrated and expressed at sufficient levels to survive . cells may be tested further to confirm stable integration of the exogenous dna . using embryogenic suspension cultures , stable transformants are recovered at a frequency of approximately 1 per 1000 transiently expressing foci . a specific embodiment of this procedure is shown in example 5 . one of the difficulties in cereal transformation , e . g ., corn , has been the lack of an effective selective agent for transformed cells , from totipotent cultures ( 36 ). stable transformants were recovered from bombarded nonembryogenic black mexican sweet ( bms ) maize suspension culture cells , using the neo gene and selection with the aminoglycoside , kanamycin ( 22 ). this approach is limited because many monocots are insensitive to high concentrations of aminoglycosides ( 12 , 19 ). the stage of cell growth , duration of exposure and concentration of the antibiotic , may be critical to the successful use of aminoglycosides as selective agents to identify transformants ( 26 , 51 , 52 ). in addition , use of the aminoglycosides , kanamycin or g418 , to select stable transformants from embryogenic maize cultures , in the inventors &# 39 ; experience , often results in the isolation of resistant calli that do not contain the neo gene . one herbicide which has been suggested in resistance studies is the broad spectrum herbicide bialaphos . bialaphos is a tripeptide antibiotic produced by streptomyces hygroscopicus and is composed of phosphinothricin ( ppt ), an analogue of l - glutamic acid , and two l - alanine residues . upon removal of the l - alanine residues by intracellular peptidases , the ppt is released and is a potent inhibitor of glutamine synthetase ( gs ), a pivotal enzyme involved in ammonia assimilation and nitrogen metabolism ( 33 ). inhibition of gs in plants by ppt causes the rapid accumulation of ammonia and death of the plant cells . the organism producing bialaphos also synthesizes an enzyme phosphinothricin acetyl transferase ( pat ) which is encoded by the bar gene . the use of the herbicide resistance gene encoding phosphinothricin acetyl transferase ( pat ) is referred to in de 3642 829 a wherein the gene is isolated from streptomyces viridochromogenes . this enzyme acetylates the free amino group of ppt preventing autotoxicity ( 45 ). the bar gene has been cloned ( 29 , 45 ) and expressed in transgenic tobacco , tomato and potato plants ( 10 ) and brassica ( 11 ). in previous reports , some transgenic plants which expressed the resistance gene were completely resistant to commercial ppt and bialaphos in greenhouses . pct application no . wo 87 / 00141 refers to the use of a process for protecting plant cells and plants against the action of glutamine synthetase inhibitors . this application also refers to the use of such of a process to develop herbicide resistance in determined plants . the gene encoding resistance to the herbicide basta ( hoechst phosphinothricin ) or herbiace ( meiji seika bialaphos ) was said to be introduced by agrobacterium infection into tobacco ( nicotiana tabacum cv petit havan sri ), potato ( solanum tuberosum cv benolima ) and tomato ( lycopersicum esculentum ) and conferred on plants resistance to application of herbicides . an exemplary embodiment of vectors capable of delivering dna to plant host cells is the plasmid , pdpg165 . this plasmid is illustrated in fig1 a and ic . a very important component of this plasmid for purposes of genetic transformation is the bar gene which acts as a marker for selection of transformed cells . the suspension culture ( designated sc82 ) used in the initial experiments ( see example 3 ) was derived from embryogenic type - ii callus of a188 × b73 . following bombardment ( see example 3 ), cells on filters were resuspended in nonselective liquid medium , cultured for 1 to 2 weeks and transferred to filters overlaying solid medium containing 1 or 3 mg / l bialaphos . the degree of inhibition of tissue growth during selection was dependent upon the density of the cells on the filter and on the concentration of bialaphos used . at the density plated ( 0 . 5 pcv / filter ), the growth of the cells cultured on 1 mg / l bialaphos was only partially inhibited (· 30 - 50 % of nonselected growth ) and after 3 to 4 weeks much of this tissue was transferred as discrete clumps (· 5 mm in diameter ) to identical medium . on medium containing 3 mg / l bialaphos , the growth of cells on the original selection filter was severely inhibited (· 10 % of nonselected growth ) and selection was carried out without removing the tissue from the original filter . using either selection protocol ( 1 or 3 mg / l bialaphos ), resistant cell colonies emerged on the selection plates of sc82 bombarded with pdpg165 approximately 6 to 7 weeks after bombardment ( fig2 a ). bialaphos - resistant calli were maintained and expanded on selection medium . much of this tissue was embryogenic ( fig2 b ). no colony growth occurred on plates to which cells were added from suspension cultures on which no transforming attempts were made . these are controls which confirm the prediction that cells without the bar gene are not resistant to bialaphos . colonies on solid supports are visible groups of cells formed by growth and division of cells plated on such support . colonies can be seen in fig2 a on a petri dish . in this figure , the cells capable of growth are those that are resistant to the presence of the herbicide bialaphos , said resistance resulting from integration and expression of the bar gene . exposure of cells was to 1 mg / l bialaphos . fig2 b is a magnification showing the morphology of one bialaphos - resistant culture maintained on selection media indicating that growth is embryogenic . as a confirmation that the cells forming the colonies shown in fig2 had indeed incorporated the bar gene and were expressing it , bialaphos - resistant callus lines were analyzed for activity of the bar gene product , phosphinothricin acetyl transferase ( pat ), by thin - layer chromatography . protein extracts from eleven callus lines ( e1 - 11 ) isolated from sc82 bombardment experiments contained pat activity as shown in fig3 and activity levels varied approximately 10 - fold among the isolates . still further and more direct confirmation of the presence of the bar gene was obtained by analysis of the genomic dna of potential transformants by dna gel blots ( fig4 ). the sources of dna which were electrophoresed through the gel were the bialaphos - resistant callus lines designated e1 - e11 and a non - selected control , e0 . ( fig1 indicates the cleavage sites of those enzymes within the bar gene plasmid .) after the dna was electrophoresed through the gel and transferred to nylon membranes , the resulting blot was hybridized with a 32 p - labeled bar gene sequence from the plasmid pdpgl65 . the radioactivity used per blot was approximately 25 × 10 6 cerenkov cpm . the lane in fig4 designated &# 34 ; 1 &# 34 ; and &# 34 ; 5 &# 34 ; copies contain 1 . 9 and 9 . 5 pg respectively of the 1 . 9 kb bar expression unit released from the plasmid pdpgl65 by application of the ecori and hindiii enzymes ; these amounts represent about 1 and 5 copies per diploid genome . genomic dna from all eleven bialaphos - resistant isolates contained bar - hybridizing sequences as shown in fig4 . the hybridization in all isolates to a fragment migrating slightly larger than 2 kb may be due to contaminating puc19 sequences contained in this bar probe preparation ; no such hybridization occurred in subsequent experiments using the same genomic dna and a different preparation of the bar probe . hybridization to a 1 . 9 kb fragment in eight of the eleven isolates indicated that these isolates contained intact copies of the 1 . 9 kb bar expression unit . the estimated copy numbers of the intact unit ranged from one or two ( e1 , e7 , e8 , e10 , e11 ) to approximately 20 ( e3 , e4 , e6 ). hybridization with the bar probe in isolates e2 and e5 occurred only to a single , higher molecular weight fragment (· 3 kb ). to establish that the pat coding sequence was intact in isolates e2 and e5 , genomic dna was digested with smai , which releases a 559 bp fragment containing the pat structural gene ( fig1 a ), and subjected to dna gel blot analysis using 32 p - labeled bar . this analysis confirmed the presence of a single intact copy of bar . expression of pat in these isolates may not be dependent on the 35s promoter or the tr7 3 &# 39 ; end . the hybridization patterns of some of the isolates were identical ( e2 and e5 ; e7 and e8 ; e3 , e4 , and e6 ); therefore , it is probable that some isolates did not arise from independent transformation events but represent transformants that were separated during selection . seven hybridization patterns were unique , likely representing seven independent single - cell transformation events . the patterns and intensities of hybridization for the seven transformants were unchanged during four months in culture , providing evidence for the stability of the integrated sequences . the seven independent transformants were derived from two separate bombardment experiments . four independent transformants representing isolates e2 / e5 , e3 / e4 / e6 , e1 and e7 / e8 , were recovered from a total of four original filters from bombardment experiment # 1 and the three additional independent transformants , e9 , e10 , and e11 , were selected from tissue originating from six bombarded filters in experiment # 2 . these data are summarized in table 2 . table 2__________________________________________________________________________summary of maize transformation experiments # of # with independent intact # of bar bar # with gus # with cointegration coexpressionexp . culture filters transformants expression coding gus frequency frequency # bombarded bombarded recovered units sequence activity (%) (%) __________________________________________________________________________1 sc82 4 4 3 n . a . 2 sc82 6 3 2 n . a . 3 sc94 10 8 6 n . a . 4 sc716 * 8 13 8 11 3 85 235 sc716 * 8 7 4 6 1 86 146 sc82 * 4 19 17 13 3 68 16 totals 40 54 40 30 7 77 18 ( 30 / 39 ) ( 7 / 39 ) __________________________________________________________________________ * culture reinitiated from cryopreserved cells n . a . not applicable ; only pdpg165 dna used or cotransformation analysis not done studies with other embryogenic suspension cultures produced similar results . using either an sc82 culture that was reinitiated from cryopreserved cells ( experiment # 6 ) or an a188 × b84 ( sc94 ) suspension culture ( experiment # 3 ), numerous independent transformants were recovered ( 19 and 18 respectively ; table 2 ). all transformants contained the bar gene and expressed pat . the copy number of bar - hybridizing sequences and levels of pat expression were comparable to the studies described above . integration of the bar gene into cell lines derived from the sc716 suspension culture bombardment studies and subsequent analyses were also performed on the a188 × b73 suspension culture , termed sc716 ( see example 1 ). the resultant transformed plant cells were analyzed for integration of bar genes . to carry out this analysis , genomic dna was obtained from r1 - r21 isolates ; 6 μg of dna was digested with the restriction endonucleases ecori and hindiii , and dna gel blot analysis was performed using the bar gene as probe . in fig9 molecular weights in kb are shown to the right and left . the untransformed control is designated &# 34 ; ro ,&# 34 ; and the last column to the right contains the equivalent of two copies of the bar gene expression unit per diploid genome . for the dna load used , two copies the bar expression unit per diploid genome is 5 . 7 pg of the 1 . 9 kb ecori / hind fragment from the plasmid pdpg165 . the dna separated on the gel blot was hybridized to a 32 p - labeled bar probe . the label activity in the hybridization was approximately 10 × 10 6 cerenkov cpm . in a , the presence of an intact bar expression unit is inferred from the hybridization of the bar probe to a 1 . 9 kb band in the gel . sc716 transformants discussed in example 5 , were further analyzed for integration and expression of the gene encoding gus . as determined by histochemical assay , four of the sc716 transformants ( r5 , r7 , r16 , and r21 ) had detectable gus activity 3 months post - bombardment . expression patterns observed in the four coexpressing callus lines varied . the number of cells with gus activity within any given transformant sampled ranged from · 5 % to · 90 % and , in addition , the level of gus activity within those cells varied . the cointegration frequency was determined by washing the genomic blot hybridized with bar ( fig9 a ) and probing with 32 p - labeled gus sequence as shown in fig9 b . ecori and hindiii , which excise the bar expression unit from pdpg165 , also release from pdpg208 a 2 . 1 kb fragment containing the gus coding sequence and the nos 3 &# 39 ; end ( fig1 b ). seventeen of the independent bar transformants contained sequences that hybridized to the gus probe ; three , r2 , r14 and r19 did not . transformants in which gus activity was detected ( r5 , r7 , r16 and r21 ) had intact copies of the 2 . 1 kb ecori / hindiii fragment containing the gus structural gene ( fig9 b ). transformants that contained large numbers of fragments that hybridized to bar ( r1 , r5 , r21 ) also contained comparable number of fragments that hybridized to the gene encoding gus ( fig9 a and b ). this observation is consistent with those reported using independent plasmids in peg - mediated transformation of a188 × bms protoplasts ( lyznik , et al ., 1989 ) and in studies conducted by the inventors involving bombardment - mediated transformation of bms suspension cells . co - transformation may be achieved using a vector containing the marker and another gene or genes of interest . alternatively , different vectors , e . g ., plasmids , may contain the different genes of interest , and the plasmids may be concurrently delivered to the recipient cells . using this method , the assumption is made that a certain percentage of cells in which the marker has been introduced , have also received the other gene ( s ) of interest . as can be seen in the following examples , not all cells selected by means of the marker , will express the other genes of interest which had been presented to the cells concurrently . for instance , in example 7 , successful cotransformation occurred in 17 / 20 independent transformants ( see table 2 ), coexpression occurred in 4 / 20 . in some transformants , there was variable expression among transformed cells . co - integration and co - expression of the bar gene and the gus gene to cell lines derived from the sc82 suspension culture of the bialaphos - resistant isolates selected from a reinitiation of cryopreserved sc82 cells transformed with separate plasmids ( as described for sc716 ), nineteen independent transformants were selected in this experiment ( experiment # 6 , table 2 ). the frequency of cointegration and coexpression in those isolates was similar to that described for sc716 isolates ( table 2 ). the pattern of gus staining in these transformants varied in a manner similar to that described for coexpressing sc716 transformants . a transformant , y13 , which contained intact gus coding sequence , exhibited varying levels of gus activity as shown in fig8 . this type of expression pattern has been described previously in cotransformed bms cells ( klein , et al ., 1989 ). variable activity detected in the cells from a single transformant may be attributed to unequal penetration of the gus substrate , or differential expression , methylation , or the absence of the gene in some cells . these results show that both the bar gene and the gus gene are present in some of the cells bombarded with the two plasmids containing these genes . co - transformation has occurred . in the cotransformation examples described herein and summarized in table 2 , cotransformation frequency of the non - selected gene was 77 %; coexpression frequency was 18 %. for use in agriculture , transformation of cells in vitro is only one step toward commercial utilization of these new methods . plants must be regenerated from the transformed cells , and the regenerated plants must be developed into full plants capable of growing crops in open fields . for this purpose , fertile corn plants are required . the invention disclosed herein is the first successful production of fertile maize plants ( e . g ., see fig1 a ) from transformed cells . during suspension culture development , small cell aggregates ( 10 - 100 cells ) are formed , apparently from larger cell clusters , giving the culture a dispersed appearance . upon plating these cells to solid media , somatic embryo development can be induced , and these embryos can be matured , germinated and grown into fertile seed - bearing plants . the characteristics of embryogenicity , regenerability , and plant fertility are gradually lost as a function of time in suspension culture . cryopreservation of suspension cells arrests development of the culture and prevents loss of these characteristics during the cryopreservation period . one efficient regeneration system involves transfer of embryogenic callus to ms ( murashige and skoog , 1962 ) medium containing 0 . 25 mg / l 2 , 4 - dichlorophenoxyacetic acid and 10 . 0 mg / l 6 - benzyl - aminopurine . tissue was maintained on this medium for approximately 2 weeks and subsequently transferred to ms medium without hormones ( shillito , et al ., 1989 ). shoots that developed after 2 - 4 weeks on hormone - free medium were transferred to ms medium containing 1 % sucrose and solidified with 2 g / l gelgro r in plant con r containers where rooting occurred . another successful regeneration scheme involved transfer of embryogenic callus to n6 ( chu , et al ., 1975 ) medium containing 6 % sucrose and no hormones ( armstrong and green , 1985 ) for two weeks followed by transfer to ms medium without hormones as described above . regeneration was performed at 25 ° c . under fluorescent lights ( 250 microeinsteins • m - 2 • s - 1 . after approximately 2 weeks developing plantlets were transferred to soil , hardened off in a growth chamber ( 85 % relative humidity , 600 ppm co 2 , 250 microeinsteins • m - 2 •• s - 1 ), and grown to maturity either in a growth chamber or the greenhouse . regeneration of plants from transformed cells requires careful attention to details of tissue culture techniques . one of the major factors is the choice of tissue culture media . there are many media which will support growth of plant cells in suspension cultures , but some media give better growth than others at different stages of development . moreover , different cell lines respond to specific media in different ways . a further complication is that treatment of cells from callus initiation through transformation and ultimately to the greenhouse as plants , requires a multivariate approach . a progression consisting of various media types , representing sequential use of different media , is needed to optimize the proportion of transformed plants that result from each cell line . table 3 illustrates sequential application of combinations of tissue culture media to cells at different stages of development . successful progress is ascertained by the total number of plants regenerated . table 3__________________________________________________________________________plants to soil from bombardment of sc716 ( expts 1 , 2 ; table 2 ). regeneration media progressions 227b 227b 227b 227b 227b 227b 227b 227b # 227b 201b 52 163 205 227b 201b 205 163 227b 201b plants 227b 171 171 171 171 171 173 173 173 173 177 177 tocell line 101 101 101 101 101 101 101 101 101 101 101 101 soil__________________________________________________________________________controlsa01c - 11 x 4 x x x x 2 x x x x x 6 * a01c - 01 x 7 x x x x 27 x x x x x 34 * total x 11 x x x x 29 x x x x x 40 * transformeda01c - 11 x x x 0 0 0 x x 0 0 x x 0a01c - 12 x 2 x 0 0 0 x x 0 0 x x 2a01c - 13 x 5 1 4 0 0 1 1 1 1 x x 14 * a01c - 14 x 2 x 0 0 0 x x 1 0 x x 3 * a01c - 15 x 28 0 12 7 1 23 13 0 0 0 0 84 * a01c - 17 x 7 0 0 0 0 17 0 0 0 0 0 24a01c - 18 x 12 0 0 x 0 21 10 0 x 2 0 45 * a01c - 19 x 0 x x 0 x 0 x x 0 x 0 0a01c - 20 x 10 x 0 0 x 0 x x 0 x 0 10 * a01c - 21 x 0 x x x x 0 x x x x 0 0a01c - 24 2 4 0 0 0 0 6 5 0 0 0 0 17 * a01c - 25 x 9 x x 0 0 1 x 0 0 x x 10a01c - 27 x 0 x x x x 10 x x x x 0 10 * total 2 79 1 16 7 1 79 29 2 1 2 0 219 * combinedcontrols x 11 x x x x 29 x x x x x 40 * transformed 2 79 1 16 7 1 79 29 2 1 2 0 219 * total 2 90 1 16 7 1 108 29 2 1 2 0 259 * __________________________________________________________________________ x = regeneration not attempted by this route . *= more plants could have been taken to soil . 201b = 201 with 1 mg / 1 bialophos . 227b = 227 with 1 mg / 1 bialophos . it can be seen that using the same group of media , cell lines will vary in their success rates ( number of plants ) ( table 3 ). there was also variation in overall success rate , line a01 - 15 yielding the greatest number of plants overall . ( it should be noted , however , that because tissue was limiting not all combinations of media were used on all lines , therefore , overall comparisons are limited .) a preferred embodiment for use on cell lines in general , at least initially , is the combination shown in the second column under the regeneration media progression ( media 227 , 171 , 101 , 501 ). media 227 is a good media for the selective part of the experiments , for example , to use for growth of callus in the presence of bialaphos . this media contains the hormone dicamba . naa and 2 , 4 - d are hormones in other media . in liquid media , these are usually encapsulated for controlled release ( see example 12 hereinbelow ). thus , it can be seen from table 1 that the various media are modified so as to make them particularly applicable to the development of the transformed plant at the various stages of the transformation process . for example , subculture of cells in media 171 after applying the selective agent , yields very small embryos . moreover , it is believed that the presence of bap in the media facilitates development of shoots . myo - inositol is believed to be useful in cell wall synthesis . shoot elongation and root development proceeds after transfer to media 101 . 101 and 501 do not contain the hormones that are required for earlier stages of regeneration . transfer of regenerating plants is preferably completed in an agar - solidified media adapted from a nutrient solution developed by clark ( 1982 ; ref . 6 ), media 501 . the composition of this media facilitates the hardening of the developing plants so that they can be transferred to the greenhouse for final growth as a plant . the salt concentration of this media is significantly different from that of the three media used in the earlier stages , forcing the plant to develop its own metabolic pathways . these steps toward independent growth are required before plants can be transferred from tissue culture vessels ( e . g . petri dishes , plant cans ) to the greenhouse . approximately 50 % of transformed callus lines derived from the initial sc82 and sc716 experiments were regenerable by the routes tested . transgenic plants were regenerated from four of seven independent sc82 transformants and ten of twenty independent sc716 transformants . regeneration of thirteen independently , transformed cell lines and two control lines of sc716 was pursued . regeneration was successful from ten of thirteen transformants . although a total of 458 plantlets were regenerated , due to time and space constraints only 219 transformed plants ( representing approximately 48 % of the total number of regenerants ) were transferred to a soilless mix ( see below ). approximately 185 plants survived . twelve regeneration protocols were investigated and the number of plants regenerated from each route has been quantified ( table 3 ). there appeared to be no significant advantage to maturing the tissues on 201 , 52 , 163 , or 205 ( see table 1 for media codes ) prior to transfer to medium 171 or 173 . the majority of the plants were generated by subculturing embryogenic callus directly from 227 to either 171 or 173 . these plantlets developed roots without addition of exogenous auxins , and plantlets were then transferred to a soilless mix , as was necessary for many of the transformants regenerated from sc82 . the soilless mix employed comprised pro mix , micromax , osmocote 14 - 14 - 14 and vermiculite . pro mix is a commercial product used to increase fertility and porosity as well as reduce the weight of the mixture . this is the bulk material in the mixture . osmocote is another commercial product that is a slow release fertilizer with a nitrogen - phosphorus - potassium ratio of 14 : 14 : 14 . micromax is another commercial fertilizer that contains all of the essential micronutrients . the ratio used to prepare the soilless mix was : 3 bales ( 3 ft 3 each ) pro mix ; 10 gallons ( vol .) vermiculite ; 7 pounds osmocote ; 46 ml micromax . the soilless mix may be supplemented with one or two applications of soluble fe to reduce interveinal chlorosis during early seedling and plant growth . regeneration of transformed sc82 selected cell lines yielded 76 plants transferred to the soilless mix , and 73 survived . the plants were regenerated from six bialaphos - resistant isolates , representing four of seven clonally independent transformants . eighteen protocols were used successfully to regenerate the seventy six plants ( table 4 ). differences in morphology between cell lines deemed some protocols more suitable than others for regeneration . table 4__________________________________________________________________________effects of progression of media on the number of plants regenerated ( sc82 )* 227b 227b 227b 227b 227b 227b 227b 227b 227b 227b 227b 227b 201b 227b 201b 201b 201b 201b 227b 227b 227b 227a 227a 227a 171 52 52 52 171 201b 227b 205 227b 205 1 52 142 173 171 205 209 173 173 173 173 171 173 173 178 171 177 177 178 171cell 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 # ofline 501 501 501 501 501 501 501 501 501 501 501 501 501 501 501 501 501 501 plants__________________________________________________________________________b3 - 14 - 4 1 x 14 x x x 1 1 x 2 x x 5 x 5 x x x 29b3 - 14 - 9 x x 1 1 x 4 1 x x x x x x 1 x 1 x x 9b3 - 14 - 7 x x x x x x x x x x 6 2 x x x x x 1 9b3 - 14 - 6 x x x x 1 x x x x x x x x x x x x x 1b3 - 14 - 3 x x x x x x x x x x x x x x x x x x 0b3 - 14 - 2 x x x x x x x x x x x x x x x x x x 0b3 - 14 - 1 x x x x x x x x x x x x x x x x x x 0b3 - 14 - 5 x x x x x x x x x x x x x x x x x x 0b3 - 13 - 5 x x x x x x x x x x x x x x x x x x 0b3 - 13 - 2 x 1 13 x x x 3 2 2 x x x x x 1 x x x 22b3 - 13 - 1 x 3 x 1 x x x x 1 x x x x x x x 1 x 6total 1 4 28 2 1 4 5 3 3 2 6 2 5 1 6 1 1 1 76__________________________________________________________________________ *= see table 1 for media codes . x = this media progression was either attempted and unsuccessful or not attempted . 227a = 227 with 10 . sup .- 7 m aba . 227b = 227 with 1 mg / 1 bialophos . prior to regeneration , the callus was transferred to either a ) an n6 - based medium containing either dicamba or 2 , 4 - d or b ) an ms - based medium containing 2 , 4 - d . these steps allowed further embryoid development prior to maturation . most of the maturation media contained high bap levels ( 5 - 10 mg / l ) to enhance shoot development and cause proliferation . an ms - based medium with low 2 , 4 - d ( 0 . 25 mg / l ) and high bap ( 10 mg / l ), as described by shillito , et al ., 1989 , was found to be quite effective for regeneration . likewise , an ms - based medium containing 1 μm naa , 1 μm iaa , 2 μm 2 - ip , and 5 mg / l bap ( modified from congar , et al ., 1987 ) also promoted plant regeneration of these transformants . after plantlets recovered by any of the regenerative protocols had grown to five cm , they were transferred to a nutrient solution described by clark , 1982 , which was solidified with gelgro . plantlets which were slow to develop roots were treated with 3 μl droplets of 0 . 3 % iba at the base of the shoot to stimulate rooting . plants with well developed root systems were transferred to a soilless mix and grown in controlled environmental chambers from 5 - 10 days , prior to transfer to the greenhouse . j . assays for integration of exogenous dna and expression of dna in r 0 r 1 plants studies were undertaken to determine the expression of the transformed gene ( s ) in transgenic r 0 and r 1 plants . functional activity of pat was assessed by localized application of a commercial herbicide formulation containing ppt to leaves of sc82 r 0 and r 1 plants . no necrosis was observed on leaves of r 0 plants containing either high levels ( e2 / e5 ), or low levels ( e3 / e4 ) of pat . herbicide - treated e3 / e4 / e6 and control leaves are shown in fig1 a . herbicide was also applied to leaves of e2 / e5 progeny segregating for bar . as demonstrated in fig1 b , leaves of r 1 plants expressing bar exhibited no necrosis six days after application of the herbicide while r 1 plants without bar developed necrotic lesions . no necrosis was observed on transformed leaves up to 30 days post - application . twenty - one r 0 plants , representing each of the four regenerable transformed sc82 callus lines , were also analyzed for expression of the bar gene product , pat , by thin - layer chromatographic techniques . protein extracts from the leaves of the plants were tested . pat activity of one plant regenerated from each callus line is shown in fig5 . all 21 plants tested contained pat activity . furthermore , activity levels were comparable to levels in the callus lines from which the plants were regenerated . the nontransformed plant showed no pat activity ( no band is in the expected position for acetylated ppt in the autoradiograph from the pat chromatogram ). a band appears in the bms lane that is not in lanes containing protein extracts from the plant leaves . this extra band was believed to be an artifact . as another method of confirming that genes had been delivered to cells and integrated , genomic ( chromosomal ) dna was isolated from a nontransformed plant , the four regenerable callus lines and from two r 0 plants derived from each callus line . fig6 illustrates results of gel blot analysis of genomic dna from the four transformed calli ( c ) and the r 0 plants derived from them . the transformed callus and all plants regenerated from transformed callus contained sequences that hybridized to the bar probe , indicating the presence of dna sequences that were complementary to bar . furthermore , in all instances , hybridization patterns observed in plant dna were identical in pattern and intensity to the hybridization profiles of the corresponding callus dna . dna from e3 / e4 / e6 callus and the desired r 0 plants contained approximately twenty intact copies of the 1 . 9 kb bar expression unit ( cauliflower mosaic virus 35s promoterbar - agrobacterium transcript 7 3 &# 39 ;- end ) as well as numerous other bar - hybridizing fragments . e11 callus and plant dna contained 1 - 2 copies of the intact expression unit and 5 - 6 additional non - intact hybridizing fragments . e10 callus and plants contained 1 - 2 copies of the intact bar expression unit . e2 / e5 dna contained a single fragment of approximately 3 kb that hybridized to the probe . to confirm that the hybridizing sequence observed in all plants were integrated into the chromosomal dna , undigested genomic dna from one plant derived from each independent transformant was analyzed by dna gel blot hybridization . hybridization to bar was observed only in high molecular weight dna providing evidence for the integration of bar into the maize genome . plants were regenerated from the coexpressing callus line , y13 , shown in fig8 . plants regenerated from y13 ( experiment # 6 , table 2 ) were assayed for gus activity and histochemically stained leaf tissue from one plant is shown in fig1 c , d , e . numerous cell types including epidermal , guard , mesophyll and bundle sheath cells stained positive for gus activity . staining intensity was greatest in the vascular bundles . although all leaf samples from the regenerated plants tested ( 5 / 5 ) expressed the nonselected gene , some non - expressing leaf sectors were also observed . leaf tissue extracts from three y13 and three control plants were also assayed for gus activity by fluorometric analysis ( jefferson , 1987 ). activity detected in two opposing leaves from each of three y13 plants tested was at least 100 - fold higher than that in control leaves . a method to detect the presence of phosphinothricin acetyl transferase ( pat ) activity is to use thin layer chromatography . an example of such detection is shown in fig5 wherein various protein extracts prepared from homogenates of potentially transformed cells , and from control cells that have neither been transformed nor exposed to bialaphos selection , are assayed by incubation with ppt and 14 c - acetyl coenzyme a . 25 μg of protein extract were loaded per lane . the source in lanes e1 - e11 were sc82 transformants ; b13 is a bms ( black mexican sweet corn nonembryogenic ) bar transformant . e0 is a nonselected , nontransformed control . as can be seen at the position indicated by the arrow ( the position expected for the mobility of 14 c - n - acppt ), all lanes except the nontransformed control have activities with the appropriate mobility . variation in activity among the transformants was approximately 10 fold , as demonstrated by the relative intensity of the bands . the results of this assay provide confirmation of the expression of the bar gene which codes for pat . for analysis of pat activity in plant tissue , 100 - 200 mg of leaf tissue was extracted in sintered glass homogenizers and assayed as described previously . gus activity was assessed histochemically as described using 5 - bromo - 4 - chloro - 3 - indolyl glucuronide ( jefferson , 1987 ); tissue was scored for blue cells 18 - 24 h after addition of substrate . fluorometric analysis was performed as described by jefferson ( 1987 ) using 4 - methyl umbelliferyl glucuronide . dna gel blot analysis was performed as follows . genomic dna was isolated using a procedure modified from shure , et al ., 1983 . approximately 1 gm callus tissue was ground to a fine powder in liquid n2 using a mortar and pestle . powdered tissue was mixed thoroughly with 4 ml extraction buffer ( 7 . 0 m urea , 0 . 35 m nacl , 0 . 05 m tris - hcl ph 8 . 0 , 0 . 01 m edta , 1 % sarcosine ). tissue / buffer homogenate was extracted with 4 ml phenol / chloroform . the aqueous phase was separated by centrifugation , passed through miracloth , and precipitated twice using 1 / 10 volume of 4 . 4 m ammonium acetate , ph 5 . 2 and an equal volume of isopropanol . the precipitate was washed with 70 % ethanol and resuspended in 200 - 500 μl te ( 0 . 01 m tris - hcl , 0 . 001 m edta , ph 8 . 0 ). plant tissue may also be employed for the isolation of dna using the foregoing procedure . genomic dna was digested with a 3 - fold excess of restriction enzymes , electrophoresed through 0 . 8 % agarose ( fmc ), and transferred ( southern , 1975 ) to nytran ( schleicher and schuell ) using 10 × scp ( 20 × scp : 2 m nacl , 0 . 6 m disodium phosphate , 0 . 02 m disodium edta ). filters were prehybridized at 65 ° c . in 6 × scp , 10 % dextran sulfate , 2 % sarcosine , and 500 μg / ml heparin ( chomet , et al ., 1987 ) for 15 min . filters were hybridized overnight at 65 ° c . in 6 × scp containing 100 μg / ml denatured salmon sperm dna and 32 p - labeled probe . the 0 . 6 kb smai fragment from pdpg165 and the 1 . 8 kb bamhi / ecori fragment from pcev5 were used in random priming reactions ( feinberg and vogelstein , 1983 ; boehringer - mannheim ) to generate labeled probes for detecting sequences encoding pat or gus , respectively . filters were washed in 2 × scp , 1 % sds at 65 ° c . for 30 min . and visualized by autoradiography using kodak xar5 film . prior to rehybridization with a second probe , the filters were boiled for 10 min . in distilled h 2 o to remove the first probe and then prehybridized as described above . the herbicide formulation used , basta tx r , contains 200 g / l glufosinate , the ammonium salt of phosphinothricin . young leaves were painted with a 2 % basta solution ( v / v ) containing 0 . 1 % ( v / v ) tween - 20 . the prescribed application rate for this formulation is 0 . 5 - 1 %. in fig1 a , basta r solution was applied to a large area ( about 4 × 8 cm ) in the center of leaves of a nontransformed a188 × b73 plant ( left ) and a transgenic r 0 e3 / e4 / e6 plant ( right ). in fig1 b , basta was also applied to leaves of four r 1 plants ; two plants without bar and two plants containing bar . the herbicide was applied to r 1 plants in 1 cm circles to four locations on each leaf , two on each side of the midrib . photographs were taken six days after application . to recover progeny the regenerated , genetically transformed maize plants ( designated r 0 ), were backcrossed with pollen collected from nontransformed plants derived from seeds , and progeny ( designated r 1 ) that contained and expressed bar were recovered . an important aspect of this invention is the production for the first time of fertile , genetically transformed maize plants ( r 0 ) and progeny ( r 1 ). these were regenerated from embryogenic cells that were transformed . r 1 plants are those resulting from backcrossing of r 0 plants . pollination of transgenic r 0 ears with non - transformed b73 pollen resulted in kernel development . in addition , kernels developed from pistillate flowers on male inflorescences that were pollinated with non - transformed b73 pollen . kernels on transformed r 0 plants from sc82 developed normally for approximately 10 - 14 days post - pollination but after this period the kernels ceased development and often collapsed . most plants exhibited premature senescence at this time . a total of 153 kernels developed sporadically on numerous plants ( see table 5 ): 8 of 37 e2 / e5 plants , 2 of 22 e10 plants , and 3 of 6 e11 plants . viable progeny were recovered by embryo rescue from 11 e2 / e5 plants and one e10 plant . table 5__________________________________________________________________________regenerated plants ( r . sub . 0 ) and progeny ( r . sub . 1 ) # of independent # of bar regenerable # of # # of r . sub . 0 # of # ofexp culture transformants transformed r . sub . 0 reaching producing kernels r . sub . 1 # bombarded recovered callus lines plants maturity kernels recovered plants__________________________________________________________________________1 , 2 sc82 7 4 76 73 23 153 404 , 5 sc716 20 10 219 ( 35 ) ( 9 ) ( 51 ) ( 31 ) 3 sc94 8 . sup . 2 . sup . a . sup . 11 . sup . a ( 0 ) ( 0 ) ( 0 ) ( 0 ) 6 sc82 19 . sup . 4 . sup . a . sup . 23 . sup . a ( 0 ) ( 0 ) ( 0 ) ( 0 ) __________________________________________________________________________ . sup . a regeneration in progress . () experiment still in progress , data still being collected . sc716 r 0 plants were also backcrossed with seed - derived b73 plants . to date , from the 35 mature sc716 r 0 plants nine plants ( representing four independent callus lines ) yielded 51 kernels , 31 of which produced vigorous r 1 seedlings ( table 5 ). most kernels that developed on sc716 plants did not require embryo rescue . kernels often developed for 30 - 40 days on the plant and some were germinated in soil . the remaining seed was germinated on ms - based medium to monitor germination and transferred to soil after a few days . in addition to the improved kernel development observed on sc716 r 0 plants relative to sc82 r 0 plants , pollen dehisced from anthers of several sc716 plants and some of this pollen germinated in vitro ( pfahler 1967 ). transmission of the foreign gene has occurred both through sc716 r 1 ears and using sc716 r 1 - derived pollen on non - transformed ears . pollen obtained from transformed r 1 plants has now been successfully employed to pollinate b73 ears and a large number of seeds have been recovered ( see fig1 c ). moreover , a transformed ear from an r 1 plant crossed with pollen from a non - transformed inbred plant is shown in fig1 d . the fertility characteristics of the r 1 generation has been confirmed both from a standpoint of the pollen &# 39 ; s ability to fertilize non - transformed ears , and the ability of r 1 ears to be fertilized by pollen from non - transformed plants . analysis of progeny ( r 1 ) of transformed r 0 plants for pat and bar a total of 40 progeny of e2 / e5 r 0 plants were analyzed for pat activity , ten of which are shown in fig7 a . of 36 progeny which were assayed , 18 had pat activity . genomic dna from the same ten progeny analyzed for pat activity was analyzed by dna gel blot hybridization for the presence of bar as shown in fig7 b . the six progeny tested that expressed pat contained a single copy of bar identical in mobility to that detected in callus and r 0 plants ; the four pat - negative progeny tested did not contain bar - hybridizing sequences . in one series of assays , the presence of the bar gene product in 18 of 36 progeny indicates a 1 : 1 segregation of the single copy of bar found in e2 / e5 r 0 plants and is consistent with inheritance of pat expression as a single dominant trait . a dominant pattern of inheritance would indicate the presence in the plant of at least one copy of the gene coding for pat . the single progeny recovered from an e10 r 0 plant tested positive for pat activity . it was determined that the methods disclosed in this invention resulted in transformed r 0 and r 1 plants that produced functionally active pat . this was determined by applying basta ( ppt ) to the leaves of plants and determining whether necrosis ( tissue destruction ) resulted from this application . if functionally active pat is produced by the plants , the leaf tissue is protected from necrosis . no necrosis was observed on r 0 plants expressing high levels of pat ( e2 / e5 ) or on plants expressing low levels ( e3 / e4 / e6 ) ( fig1 a ). herbicide was also applied to leaves of r 1 progeny segregating for bar . in these studies , no necrosis was observed on r 1 plants containing and expressing bar , however , necrosis was observed on those r 1 plants lacking the bar gene . this is shown in fig1 b . segregation of bar did not correlate with the variability in phenotypic characteristics of r 1 plants such as plant height and tassel morphology . in fig9 b , the plant on the right contains bar , the plant on the left does not . in addition , most of the r 1 plants were more vigorous than the r 0 plants . of the 23 r 1 seedlings recovered to date from the sc716 transformants , ten of 16 had pat activity . pat activity was detected in four of ten progeny from r 0 plants representing callus line r18 and six of six progeny from r 0 plants representing callus line r9 . in cases where embryo rescue was required , developing embryos were excised from surface disinfected kernels 10 - 20 days post - pollination and cultured on medium containing ms salts , 2 % sucrose and 5 . 5 g / l seakem agarose . large embryos (& gt ; 3 mm ) were germinated directly on the medium described above . smaller embryos were cultured for approximately 1 week on the above medium containing 10 - 5 m abscisic acid and transferred to hormone - free medium for germination . several embryos became bacterially contaminated ; these embryos were transferred to medium containing 300 μg / ml cefoxitin . developing plants were subsequently handled as described for regeneration of r 0 plants . viable progeny , recovered from seven sc82 e2 / e5 plants and one sc82 e10 plant , were sustained by embryo rescue . this method consisted of excising embryos from kernels that developed on r 0 plants . embryos ranged in size from about 0 . 5 to 4 mm in length . small embryos were cultured on maturation medium containing abscisic acid while larger embryos were cultured directly on germination medium . two of the approximately forty viable progeny thus far recovered from sc82 r 0 plants are shown in fig1 b . most of the r 0 plants regenerated from sc82 transformants exhibited an a188 × b73 hybrid phenotype . plants were similar in height to seed derived a188 plants ( 3 - 5 feet ) but had b73 traits such as anthocyanin accumulation in stalks and prop roots , and the presence of upright leaves . many plants , regardless of the callus line from which they were regenerated , exhibited phenotypic abnormalities including leaf splitting , forked leaves , multiple ears per node , and coarse silk . although many of the phenotypic characteristics were common to all r 0 plants , some characteristics were unique to plants regenerated from specific callus lines . such characteristics were exhibited regardless of regeneration route and the time spent in culture during regeneration . nontransformed control plants were not regenerated from this culture and , therefore , cannot be compared phenotypically . pistillate flowers developed on tassels of one e11 ( 1 / 6 ), several e10 ( 3 / 22 ) and almost one - third of the e2 / e5 ( 12 / 37 ) plants with a range of three to approximately twenty ovules per tassel . primary and secondary ears developed frequently on most e2 / e5 , e10 , and e11 plants ; a mature e2 / e5 plant is shown in fig1 a . anthers rarely extruded from the tassels of plants regenerated from sc82 transformants and the limited number of anthers which were extruded did not dehisce pollen . some phenotypic characteristics observed were unique to plants regenerated from a specific callus line such as the lack of ears on e3 / e4 / e6 plants and a &# 34 ; grassy &# 34 ; phenotype ( up to 21 lone narrow leaves ) exhibited by all e11 plants . all sc82 plants senesced prematurely ; leaf necrosis began approximately two weeks after anthesis . the r 0 plants regenerated from sc82 transformed cell lines have tended to senesce prematurely ; typically before the developing kernels were mature . this has necessitated the use of embryo rescue to recover progeny ( r 1 generation ). segregation of bar in the r 1 generation does not correlate with the variability in phenotypic characteristics of r 1 plants such as plant height and tassel morphology . in fig1 b , the plant on the right contains bar , the plant on the left does not . in addition , most of the r 1 plants are more vigorous than the r 0 plants . transformed progeny ( r1 ) have now also begun to yield kernels and r2 plantlets have been recovered . of 219 plants regenerated from 10 independent sc716 transformants , approximately 35 have reached maturity ( table 5 ). the sc716 plants did not exhibit the phenotypic differences which characterized the individual callus lines of sc82 . these plants were more uniform and abnormalities less frequent . the phenotype of these plants closely resembled that of control plants regenerated from a sc716 cryopreserved culture which was not bombarded . plant height ranged from three to six feet with the majority of the plants between five and six feet . most mature plants produced large , multi - branched tassels and primary and secondary ears . pistillate flowers also developed on tassels of several sc716 plants . although anther extrusion occurred at approximately the same low frequency as in the sc82 plants , a small amount of pollen dehisced from some extruded anthers . for most of the sc716 plants that reached maturity , senescence did not commence until at least 30 days after anthesis . the improved characteristics of sc716 plants over sc82 plants indicate that differences between the suspension cultures may be responsible . while the invention is susceptible to various modifications and alternative forms , specific embodiments thereof have been shown by way of example in the drawings and herein be described in detail . it should be understood , however , that it is not intended to limit the invention to the particular forms disclosed , but on the contrary , the intention is to cover all modifications , equivalents , and alternatives falling within the spirit and scope of the invention as defined by the appended claims . the references listed below are incorporated herein by reference to the extent that they supplement , explain , provide a background for , or teach methodology , techniques , and / or compositions employed herein . reference 3 . carlsson j ., drevin h ., axen r . 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( 1978 ), proc natl acad sci usa 75 : 3737 - 3741 __________________________________________________________________________ # sequence listing - ( 1 ) general information :- ( iii ) number of sequences : 3 - ( 2 ) information for seq id no : 1 :- ( i ) sequence characteristics :# pairs ( a ) length : 36 base ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear - ( xi ) sequence description : seq id no : 1 :# 36 tggt aagcttagcg ggcccc - ( 2 ) information for seq id no : 2 :- ( i ) sequence characteristics :# pairs ( a ) length : 29 base ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear - ( xi ) sequence description : seq id no : 2 :# 29 cgct tcaagcttc - ( 2 ) information for seq id no : 3 :- ( i ) sequence characteristics :# pairs ( a ) length : 29 base ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear - ( xi ) sequence description : seq id no : 3 :# 29 agtt cgaagggct__________________________________________________________________________