Patent Application: US-35619299-A

Abstract:
the present invention provides a method for transforming plants with foreign dna by introducing foreign dna into chloroplasts . the invention provides cassettes which introduce genetic elements into chloroplast of target plants . the plants are transformed with the desired phenotype such as herbicide resistance or of other properties . transformed plants are also disclosed .

Description:
in addition to the techniques described below , the practice of the present invention will employ conventional techniques of molecular biology , microbiology , recombinant dna technology , and plant science , all of which is within the skill of the art . such techniques are explained fully in the literature . see , e . g ., maniatis et al ., molecular cloning : a laboratory manual ( 1982 ); dna cloning : volumes i and ii ( d . n . glover ed . 1985 ); oligonucleotide synthesis ( m . j . gait ed . 1984 ); nucleic acid hybridization ( b . d . hames & amp ; s . j . higgins eds . 1985 ); transcription and translation ( b . d . hames & amp ; s . j . higgins eds . 1984 ); animal cell culture ( r . i . freshney ed . 1986 ); plant cell culture ( r . a . dixon ed . 1985 ); propagation of higher plants through tissue culture ( k . w . hughes et al . eds . 1978 ); cell culture and somatic cell genetics of plants ( i . k . vasil ed . 1984 ); fraley et al . ( 1986 ) crc critical reviews in plant sciences 4 : 1 ( hereinafter plant sciences ); biotechnology in agricultural chemistry : acs symposium series 334 ( lebaron et al . eds . 1987 ). in describing the present invention , the following terminology will be used in accordance with the definitions below . a “ replicon ” is any genetic element ( e . g ., plasmid , cosmid , chromosome , virus , etc .) that behaves as an autonomous unit of dna replication in vivo ; i . e ., capable of replication under its own control within a cell . a “ vector ” is a replicon , such as a plasmid , cosmid , or bacteriophage , to which another dna segment may be attached so as to bring about replication of the attached segment . a “ dna molecule ” refers to the polymeric form of deoxyribonucleotides ( adenine , guanine , thyine , or cytosine ) in either single - or double - stranded form . when in the double - stranded form , the molecule will usually be in its normal , double - stranded helix . the term “ dna molecule ” is not limited to any particular tertiary form of dna . thus , the term includes double - stranded dna found , inter alia , in linear dna molecules , viruses , plasmids and chromosomes . when discussing the structure of particular double - stranded dna molecules , sequences may be described herein according to the normal convention of giving only the sequence in the 5 ′ to 3 ′ direction ( left to right ) along the nontranscribed ( anti - sense ) strand of dna ( i . e , the strand having a sequence homologous to the mrna ). if both strands are shown , the anti - sense strand will be on top . a dna “ coding sequence ” is a dna sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences . the boundaries of the coding sequence are determined by the start codon at the 5 ′ terminus and a translation stop codon at the 3 ′ terminus . examples of coding sequences include cdna reverse transcribed from eukaryotic mrna , genomic dna sequences from eukaryotic cells , and synthetic dna sequences . a cell which has been “ transformed ” by an exogenous dna sequence is a cell into which the exogenous dna has been introduced . the exogenous dna may be integrated ( covalently linked ) to chromosomal dna making up the genome of the cell , or it may remain extrachromosomal . “ stably ” integrated dna sequences are those which are inherited through chromosome replication by daughter cells or organisms ( accounting for loss by mendelian segregation ). this stability is exhibited by the ability to establish cell lines or clones comprised of a population containing the exogenous dna . a “ clone ” or “ cell line ” is a population of cells descended from a single cell or common ancestor by mitosis and is capable of stable growth in vitro for many generations . a composition of a first type of matter ( e . g ., a dna molecule containing a coding region ) is “ substantially free ” of a second type of matter ( e . g ., dna molecules which do not contain a coding region ) if the composition is comprised of less than about 10 % ( weight / weight ) of the second type of matter relative to the sum of the first and second types of matter . preferably , the composition contains less than about 5 % of the second type of matter , most preferably less than about 1 %. a cell organelle , such as a chloroplast , has been “ transformed ” by an exogenous ( foreign ) dna sequence if exogenous or foreign dna has been introduced inside the organelle membrane . the foreign dna may be integrated ( covalently linked ) into chloroplast dna making up the genome of the organelle , or it may remain unintegrated ( e . g ., by including a chloroplast origin or replication ). “ stably ” integrated dna sequences are those which are inherited through organelle replication , thereby transferring new organelles through the cytoplasm to daughter cells . the term “ chloroplast ” includes all developmental stages of a chloroplast , such as proplastids , etioplasts , and mature chloroplasts . in the practice of the present invention , it is preferred to transform etioplasts . “ plant ” refers to any higher or lower plant , particularly dicots and monocots . in particular , the present invention is directed to important dicotyledons such as peas , lentils , watermelon , sunflower , soybeans , and cucumbers , as well as important monocotyledons , such as wheat , corn , barley , and rice . chloroplasts from all lower and higher plants are very similar in properties , and the present invention is therefore directed to all such organisms and their chloroplasts . in the practice of the present invention , foreign dna is provided for transformation into a plant chloroplast . foreign or exogenous dna refers to any dna which is not found within the chloroplast in nature . thus , foreign dna can encompass a wide variety of dna molecules . particularly referred are dna molecules containing an expression cassette ; i . e ., a dna construct comprising a coding sequence and appropriate control sequences ( e . g ., promoter and appropriately matched transcription termination sequence ) to provide for the proper expression of the coding sequence in the chloroplast . typically , the expression cassette is flanked by convenient restriction sites to facilitate cloning . in a preferred embodiment , the foreign dna used for transformation comprises an expression cassette flanked by chloroplast dna to facilitate the stable integration of the expression cassette into the chloroplast genome by homologous recombination . in constructing the above expression cassettes , one will employ a promoter operable in the chloroplast of interest . typically , chloroplast promoters will be employed , although many procaryotic ( e . g ., bacterial ) promoters are functional in chloroplasts . important coding sequences which can be used in the above expression cassettes include genes for herbicide resistance , such as the atrazine resistance gene psiib ( cheung et al ., 1986 , proc . vii intl . cong . on photosynthesis , pp . 210 - 494 ) and the glyphosate resistance gene es - 3 - p synthetase ( comai , u . s . pat . no . 4 , 535 , 060 ). additional coding sequence which may be of interest include mutants of the gene for ribulose bisphosphate carboxylase / oxygenase ( rubisco ), which may increase catalytic activity and thereby elevate the level of photosynthesis and plant growth . it is usually desirable to include with the expression cassette a selectable marker gene which encodes a selectable phenotype ; i . e ., a phenotype of a cell or organism which allows for the identification or selection of cells expressing the selectable marker gene . well - known marker genes include , but are not limited to , the gene for chloramphenicol acetyltransferase ( cat ), neomycin phosphotransferase ( neo r ), neomycin phosphotransferase ii ( npt - ii ), nopaline synthase ( nos ), dihydrofolate reductase ( mtx r ), hypoxanthine phosphoribosyl transferase ( hpt ), and thymidine kinase ( tk ). the expression cassette is constructed so that the coding sequence is loaded within the cassette along with the appropriate control sequences , the positioning and orientation of the coding sequence with respect to the control sequences being such that the coding sequence can be transcribed under the control of the regulatory sequences ( i . e ., by rna polymerase , which attaches to the dna molecule at the control sequences ) in the host chloroplast transformed by the expression cassette . it is possible to assemble the expression cassette prior to inserting it into a cloning vector . alternatively , an expression cassette can be constructed by cloning the coding sequence directly into an expression vector already containing the appropriate regulatory sequences and the restriction site downstream from the promoter . it is particularly preferred to use , as foreign dna for chloroplast transformation , dna molecules containing an expression cassette using a chloroplast promoter . examples of such promoters include the psba promoter , the rbcl promoter , and the atpb promoter . see , e . g ., gruissem et al . ( 1985 ) embo j 4 : 3375 - 3383 . an is example of such foreign dna is expression vector phd407 , which comprises the psba promoter inserted as a bamhi - hindiii fragment into the pkk232 - 8 vector ( brosius , 1984 , gene 27 : 151 , commercially available from pharmacia ) at the appropriate restriction site upstream from the promoter - less cat coding sequence . according to one embodiment of the present invention , foreign dna , such as dna molecules containing the expression cassettes described above , are used to transform isolated chloroplasts by direct uptake . this method takes advantage of the fact that the chloroplast membrane is believed to be porous , thus allowing for the entry of exogenous dna into the organelle . it has been found to be critical to pretreat isolated chloroplasts with a metal chelating agent , such as edta , to bind the metal ions on which the chloroplast nucleases are dependent . in the absence of pre - treatment with a metal chelating agent , chloroplast nucleases are available to degrade foreign dna as it enters through the chloroplast membrane . the particular chelating agent and the protocol ( e . g ., time , temperature , concentration , etc .) for pretreatment are not critical within wide parameters . numerous chelating agents and pretreatment protocols are within the skill of the art . all that is required is that the protocol be such that the chloroplasts survive and that a sufficient inhibition of nuclease activity occurs to permit a high level of transformation to occur relative to untreated chloroplasts ( e . g ., at least 10 - fold ). the preferred chelating agent is edta , and a preferred protocol is described below . as an alternative method of transforming chloroplasts , foreign dna comprising expression cassettes as described above can be coated on metal particles and inserted by high - velocity impact on cultured cells or callus tissue of intact plant cells . it is believed that a certain proportion of the foreign dna will find its way into chloroplasts and be expressed there . see , e . g ., kline et al . ( 1987 ) nature 327 : 70 . when isolated chloroplasts are transformed directly , it will be necessary to reintroduce the chloroplast into plant cells . large numbers of chloroplasts can be taken up by plant protoplasts , particularly when the protoplasts have been treated with peg . see , e . g ., carlson ( 1973 ) proc . natl . acad . sci . usa 70 : 598 - 602 ; bonnett et al . ( 1974 ) planta 120 : 71 - 79 ; bonnett ( 1976 ) planta 131 : 229 - 233 . once transformed plant cells have been produced , it is usually desirable to grow the cells into callus or a cell line in suspension . techniques for regenerating callus and for producing cell lines are known in the art . transformed cells can also be induced to undergo organogenesis . alternatively , the transformed cells can be used to regenerate plants from tissue culture . various techniques of callus tissue culture , organ culture , and plant regeneration are known . the selection of the appropriate method for a plant species is within the skill of the art . set forth below are specific examples of the present invention which are intended for illustrative purposes only . these examples are not intended to limit the present invention in any manner . the references cited herein , as well as daniell & amp ; mcfadden ( 1987 ) proc . natl . acad . sci . usa 84 : 6349 - 6353 , are incorporated by reference herein . cucumber seeds ( cucumis sativus linnaeus ) were germinated in moist vermiculite at 32 ° c . for 3 days in the dark . e . coli strain hb101 harboring the plasmid pcs75 was a gift from f . r . tabita ( university of texas at austin ) and was grown in tye medium at 37 ° c . ( 19 ). the plasmid puc9 - cm ( incorrectly printed as puc9m in ref . 20 ), a derivative of puc9 with an insert of a nucleotide 1192 - 2480 hae ii - sau3a fragment from pacyc 184 containing the coding sequence of the cat gene with its promoter , was a gift from g . an ( washington state university , pullman ). plasmids pcs75 and puc9 - cm were isolated as described ( 21 ). all solutions used for treatment of cotyledons and isolation or treatment of etioplasts were sterile and manipulations were carried out under aseptic conditions . all ph adjustments were done at 25 ° c . and centrifugations were carried out at 4 ° c . four to eight batches ( 50 each batch ) of 3 - day germinated cotyledons were excised with hypocotyl hooks and each batch was incubated in the dark at 32 ° c . in 10 ml of 0 . 5 mm kinetin ( prepared as a 15 mm stock , solubilized with naoh ) and 2 mm gibberellic acid in large ( 10 - cm ) deep petri dishes for 20 hr . after the hooks had been removed , each batch was hand homogenized with 10 pestle strokes in a prechilled mortar and pestle in 20 ml of the isolation medium , which consisted of 0 . 5 m sucrose , 15 mm hepes , 30 mm 2 -{[ tris ( hydroxymethyl ) methyl ] amino } ethanesulfonic acid ( tes ), 1 mm mgcl 2 , 1 mm edta , 5 mm cysteine , 2 mm dithiothreitol , and 2 % bovine serum albumin at a final ph of 7 . 7 . the homogenate was passed through four layers of autoclaved chilled cheesecloth . the homogenate was then centrifuged at 200 × g for 3 min . at 4 ° c . and the plastids were sedimented by centrifuging the resultant supernatant for 7 min . at 1500 × g . suspensions of etioplasts from different batches were pooled and purified by layering on 35 ml of 0 . 6 m sucrose prepared in the homogenization medium and centrifuging at 500 × g for 15 min ( 22 ). the plastids , after treatment in the presence or absence of edta ( as described in the next section ), were suspended in an ice - cold medium a , which consisted of 0 . 5 m sucrose , 15 mm hepes , 30 mm tes , 40 mm nad , 2 . 5 mm edta , and 1 % bovine serum albumin with a final ph of 7 . 7 . this medium was derived from the previously described translation medium for cucumber etioplasts ( 18 ), after deletion of atp and metallic ions , which we found to inhibit dna uptake . edta - washed etioplasts were prepared according to daniell and rebeiz ( 13 ) by suspending etioplasts pelleted from the 0 . 6 m sucrose homogenization buffer in an edta wash buffer , ph 7 . 7 , which consisted of 0 . 5 m sucrose , 15 mm hepes , and 30 mm tes containing 10 mm edta . after 10 min . of incubation at 0 - 4 ° c . in the dark , the suspended etioplasts were pelleted at 1500 × g for 7 min . and resuspended in medium a as described in the preceding section . etioplasts , untreated or treated with edta , were incubated at 3 . 0 ml of medium a containing 1 μg of [ 32 p ] dna per ml , which had been labeled by nick - translation of pcs75 using [ α - 32 p ] dctp and dna polymerase 1 ( 23 ). incubations were carried out at 27 ° c . for different durations with cool white fluorescent light [ intensity : 30 μe - m - − 2 s − 1 ; 1 e ( einstein )= 1 ml of photons ] on a reciprocating shaker operated at 60 oscillations per min . in a water bath . binding , uptake and breakdown of dna were measured as described previously ( 23 ). edta - treated etioplasts were incubated in a water bath in resuspension medium a without or with plasmid dna ( 50 μg / ml ) at 27 ° c . for 2 hr . with cool white fluorescent light ( 30 μe - m - − 2 s − 1 ) on a reciprocating shaker operated at 60 oscillations per min . at the end of the incubation , the plastids were pelleted at 1500 × g for 7 min . and the incubation medium was discarded . etioplasts were washed thoroughly with ice - cold medium b ( 0 . 5 m sucrose / 15 mm hepes / 30 mm tes , ph 7 . 7 ) and were resuspended in transcription - translation medium , which contains the following components at a final ph of 8 . 2 : 0 . 4 m sucrose [ rnase -, dnase -, and protease - free ( schwartz / mann )], 50 mm tris acetate , 60 mm potassium acetate , 11 mm ammonium acetate , 14 mm magnesium acetate , 20 mm atp , 0 . 5 mm gtp , 0 . 5 mm ctp , 0 . 5 mm utp , 0 . 5 mm camp , 10 mm phosphoenolpyruvate , type iii pyruvate kinase ( sigma , 6 . 4 units / ml of final incubation mixture ); e . coli trna ( 0 . 125 μg / ml ), 19 amino acids ( 0 . 2 mm each ), 0 . 14 mm pyridoxine - hcl , 0 . 1 mm fad , 0 . 1 mm nadp , 0 . 06 mm p - aminobenzoic acid , 1 . 6 mm dithiothreitol , 1 % polyethylene glycol 6000 , and 20 μci 1 ( 1 ci = 37 gbq ) of [ 35 s ] methionine per 100 - μl reaction mixture . each component was stored independently as a 10 × stock solution at − 20 ° c . storage of combined reaction mixture at − 20 ° c . resulted in rapid loss of transcription - translation . incubations were carried out for 2 hr . at 27 ° c . at the end of each incubation , the plastids were centrifuged , washed once with ice - cold medium b , and finally frozen in liquid nitrogen in suitable aliquots . β - lactamase was assayed by a spectrophotometric method ( 21 ) in which the β - lactam ring of nitrocefin ( λ max = 390 nm ) is hydrolyzed to the ring - opened product ( λ max = 490 nm ). immunoprecipitation of small subunits of rubisco with antiserum to the a . nidulans protein ( raised in rabbits by jose torres - ruiz of our laboratory ) was done essentially as described previously ( 21 ). cat was assayed in etioplast sonic extracts as reported in ref . 24 except that the amount of [ 14 c ] chloramphenicol was decreased to 50 nci per sample . preliminary experiments on incubation of etioplasts with nick - translated 32 p - labeled pcs75 ( 3 . 8 × 10 8 cpm / μg of plasmid ) showed a linear increase in uptake or binding of dna at 27 ° c . for at least 120 min . therefore , all incubations of dna with etioplasts were carried out for 2 hr . dnase treatment of etioplasts ensured that the high ( table 1 ) and continuing uptake of dna observed was due to intact organelles , confirming earlier results ( 15 ) with an organelle fraction isolated identically . the rate of uptake of [ 32 p ] pcs75 by etioplasts in the light , 0 . 86 × 10 4 cpm / μg of protein , was comparable to a value of 1 . 35 × 10 4 cpm / μg of protein calculated for “ permeaplasts ” of a . nidulans when [ 32 p ]- pbr322 of approximately identical specific radioactivity were used ( 22 ). permeaplasts are cells with partially digested cell wall obtained by treatment of cells with lysozyme and edta ( 22 ). etioplasts that had been subjected to 10 - min incubation at 0 - 4 ° c . in the dark in the sucrose / hepes / tes buffer lacking edta showed only 3 % of as much uptake dna as was observed with edta - washed etioplasts after 2 hr of incubation with nick - translated [ 32 p ] pcs75 ( table 1 ). on the other hand , 42 % as much binding of dna as had been observed with edta - washed etioplasts was observed with etioplasts washed in the absence of edta under identical conditions of incubation . the presence or absence of light did not affect dna uptake , binding , or breakdown in etioplasts washed in the presence or absence of edta ( table 1 ). cations such as calcium or magnesium ions significantly inhibited dna uptake ( 86 %) in edta - washed etioplasts enhanced binding ( 23 - 200 %) and breakdown ( 163 - 235 %) dna ( table 1 ). it has been previously suggested that the calcium - dependent breakdown of donor dna , reflected in the perchloric etioplasts were incubated in medium a with 1 μg of nick translated 32 p - labeled pcs75 under various conditions . the additional components were added at the following concentrations : gramicidin , 10 μm ; nh 4 cl , 5 mm : valinomycin , 10 μm ; atp 10 mm ; magnesium acetate , 20 mm ; mgcl 2 , 20 mm ; and cacl 2 , 20 mm acid - soluble fraction , may be an essential requirement for transformation of bacillus subtilis ( 25 - 27 ). however , in studies of yeast protoplasts , binding of plasmid dna was not accompanied by its degradation ( 28 ). we have demonstrated recently that dna uptake in permeaplasts of a . nidulans is unrelated to the breakdown of donor dna ( 22 ). in the present work on etioplasts , cations inhibited dna uptake but enhanced breakdown ( table 1 ), suggesting that the two processes may be unrelated in this case also . protonmotive force , which consists of a membrane potential ( δψ ) and proton gradient ( δph ), has been shown to be the driving force for dna uptake in the transformation of some bacteria ( 29 ). therefore , the effect of various reagents that inhibit atp synthesis or uncouple photophosphorylation from photoelectron transport was investigated . gramicidin forms an aqueous transmembrane channel through which protons pass slowly but does not inhibit electron transfer even at very high concentrations ; gramicidin inhibits phosphorylation by abolishing δψ without having a major effect upon the transmembrane δph ( 30 ). to achieve total uncoupling by abolishing both δψ and δph , we used the “ uncoupler couple ” nh 4 cl plus gramicidin . neither gramicidin alone nor gramicidin together with nh 4 cl had any effect on dna uptake , binding , or breakdown by etioplasts ( table 1 ). in b . subtilis , valinomycin , a dissipator of the δψ component of the protonmotive force , inhibited dna binding ( 29 ), but this compound had no effect on dna uptake or binding by etioplasts ( table 1 ). in b . subtilis , dna uptake or binding is an active process driven by protonmotive force ( 29 ), whereas in yeast protoplasts ( 28 ), cyanobacterial permeaplasts ( 22 ), or etioplasts ( table 1 ) each is a passive process . both dna uptake and dna binding were severely inhibited in etioplasts by atp , but the breakdown of dna was essentially unaffected ( table 1 ). the effect was almost certainly due to the hydrolysis of atp , because the nonhydrolyzable ( or very poorly hydrolyzable ) atp analog adenyl - 5 ′- yl imidodiphosphate did not inhibit uptake or binding of dna ( fig1 ). the striking inhibition of dna binding ( and binding - dependent uptake ) by atp is reminiscent of the effect of atp on the same phenomena observed with permeaplasts of a . nidulans ( 22 ). because this atp analog is noninhibitory , we conclude that externally added atp may phosphorylate one or more etioplast membrane components that are involved in dna binding . the introduction and expression of foreign dna was measured by assaying β - lactamase encoded by the plasmid pcs75 , using a spectrophotometric method in which a spectral shift from 390 nm to 490 nm accompanies hydrolysis of the β - lactam ring of nitrocefin ( 23 ). whereas sonic extracts of etioplasts incubated in the absence of dna did not hydrolyze nitrocefin , those incubated with dna hydrolyzed nitrocefin ( fig2 ). the initial rate of nitrocefin hydrolysis by transformed etioplasts with 0 . 12 a 490 unit / min per mg of protein , may be compared with a value of 0 . 4 obtained for pbr322 transformants of permeaplasts of a . nidulans ( 23 ). in transformed higher plant protoplasts , nitrocefin hydrolysis was extremely slow and could be detected only after an 18 - hr incubation ( 31 ). in these experiments , special precautions were taken to minimize the possibility that bacterial contamination of etioplasts could account for these results . to address this questions bacteria were enumerated at 30 ° c . and 37 ° in etioplast fractions ( with or without edta treatment ) incubated in the presence or absence of pcs75 . bacterial contamination was estimated in terms of the ratio of bacterial protein to etioplast protein . the high ratios of etioplast bacterial protein ( data not shown ) established that bacterial contamination was minimal . moreover , ampicillin - resistant bacterial could not be detected after plating etioplast fractions on lb medium containing ampicillin . in this connection , it should also be pointed out that in b . subtilis and e . coli , dna uptake and binding are quenched by edta and uncouplers ( 29 ), in contrast to our results with etioplasts ( table 1 ). moreover , with those bacteria , ca 2 + - dependent breakdown of donor dna may be required for transformation ( 25 - 27 ). the plasmid pcs75 carries the genes for the large and small subunits of rubisco from a . nidulans . immunoprecipitation of the translation products with antiserum to the small subunit of rubisco from a . nidulans suggested the presence of labeled 12 - kda small subunits in etioplasts incubated with pcs75 ( fig3 a ), a molecular mass that is in accord with that for the small subunit of rubisco from a . nidulans ( 19 ). to check for contamination of etioplasts by cytoplasmic messages , experiments were carried out in cotyledons labeled with [ 35 s ] methionine in vivo . etioplasts preincubated with pcs75 showed a translation product 2 kda smaller than that of cucumber encoded by its nuclear genome ( data not shown ). our immunoblots further revealed that cucumber small subunit strongly crossreacts with the small subunit of rubisco from chromatium vinosum but not with that from a . nidulans . it is known that newly synthesized stromal proteins of low molecular mass are almost completely degraded in chloroplasts in the presence to atp ( 32 , 33 ). the imported rubisco small subunit is also destroyed rapidly by proteolysis when conditions for assembly with the large subunit are limiting ( 34 ). therefore , studies of the expression of a . nidulans small subunit ( fig3 a ) were not pursued . etioplasts treated with edta ( 10 mm ) for various times showed that 10 min . was optimal for the expression of cat ( fig3 b ). the background activity observed in etioplasts in the absence of dna ( fig3 c , lane 1 ) could have been due to a low level of endogenous acetylase activity found to be present in all plant species tested ( 35 ). a progressive increase in the expression of cat was observed with an increase in the concentration of puc9 - cm in the dna uptake medium ( fig3 c ). furthermore , treatment of etioplasts with ribonuclease during incubation in the transcription - translation reaction mixture or with thermolysin after coupled transcription - translation did not affect the expression of cat ( fig3 c ) or incorporation of [ 35 s ] methionine ( data not shown ). these results with thermolysin and ribonuclease confirm our conclusion that etioplasts are , indeed intact during translation . all of the present studies ( fig1 - 3 ) were conducted on etioplasts isolated at zero - time greening . in organello translation of endogenous mrna in ethiochloroplasts isolated from cucumber at different stages of greening showed a continuous increase as greening progressed ( fig4 ). in contrast , dna dependent incorporation of [ 35 s ] methionine decreased dramatically as the tissue matured , and only 5 % of transcription / translation activity towards foreign dna remained after 24 hr . of greening . it seems likely that the overall enhancement in translation of 60 % observed in pcs75 - treated etioplasts ( fig4 ) was due to protection of endogenous mrna by incorporated plasmid . in this connection , carrier dna has been used in addition to plasmid dna to increase translation by plant protoplasts that were subjected to electroporation ( 35 , 36 ). 1 . nester , e . w ., gordon , m . p ., amasinc , r . m . & amp ; yanofsky , m . f . 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