Proc. Natl. Acad. Sci. USAVol. 90, pp. 913-917, February 1993Genetics

High-frequency plastid transformation in tobacco by selection for achimeric aadA gene

(homologous recomblnation/homoplamy/plastome eng /plastome sortng/transaomlc)

ZORA SVAB AND PAL MALIGAWaksman Institute, Rutgers, The State University of New Jersey, Piscataway, NJ 08855-0759

Communicated by Charles S. Levings Iii, October 19, 1992 (received for review August 17, 1992)

ABSTRACT We report here a 100-fold increased fre-quency of plastid transformation in tobacco by selection for achimeric aadA gene encoding aminoglycoside 3"-adenylyltrans-ferase, as compared with that obtained with mutant 16S rRNAgenes. Expression of aad4 confers resistance to spectinomycinand streptomycin. In transforming plasmid pZS197, a chimericaadA is cloned between rbcL and open reading frame ORF512plastid gene sequences. Selection was for spectinomycin resis-tance after biolistic delivery of pZS197 DNA into leaf cells.DNA gel-blot analysis cofrmed incorporation of the chimericaadA gene into the plastid genome by two homologous recom-bination events via the flanking plastid gene sequences. Thechimeric gene became homoplasmic in the recipient cells and isuniformly transmitted to the maternal seed progeny. Theability to transform routinely plastids of land plants opens theway to manipulate the process of photosynthesis and to incor-porate novel genes into the plastid genome of crops.

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Plastids of land plants, best known when differentiated aschloroplasts, the green organelles of photosynthesis, carry500-10,000 copies ofa small 120- to 160-kilobase (kb) circulargenome per cell (1, 2). Stable transplastomic lines are ob-tained when each of the genome copies are uniformly trans-formed. Transformation of the plastid genome in the unicel-lular alga, Chlamydomonas reinhardtii (3), and the land planttobacco (Nicotiana tabacum; ref. 4) has been achieved onlyrecently. Plastid transformation in Chlamydomonas is effi-cient and has been successfully applied to address questionsof plastid biology (5).

Progress in plastid transformation in land plants is cur-rently hampered by the inefficiency of the process, which isless frequent by 2-3 orders of magnitude than biolistictransformation of nuclear genes: one bombardment withDNA-coated microscopic tungsten particles yields 2-20 nu-clear gene transformants (6, 7), whereas one plastid trans-formant is recovered in about 100 bombarded samples (4, 8).Selection of plastid transformants in these experiments hasbeen by spectinomycin resistance encoded in mutant 16SrRNA genes. We report here that transformation with achimeric bacterial aadA gene encoding aminoglycoside 3"-adenylyltransferase dramatically improved the recovery ofplastid transformants, yielding frequencies typical of thosefor nuclear genes.

MATERIALS AND METHODSConstruction of Vector pZS197. Plasmid pZS197 (Fig. 1)

carries a chimeric aadA gene between the tobacco rbcL geneand open reading frame ORF512 (BamHI site at nucleotide59,286; ref. 11) in a Sac II-EcoRV plastid fragment between

- 3 kb

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FIG. 1. (Lower) The pZS197 transformation vector. The chimericaadA gene was cloned into a Sac II/EcoRV ptDNA fragmentbetween the rbcL and ORF512 genes (coding regions are boxed) inthe 3-kb pBluescript vector (thin line). In the chimeric gene, expres-sion of the aadA coding region (9) is controlled by a modified plastidribosomal RNA operon promoter, Pan, and the 3' region of theplastid psbA gene (psbA3'). (Upper) Prrn includes sequences of theplastid ribosomal RNA operon promoter (nucleotides 1-117; ref. 10).G at position 1 corresponds to nucleotide 102,561 in ref. 11. Muta-tions generated to eliminate upstream AUGs in the mRNA are inlowercase letters. A synthetic leader sequence (nucleotides 118-135), including a ribosome binding site (RBS), are in lowercaseletters. The cptl and cpt2 regulatory sequences (underlined) aresimilar to the Escherichia coli -35/-10 upstream promoter elements(12). ATG at nucleotide 136 (boldface letters) is the aadA transla-tional initiation codon. Fragment sizes are shown inside the map.

nucleotides 57,750 and 60,593, respectively (11) cloned intoa pBluescript KS(+) phagemid vector (Stratagene).The 16S rRNA promoter region, P.=,, was cloned from

plasmid pJS71 (8) as a 252-base-pair (bp) Dra I-Sph I frag-ment. Nucleotides at positions 101 and 102 were changedfrom AT to TC by site-directed mutagenesis (13) usingoligonucleotide 5'-CTTGTATCCATGCGCTTCgaATTC-GCCCGGAGTTCG-3' to create an EcoRP restrictfion site(underlined; mismatches are in lower case). The newly cre-ated EcoRI site at the 3' end of the promoter region waslinked to the Nco I site at the 5' end ofthe aadA coding regionfrom plasmid pHC1 (14) with a synthetic linker obtained byannealing the single-stranded oligonucleotides 5'-AATTC-GAAGCGCTTGGATACAGTTGTAGGGAGGGATC-3'and 5'-CATGGATCCCTCCCTACAACTGTATCCAAG-

Abbreviations: ptDNA, plastid DNA; ORF, open reading frame;Prr,, 16S ribosomal RNA promoter.

913

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914 Genetics: Svab and Maliga

CGCTTCG-3'. Subsequently, the EcoRI site was eliminated,and the original sequence was partially restored by a T - Achange (position 101 in Fig. 1) with oligonucleotide 5'-GTATCCAaGCGCTTCGtATTCGCCCGGAG-3'. This oli-gonucleotide also introduced an A -+ T mutation at position110 to remove an upstream AUG (mismatches are in lowercase). Nucleotides at the -1 and -2 positions were changedfrom CC to TT to reproduce the rbcL leader sequence (15)with oligonucleotide 5'-GGCGATCACCGCTTCtgCCAT-aaATCCCTCCCTAC-3' (mismatches are in lower case). Thesame oligonucleotide changed GG to CA at positions 5 and 6to make the context around the translational initiation codonAUG similar to that of plastid mRNAs (16).

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FIG. 2. Selection of transplastomic lines by spectinomycin resis-tance. (A) Leaves are bombarded with tungsten microprojectiles coatedwith pZS197 DNA. (B) After 2 days the leaves are dissected into 5 mmx 5mm sections and transferred onto an RMOP medium (4) containing500 yg of spectinomycin sulfate per ml. Recipient leaf sections bleach.Resistant green shoots and calli appear in 3-8 weeks. (C) Shoots are

regeneratedfrom resistant leafsections during a second culture cycle onselective medium. Homoplasmic shoots are identified by DNA gel blotsat this stage. (D) Homoplasmic shoots are rooted and transferred to thegreenhouse (for details, see refs. 4 and 8). m, Macroprojectile; sp,stopping plate.

Proc. Natl. Acad. Sci. USA 90 (1993)

The psbA 3' regulatory region contains nucleotides 533-141 ofthe plastid genome (11). A Sau3AI site at the 5' end anda Taq I site at the 3' end were converted into Xba I andHindIII sites, respectively, by linker ligation. The psbA 3'region was cloned downstream of the aadA coding sequenceas an Xba I-HindIII fragment in plasmid pHC1 (14) togenerate the aadA 3' region.

Transformation and Regeneration of Transgenic Plants.Tobacco (N. tabacum) plants were grown aseptically onagar-solidified medium containing MS salts (17) and sucrose(30 g/liter). Leaves were placed abaxial side up on RMOPmedium (4) for bombardment. Tungsten microprojectiles (1gum) were coated with DNA, and the bombardment wasperformed with the DuPont PDS1000 gun-powder chargeBiolistic gun as described by Klein et al. (6). Spectinomycin-

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

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FIG. 3. Probing of DNA gel blots confirms plastid transforma-tion. Total cellular DNA was digested with EcoRI and EcoRVrestriction enzymes for which there are no sites in plasmid pZS197.Blots were probed with the targeting plastid DNA fragment encodingthe rbcL and ORF512 genes (P1 in Fig. 3C) (A) and the aadA codingregion (P2 in Fig. 3C) (B). Lanes: 1 and 2, DNA from wild-typerecipient tobacco leaves; 3-19, DNA extracted from the spectino-mycin-resistant lines pZS197-159, -160, -162, -166, -172, -175, -177,-179, -182, -183, -193, -185, -186, -187, -188, -189, and -173. (C) DNAprobes. (D) T1ptDNA formed by recombination (dashed lines) viarbcL and ORF512 sequences. (E) Lineup of regions of donor DNAand of the wild-type plastid genome that were involved in formingT2ptDNA by recombination via rbcL and psbA 3'-end sequences.The bold line in T2ptDNA indicates the sequence in the repeatedregion. Restriction endonuclease recognition sites: RI, EcoRI; RV,EcoRV; NI, Nco I; SII, Sac II; XI, Xba I. Maps are to scale.

Proc. Natl. Acad. Sci. USA 90 (1993) 915

resistant calli and shoots were selected on RMOP mediumcontaining 500 ,ug of spectinomycin dihydrochloride per ml.Resistant shoots were regenerated on the same selectivemedium and rooted on MS agar to obtain To plants. The first-and second-seed generations of To plants are the T1 and T2generations, respectively.DNA Gel-Blot Analysis ofTotal Cellular DNA. Total cellular

DNA was prepared by the method ofMettler (18). Restrictionenzyme-digested DNA was electrophoresed on 0.7% agarosegels and transferred to nylon membrane (Amersham) by usingthe PosiBlot Transfer apparatus (Stratagene). Blots wereprobed by using rapid hybridization buffer (Amersham) with32P-labeled probes generated by random priming (BoehringerMannheim).

Testing of Seedling Phenotypes. Seedling phenotype wasdetermined by plating surface-sterilized seeds on MS me-dium. On selective medium (spectinomycin or streptomycinat 500 ,ug/ml, or both at 500 gg/ml) resistant progeny aregreen, whereas sensitive progeny are white (4, 8).

RESULTSpZS197 Plastid Vector. The aadA gene, encoding amino-

glycoside 3"-adenyltransferase, causes spectinomycin andstreptomycin resistance in bacteria (9). Chimeric aadA genesconfer resistance to the same antibiotics in tobacco nuclei(19) and Chlamydomonas chloroplasts (20). To express theaadA gene in tobacco plastids, we placed the aadA codingregion under control of the strong, constitutive Pn of theplastid rRNA operon and the 3' untranslated region of theplastid psbA photosynthetic gene. To facilitate translation ofthe chimeric mRNA a synthetic ribosome binding site (RBS),designed after the RBS of the abundant rubisco large subunitpolypeptide, was incorporated in the 5' regulatory region(Fig. 1). The chimeric aadA gene was cloned between theplastid rbcL and ORF512 genes. These homologous flankingplastid DNA sequences direct the insertion of aadA betweenrbcL and ORF512 in the plastid genome by two homologousrecombination events (see below).

Transformation and Selection of Transplastomic Lines.Transformation with plasmid pZS197 by the biolistic processand selection of spectinomycin-resistant lines is shown inFig. 2. Resistant shoots and calli appeared in 3-8 weeks onthe selective medium (Fig. 2B). Shoots and calli formed atone site were termed a clone. Plastids in the leaves of theseshoots were typically heteroplasmic and contained wild-typeand transformed genome copies (see below). Shoots regen-erated from leaf sections during a second cycle on spectino-

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mycin-containing medium were termed subclones (Fig. 2C).Typically, half ofthe shoots obtained during the second cycleof regeneration were homoplasmic for the transgenome.Since shoot apices form from one or very few cells, plantregeneration is tantamount to single-cell cloning of the chi-meric tissue. Homoplasmic shoots at this stage were rooted(Fig. 2D) and transferred to the greenhouse. By this protocolhomoplasmic shoots were obtained in 3-5 months.

Spectinomycin resistance may be due to (i) integration ofaadA into the plastid genome, (ii) integration ofaadA into thenuclear genome and fortuitous expression from an upstreampromoter, or (iii) occurrence of a spontaneous plastomemutation. In 79 bombarded leaf cultures, 84 spectinomycin-resistant clones were isolated. DNA gel-blot analysis con-firmed that 40 of 50 clones had integrated aadA into theplastid genome (see below). The remaining 10 clones did nothybridize with the aadA probe and are spontaneous mutants.

Integration of aadA into Plastid DNA (ptDNA) and Sortingof Wild-Type and Transformed Genome Copies. Screening ofsubclones after the second selection cycle is shown in Fig. 3.Total cellular DNA was extracted from leaves and probedwith wild-type rbcL/ORF52 (probe P1) and aadA (probe P2)sequences. DNA gel blots indicated two types of integrationevents, yielding genomes TlptDNA and T2ptDNA.TlptDNA resulted from incorporation of the 1.3-kb aadA

gene into the 3.0-kb fragment containing rbcL and ORF512 bytwo homologous recombination events (Fig. 3D and Fig. 4 Aand B). Digestion of the TlptDNA with EcoRI and EcoRVrestriction endonucleases yielded a 4.3-kb fragment hybrid-izing with both the targeting sequences and the aadA probe.Some of the lines carried only transformed genome copiesand therefore were homoplasmic (for example, lanes 4, 5, and6 in Fig. 3 A and B). Others were heteroplasmic and carriedboth wild-type (3.0 kb) and transgenic (4.3 kb) fragmentswhen probed with the targeting sequences (for example, lane3 in Fig. 3A). Homoplasmic shoots from such heteroplasmicclones were readily obtained by regenerating plants on aselective medium.

In about half of the clones, in addition to TlptDNA-typeintegration events, an unexpected 3.4-kb fragment was alsopresent. The 3.4-kb fragment is a minor component in mostclones, except pZS197-173 plastids (lane 19 in Fig. 3 A and B)in which this was the only fragment hybridizing with the aadAprobe. To determine its origin, the fragment was cloned frompZS197-173 plastids and sequenced. Sequencing revealedthat the aadA 3' region (derived from the endogenous psbAgene) is followed by trnH and the inverted repeat (Fig. 3E)as in the wild-type plastid genome. Therefore, the 3.4-kb

B

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FIG. 4. Wild-type (A) and transformed (B and C) plastid genomes. (A) Recombination sites between homologous flanks in pZS197 and thewild-type plastid genome (wtptDNA) are shown by arrows. (B) TlptDNA is the product of recombination shown in A. (C) T2ptDNA has a 26-kbdeletion and duplication of the psbA 3' region and trnH. Note that the 5'-end of psbA is distal to trnH. Inverted repeat sequences are pairedso that the large and small single-copy regions form loops. Genomes are not drawn to scale. psbA, trnH, rbcL, and ORF512 are plastid genes.Maps are based on ref. 11.

Genetics: Svab and Maliga

Proc. Natl. Acad. Sci. USA 90 (1993)

FIG. 5. Maternal inheritance of spectinomycin/streptomycin resistance in the pZS197-162 seed progeny. Resistant seedlings are green (blackin photograph), and sensitive seedlings are white on the selective medium. (Left) Selfed seed progeny. (Center) pZS197-162 female x wild-typemale. (Right) Wild-type female x pZS197-162 male.

fragment is also a product of two homologous recombinationevents via (i) the endogenous and donor 1.55-kb rbcL se-quences and (ii) the 0.4-kb aadA 3' region and the endoge-nous psbA gene 3' region. The proposed T2ptDNA-structureis shown in Fig. 4C. Regions of the donor DNA and of thewild-type plastid genome involved in forming T2ptDNA areshown in Fig. 3E. The molecular events yielding T2ptDNAare not understood. It may have been formed by a multistepprocess immediately after the introduction of the donorDNAor later, from TlptDNA, by recombination via the homolo-gous aadA and psbA 3' regions and copy correction.The proposed T2ptDNA (Fig. 4C) would lack a 26-kb

region between aadA and the inverted repeat, as comparedwith T1ptDNA (Fig. 4B). The putative deleted region con-tains ribosomal protein genes (11) required for plastid main-tenance (21). Evidence supporting formation of defectivetransgenomes are (i) rapid loss ofT2ptDNA in the absence ofselection and (ii) failure of repeated efforts to purifyT2ptDNA to homoplasmy. Accordingly, the T2ptDNA ge-nome in the pZS197-173 line could be maintained only inbalanced heteroplasmic plants, in which the transgenomesconferred phenotypic resistance and the wild-type genomescomplemented the deleted functions.

Presence of the Chimeric aadA Gene in the Seed Progeny.Seed transmission of the chimeric aadA was studied inpopulations of 500-800 seedlings of the TlptDNA-typeclones pZS197-159, pZS197-160, and pZS197-162. The resis-tance phenotype was determined by germinating T1 genera-tion seedlings on selective medium. Fig. 5 shows differenti-ation by color: resistant seedlings are green, whereas sensi-tive seedlings are white. Uniform spectinomycin resistance inthe selfed seed progeny indicates that aadA is maintained inplants grown to maturity in greenhouse. The F1 progenyobtained by pollinating transplastomic flowers with wild-typepollen were also uniformly resistant, whereas progeny fromthe reciprocal crosses (wild-type female, transplastomic pol-len) were sensitive to spectinomycin (Fig. 5). Lack of pollentransmission of the transplastomic aadA gene is expected intobacco, a species with strict maternal inheritance of itsplastids.

Stability ofthe aadA gene in ptDNA was further confirmedby uniform resistance of 21,000 T2 generation seedlings.About 7000 seedlings were tested from each of the threetransplastomic lines.

DISCUSSIONTransformation efficiency with the chimeric aadA gene isabout 100-fold greater than with antibiotic resistance encodedby mutations in 16S ribosomal RNA genes (4, 8) and ap-proaches the frequency of nuclear gene transformation by the

biolistic process (6, 7). We believe that the increase in thetransformation frequency is due to an improved recovery ofthe newly formed transgenomes by the dominant aadA gene(see below).We propose that the 100-fold lower transformation fre-

quency with spectinomycin-resistant ribosomal RNA genes(4, 8) is due to the recessive nature of the markers. Ourassumption that the markers are recessive is based on ex-periments with ribosomal RNA mutants in E. coli (22-24) thatare similar to the plastid mutants (25, 26). Due to therecessive nature of the markers, phenotypic resistance is notexpressed until sorting out of the transgenomes is essentiallycomplete. Lack of phenotypic resistance permits the loss ofthe resistant ribosomal RNA gene in 99 of 100 potentialtransformation events. Elimination of the transgene mayoccur by gene conversion, an active mechanism that keepsboth copies of the -25-kb inverted repeat identical at theDNA sequence level (27).

Integration of aadA was by two homologous recombina-tion events via (i) the rbcL (1.55 kb) and ORF512 (1.29 kb)genes or (ii) the rbcL (1.55 kb) and psbA 3' (383 bp) regions.Interestingly, no recombination was detected between theaadA promoter region and its cognate source. This may bedue to the short sequence homology (120 bp), relative posi-tion, or nature of homologous sequences. Understanding therules of recombination between transgene expression signalsand cognate endogenous sequences will facilitate the designof stable transgenomes.

High-frequency transformation makes manipulation of theplastid genome a practical experimental tool in land plants.The plastid aadA gene can now be used to introduce effi-ciently linked photosynthetic and passenger genes. Routinetransformation should lead to rapid progress in understandingplastid gene regulation (12, 28, 35), interaction of plastid andnuclear genomes (29-31), identification of the function ofconserved open reading frames (27, 32), plastid evolution(33), and applications of genetic engineering to the plastidgenome of crop plants (34).

We thank Jeffrey Staub for discussions, and Carl Price for a criticalreading of the manuscript. This work was supported by NationalScience Foundation Grant DMB 9004054 to P.M.

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Genetics: Svab and Maliga