Supporting InformationNaz et al. 10.1073/pnas.1214300110SI Materials and MethodsPlant Materials and Growth Conditions. Seeds of Solanum lyco-persicum cultivar (cv.) Condine Red (CR), cv. Rheinland Ruhm(RR), tf-2 (cv. CR), tf-3 (cv. RR), and entire (e) (MLE 434, cv.Ailsa Craig) were obtained from the Genbank, Institute for PlantGenetics and Crop Plant Research (Gatersleben, Germany); tf-z(e0761B, cv. M82) from http://zamir.sgn.cornell.edu/mutants/;(1); Moneymaker (MM) from Kiepenkerl (Everswinkel, Ger-many); tf-1 (LA0512, cv unknown), Me (LA0324, cv. Rutgers),clau (LA0591, cv. Lukullus), and bip (LA0663, cv. Lukullus) fromthe Tomato Genetics Resource Center (Davis, CA). tf-4 seedswere kindly provided by Yuval Eshed (Weizmann Institute ofScience, Rehovot, Israel). For double-mutant analysis, tf-z wascrossed to Me/+, e, bip, and clau, and F1 plants were self-polli-nated to produce segregating F2 populations, from which specificgenotypes were selected via PCR or DNA sequencing for phe-notypic evaluations. Plants were grown under standard glasshouseconditions with additional artificial light (16-h photoperiod).Two different loss-of-function alleles (tf-2 and tf-z) in different

genetic backgrounds showed very similar phenotypic defects, sug-gesting that phenotypic alterations were not a consequence of thegenetic background. Therefore, phenotypic defects associated withtf mutations were investigated and compared in tf-z and tf-2 mu-tants in comparison with corresponding WT plants as controls.Shoot length was measured after appearance of the first inflo-

rescence as the length of the primary shoot from the cotyledons tothe first sympodial shoot.

Mutant Selection and Positional Cloning. To identify a differentbranching mutant, e0761B [selected from the mutant populationestablished in the field tomato cultivar M82 (http://zamir.sgn.cornell.edu/mutants/; (1)] was tested for defects in lateral meri-stem initiation. e0761B plants were completely unbranched,plant stature and leaf complexity were reduced, and fruits didnot contain seeds under our greenhouse conditions. To rescuethis mutant line, plants were pollinated with WT pollen from cv.MM. F1 plants displayed WT leaf form and shoot branching aswell as a mixture of other phenotypic aspects of MM and M82.In the F2 generation, the recessive branching defect cose-gregated with a trifoliate (tf) leaf phenotype, whereas the reducedplant height and the self pruning (sp)-dependent terminationphenotype segregated independently. Plants displaying the tfphenotype produced fruits with very few seeds, which partiallyimproved after a second backcross to MM. Because the leaves ofthe newly developed mutant line (75% MM + 25% e0761B)showed the tf phenotype, an allelism test was performed bycrossing it to tf-2. All F1 and F2 progeny from this cross dis-played a tf leaf phenotype, suggesting that e0761B contains a newtf allele, which we named tf-z (for Zamir).Initial mapping of tf in an F2 population (545 F2 plants) from a

cross between S. lycopersicum (tf-z) and Solanum pennellii identi-fied a region of about 1.0 cM on chromosome 5 between markersBs4 (2) and C2_At1g026810 (3). Around 1,248 additional F2plants were screened to select informative recombinants (IR) be-tween markers C2_At1g14300 and C2_At1g026810 (3). Furthergenotyping using IRs revealed that tf is positioned in an interval of0.3 cM between the markers Bs4 and Y45R (4), flanked by 7 and 9IRs, respectively. A single Bacterial Artificial Chromosome (BAC)clone (Hba-0143B20) harboring both markers was identified byPCR screening of the tomato BAC library Heinz1706-HindIII (5).Sequencing of Hba-0143B20 (123,941 kb) revealed a region ofabout 41 kb between Bs4 and Y45R containing two genes, coding

for an R2R3 MYB and a DOF2 transcription factor. Sequenceanalysis of the R2R3MYB transcription factor gene inWT, tf1, tf2,tf3, tf4, and tfz revealed five independent mutations (Table S2).Primers used are listed in Table S4.

DNA Sequencing and Analysis. Plant DNA was prepared as de-scribed (6) or using the DNeasy 96 Plant Kit and BioSprint 96automated DNA extraction apparatus (Qiagen). Standard tech-niques were carried out according to Sambrook and Russel (7),unless otherwise stated.PCR and inverse PCR products were treated with ExoSAP-IT

(USB Corp.) before sequencing. DNA sequences were determinedby theMax PlanckGenomeCentreCologne onApplied BiosystemsAbi Prism 377, 3100, and 3730 sequencers using BigDye-terminatorv3.1 chemistry. Premixed reagents were from Applied Biosystems.Oligonucleotides were purchased from Eurofins MWG Operon.Hba-0143B20 sequencing was outsourced to Qiagen. BLAST an-alyses were performed on Sol Genomics Network (8) andGenBankdatabases (National Center for Biotechnology Information).The phylogenetic tree was calculated by comparing amino acidsequences of the conserved MYB domain using the neighborjoining method with the software programs MAFFT (9) andArchaeopteryx (10).

Rapid Amplification of cDNA Ends (5′ RACE). RACE experimentswere performed using the 5′/3′RACE kit (Roche). For 5′RACE,first-strand cDNA synthesis was performed using a gene-specificprimer (SP1). cDNA was tailed with oligo-dA and amplified byPCR using a gene-specific primer (SP2) and the anchored olig-dT primers. A second PCR amplification was done using a nes-ted gene-specific primer (SP3) and the universal anchored PCRprimers. The PCR product was cloned into pCR-Blunt IITOPO(Invitrogen Life Technologies) and sequenced. Primers sequencesare listed in Table S4.

Tf Complementation and Overexpression. For complementation, aDNA fragment of 6.2 kb containing the Tf gene (nucleotides−2.388to +3801 relative to the ATG) was isolated from BAC cloneHba-0143B20 and cloned into pUC19 (11). Subsequently, thefull-length genomic cassette of Tf was cloned from pUC19 intothe binary vector pGPTV-Kan (12). For overexpression, Tffragment was amplified from cDNA using gene-specific primers(pFK-PciI-for and pFK-XhoI-rev), restricted with PciI and XhoI,and cloned into the vector pFK026 containing cauliflower mosaicvirus 35S promoter (13). Later, a DNA fragment containing 35Spromoter and the Tf coding sequence was isolated and trans-ferred into the binary vector pTP05 (13) using XbaI and SalIrestriction enzymes. pFK026 and pTP05 were kindly provided byFarnusch Kaschani (Max Planck Institute for Plant BreedingResearch, Cologne, Germany). The full-length Tf genomicfragment was introduced into tf-z, whereas overexpression con-struct was introduced into tf-z and into tomato cv. MM as de-scribed (14). Primer sequences are listed in Table S4.

RNA Isolation and Quantitative RT-PCR. RNA was isolated from theshoot apex (SAM with five-leaf primordia from 15 d-old-plants),leaf1 (fifth leaf, length: 1 cm, from 25-d-old plants), leaf2 (fifthleaf, length: 2 cm, from 28-d-old plants), stem (internode betweenfourth and fifth leaf, from 28-d-old plants), flower bud (first in-florescence, from 28-d-old plants), and root (complete root, 28-d-old plants) of MM. RNA was extracted with the RNeasy PlantMini Kit (Qiagen) including on-column DNase treatment. cDNAsynthesis was done using RevertAid H Minus M-MuLV Reverse

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TranscriptaseKit (Fermentas). cDNA concentrations were quan-tified using actin as control. A semiquantitative RT-PCR analysiswas carried out at 28, 30, 33, and at 35 cycles using a primer pairencompassing the first intron. RNA for real-time quantitative RT-PCR was isolated from the first five leaves, each harvested ata similar primordial stage of less than 1 mm in size. QuantitativeRT-PCR analysis was done on Roche LightCycler480 using SYBRGreen PCR Master Mix (Applied Biosystems/Life Technologies).Relative quantification was done using internal standard curves andcorrecting with reference gene CAC (15). The primer sequencesare given in Table S4.

RNA in Situ Hybridization. Sample preparations and in situ hy-bridizations were performed as described (16) with minor mod-ifications. Plant material was embedded in Paraplast (Kendall)using the ASP300 tissue processor (Leica). Antisense probes con-tained the following nucleotides relative to the ATG: Tf, 2–560;

TKn2, 2–902. These sequences were PCR amplified using T7promoter linkers and in vitro transcribed using ROCHE Linkedin vitro Transcription Kit (Roche). After the color reaction, slideswere mounted in 30% (vol/vol) glycerol and photographed usingdifferential interference contrast microscopy.

Histology and SEM Analysis. Sectioning of leaf rachis was carried outby hand on young fresh leaves of 28-d-old plants. Fine cuts weremadewith the help of a sharp razor blade. The excised tissues werestainedwith a solution of fuchsine, chrysoidin, andAstraBlue (17).SEM analysis of shoot apices, leaf primordia, and leaves wasperformed on a DSM 940 (Zeiss) using fresh tissue; cell sizemeasurements were done using software ImageJ (18).

Accession Numbers. Sequence data from this article can be foundin the Sol Genomics Network data library under the followingaccession numbers: Bl, Solyc11g069030; Ls, Solyc07g066250.

1. Menda N, Semel Y, Peled D, Eshed Y, Zamir D (2004) In silico screening of a saturatedmutation library of tomato. Plant J 38(5):861–872.

2. Schornack S, et al. (2004) The tomato resistance protein Bs4 is a predicted non-nuclearTIR-NB-LRR protein that mediates defense responses to severely truncated derivativesof AvrBs4 and overexpressed AvrBs3. Plant J 37(1):46–60.

3. Fulton TM, Van der Hoeven R, Eannetta NT, Tanksley SD (2002) Identification,analysis, and utilization of conserved ortholog set markers for comparative genomicsin higher plants. Plant Cell 14(7):1457–1467.

4. Ballvora AB, et al. (2001) Chromosome landing at the tomato Bs4 locus. Mol GenetGenomics 266(4):639–645.

5. Budiman MA, Mao L, Wood TC, Wing RA (2000) A deep-coverage tomato BAC libraryand prospects toward development of an STC framework for genome sequencing.Genome Res 10(1):129–136.

6. Brandstädter J, Rossbach C, Theres K (1994) The pattern of histone H4 expression inthe tomato shoot apex changes during development. Planta 192(1):69–74.

7. Sambrook J, Russell DW (2001) Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory, Cold Spring Harbor, NY), 3rd Ed.

8. Mueller LA, et al. (2005) The SOL Genomics Network: A comparative resource forSolanaceae biology and beyond. Plant Physiol 138(3):1310–1317.

9. Katoh K, Toh H (2010) Parallelization of the MAFFT multiple sequence alignmentprogram. Bioinformatics 26(15):1899–1900.

10. Han MV, Zmasek CM (2009) phyloXML: XML for evolutionary biology and comparativegenomics. BMC Bioinformatics 10:356.

11. Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors andhost strains: Nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33(1):103–119.

12. Becker D, Kemper E, Schell J, Masterson R (1992) New plant binary vectors with selectablemarkers located proximal to the left T-DNA border. Plant Mol Biol 20(6):1195–1197.

13. Shabab M, et al. (2008) Fungal effector protein AVR2 targets diversifying defense-related cys proteases of tomato. Plant Cell 20(4):1169–1183.

14. Knapp S, Larondelle Y, Rossberg M, Furtek D, Theres K (1994) Transgenic tomato linescontaining Ds elements at defined genomic positions as tools for targeted transposontagging. Mol Gen Genet 243(6):666–673.

15. Expósito-Rodríguez M, Borges AA, Borges-Pérez A, Pérez JA (2008) Selection ofinternal control genes for quantitative real-time RT-PCR studies during tomatodevelopment process. BMC Plant Biol 8:131.

16. Müller D, Schmitz G, Theres K (2006) Blind homologous R2R3 Myb genes control thepattern of lateral meristem initiation in Arabidopsis. Plant Cell 18(3):586–597.

17. Etzold H (2002) Simultanfärbung von Pflanzenschnitten mit Fuchsin, Chrysoidin undAstrablau. Mikrokosmos 91:316.

18. Abramoff MD, Magelhaes PJ, Ram SJ (2004) Image processing with ImageJ. BiophotonicsInt 11:36–42.

Fig. S1. tf mutants are compromised in axillary meristem formation. (A and B) SEM micrographs showing the formation of an axillary bud in a leaf axil of MM(A) and the absence of any such bud initiation in tf-z mutant (B). (C and D) A focused expression of TKn2 (arrow) marks the initiation of a new meristem in theaxil of CR WT (C), whereas this expression is absent is tf-2 (D). P8, primordium 8; S, stem. (Scale bars: A, C, and D: 100 mm; B, 200 mm.)

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Fig. S2. Comparison of trichome development in MM and tf-z. (A–D) SEMmicrographs of shoot apices of theWTMM (A and C) and the tf-zmutant (B andD). (Cand D) A higher magnification of (A and B), respectively. tf-z mutants show enhanced trichome development from young leaf primordia. (Scale bars: 100 mm.)

A

B

LOF1

LOF2

AtMYB69

AtMYB56

AtMYB54

AtMYB52

Sl03g098260

Sl10g081320

Sl04g064540

Tf

96

83

57

84

7276

66

TF.Sl05g007870 CARGHWRPAEDAKLKELVAIYGPQNWNLIAEKLEGRSGKSCRLRWFNQLDPRINRRAFSEEEEERLMAAHRLYGNKWAMIARLFPGRTDNAVKNHWHVIMARKLOF1.AtMYB117 LGRGHWRPAEDVKLKELVSIYGPQNWNLIAEKLQGRSGKSCRLRWFNQLDPRINRRAFTEEEEERLMQAHRLYGNKWAMIARLFPGRTDNSVKNHWHVVMARKLOF2.AtMYB105 VSRGHWRPAEDTKLKELVAVYGPQNWNLIAEKLQGRSGKSCRLRWFNQLDPRINRRAFTEEEEERLMQAHRLYGNKWAMIARLFPGRTDNSVKNHWHVIMARKSl10g081320 CARGHWRPHEDAKLKELVGQYGPQNWNLIAEKLEGRSGKSCRLRWFNQLDPRINRKAFSEEEEERLLSAHKMYGNKWSMIARLFPGRTDNAVKNHWHVIMARKAtMYB56 CSRGHWRPTEDAKLKELVAQFGPQNWNLISNHLLGRSGKSCRLRWFNQLDPRINKRAFTEEEEFRLLAAHRAYGNKWALISRLFPGRTDNAVKNHWHVIMARRSl04g064540 CPRGHWRPAEDEKLRQLVEQYGPQNWNSIAEKLQGRSGKSCRLRWFNQLDPRINRRPFSEDEEEKLIGAHRIHGNKWALISRLFPGRTDNAVKNHWHVLMARKAtMYB52 CSRGHWRPAEDEKLRELVEQFGPHNWNAIAQKLSGRSGKSCRLRWFNQLDPRINRNPFTEEEEERLLASHRIHGNRWSVIARFFPGRTDNAVKNHWHVIMARRAtMYB54 CSRGHWRPAEDEKLKDLVEQYGPHNWNAIALKLPGRSGKSCRLRWFNQLDPRINRNPFTEEEEERLLAAHRIHGNRWSIIARLFPGRTDNAVKNHWHVIMARRSl03g098260 CSRGHWRPHEDEKLRELVAKYGPHNWNAIALNLQGRSGKSCRLRWYNQLDPRINRSPFTEEEEERLLSSHRIHGNRWAMIARLFPGRTDNAVKNHWHVIMSRKAtMYB69 CQRGHWRPVEDDNLRQLVEQYGPKNWNFIAQHLYGRSGKSCRLRWYNQLDPNITKKPFTEEEEERLLKAHRIQGNRWASIARLFPGRTDNAVKNHFHVIMARR

Fig. S3. Phylogenetic tree of Tf-related proteins. (A) Amino acid sequence alignment of the MYB domains (aa 118–220) of Tf-related proteins from tomatoand Arabidopsis. Amino acid changes are indicated in blue. (B) The MYB domains (aa 118–220) of Tf-related proteins were compared by the neighbor joiningmethod using the software programs MAFFT and Archaeopteryx. Bootstrap values are indicated in the tree. LOF, LATERAL ORGAN FUSION; Tf, Trifoliate.

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Fig. S4. Expression of Tf mRNA in different tomato plant organs. Semiquantitative PCR analysis of RNA isolated from different plant organs. F, flowerbuds; L, developed leaf (2 cm); L5, fifth leaf of a tomato seedling (1 cm); R, root; S, stem; SA, shoot apex including shoot apical meristem and five youngestleaf primordia.

Fig. S5. Tf promotes axillary bud formation and leaf dissection. (A and B) Shoot branching phenotype of tf-z plants (A) and MM WT plants (B) expressing Tffrom an endogenous promoter fragment (pTf:Tf) or from the 35S promoter (35S:Tf). Every column denotes the primary shoot of a plant, with every boxrepresenting a leaf axil. Green, leaf axil with a side shoot, light green, leaf axil with two side shoots; yellow, empty leaf axil, (C) Leaf morphology in tf-z plants(T1 generation) expressing Tf from the endogenous promoter (pTf:Tf) or from a 35S promoter (35S:Tf). Every column denotes the primary shoot of a plant, withevery box representing a leaf. Green, leaf with >10 leaflets; orange, leaf with >3 and <10 total leaflets; yellow, leaf with < 4 leaflets. (D–F) Leaf morphology ofseventh leaf of a MM (D) and a 35S::Tf (E) plant (T1 generation). (F) Average number of leaflets in the seventh leaf of four 35S::Tf lines (T1 generation)compared with those in MM. Gray bars, primary leaflets; green bars, secondary-, tertiary-, and intercalary leaflets together (n = 10). (Scale bars: D and E, 5 cm.)

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T1 T2 T3 T4 T5 MM0

1

2

3

4

5

6

7

8

9

10

Rel

ativ

e Tf m

RN

A ac

cum

ulat

ion

Fig. S6. Tf mRNA accumulation in leaf primordia of 35S:Tf plants. RNA was extracted from leaf primordia (P5) of 2-wk-old seedlings of five transgenic linesand the MM WT. Tf mRNA levels of were determined by quantitative RT-PCR analysis. Error bars indicate SD.

L7L1 L5L30

3

45

21

8

7

6

9

10

Rel

ativ

e Tf m

RN

A ac

cum

ulat

ion

Fig. S7. Tf mRNA accumulation in tf-2 leaf primordia. RNA was extracted from leaf primordia (≤1 mm) of leaf 1 (L1), L3, L5, and L7. tf-2 transcript levels weredetermined by quantitative RT-PCR analysis.

L1 L3 L5 L70

5

10

15

20

25

30

35

40

45

Rel

ativ

e TKn2

mR

NA

accu

mul

atio

n

CR

Fig. S8. Comparison of LeT6/Tkn2 mRNA accumulation in leaf primordia of WT and tf-2 plants. RNA was extracted from leaf primordia (∼1 mm) of leaf 1 (L1),L3, L5, and L7 of WT CR (blue) and tf-2 (red) plants. LeT6/TKn2 transcript accumulation, as determined by quantitative RT-PCR analysis, was similar in WT andtf-2 leaf primordia, except for leaf 5, where we found an up-regulation in the WT. Error bars indicate SD.

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Fig. S9. Model of how auxin application in tf-2 results in missing leaflets. Green circle, auxin maxima; red arrows, directional auxin flow. Gray regionsrepresent hypothesized region in tf-2, which is unresponsive in auxin. Black square indicates site of auxin application.

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Table S1. Comparison of cell area size (μm2) in Condine Redand tf-2

Cell no. Condine Red tf-2

1 595 1.7802 660 2.2693 442 2.0374 647 1.4895 540 1.4236 503 2.0917 589 1.2228 804 1.7099 777 2.17110 651 1.65711 587 1.16012 714 1.53713 525 1.74014 657 1.31115 620 1.72416 855 1.36517 524 1.82918 548 1.66019 656 1.60520 556 1.51821 520 1.30122 444 1.98623 404 1.85324 514 1.53125 537 2.23026 524 1.58127 576 1.54328 575 1.37829 406 1.46830 446 2.59831 414 2.10132 698 2.14633 474 1.75734 727 2.04835 507 1.60636 534 1.84437 476 1.88638 488 1.83639 594 1.77140 473 1.895Average 570 1.741Minimum 404 1.160Maximum 855 2.598

Table S2. Mutations in tf alleles

Allele DNA Protein

tf-1 Gene cannot be amplified No proteintf-2 Frame shift resulting from deletion of T

at nucleotide 637Tryptophan to Stop at position 213

tf-3 Splicing defect caused by T to C at nucleotide 963followed by the deletion of 5 nucleotides

Glycine to Stop at position 321

tf-4 G to A at nucleotide 470 Serine to Asparagine at position 157 (MYB domain)tf-z G to A at nucleotide 368 Tryptophan to Stop at position 123

A macro lesion was detected in tf-1, where we were unable to amplify genomic fragments including exon 1, exon 2, and part of thepromoter region.

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Table S4. List of primers

Target gene/experiment Primer name Forward/reverse DNA sequence

Tf/genetic mapping Bs4-F Forward 5′-GGAGCTGAATACGGATTGGA-3′Bs4-R Reverse 5′-ATCGTTCCGATGATTTCTGG-3′Y45R-F Forward 5′-CAGTAGACACGTCTTATAGTTAG-3′Y45R-R Reverse 5′-CTTGTGTAATTATTATTATTGTTGC-3′C2_At1g026810-F Forward 5′-AGTTGCGGTGCGTTTTTTTGTTGG-3′C2_At1g026810-R Reverse 5′-ACTCGAACAAATGCATCATCATCTGT-3′C2_At1g14300-F Forward 5′-AGGCGCTAGAGGCTATTTATTTGC-3′C2_At1g14300-R Reverse 5′-TCACTGACCAAAATGCTCTTCTGCC-3′

Tf/5′-RACE PCR SP1 Reverse 5′-CCAATTCTGAGGGCCATAAA-3′SP2 Reverse 5′-TCCTTGCCATTACAGCCACT-3′SP3 Reverse 5′-TGGGCTTCATCAAGACTACG-3′

Actin/sqRT-PCR Actin-F Forward 5′-TGTTGCTATTCAGGCTGTGC-3′Actin-R Reverse 5′-CTGCTCCTGGCAGTTTCAAT-3′

Tf/sqRT-PCR Tfsq-F Forward 5′-CATTGGAGACCAGCTGAAGA-3′Tfsq-R Reverse 5′-TTGGCCACTTGAATTATCTGC-3′

CAC/qRT-PCR CACq-F Forward 5′-CCTCCGTTGTGATGTAACTGG-3′CACq-R Reverse 5′-ATTGGTGGAAAGTAACATCATCG-3′

Tf/qRT-PCR Tfq-F Forward 5′-AGGAGAAGAAAAATGGGACAA-3′Tfq-R Reverse 5′-GCAGCAAATGGTGAGTATGG-3′

TKn2/qRT-PCR TKn2q-F Forward 5′-CCTAAGGAAGCAAGGCAACA-3′TKn2q-R Reverse 5′-AGCAGCATCCATCACAACAA-3′

Tf/probe synthesis Tfpr-F Forward 5′-AGGAGAAGAAAAATGGGACAA-3′Tfpr-R Reverse 5′-ATTGTGATTGATCTTGTTGCAT-3′

TKn2/probe synthesis TKn2-F Forward 5′-TGGAGGGTGGTTCTAGTGGA-3′T7-TKn2-R Reverse 5′TAATACGACTCACTATAGGGAGACCCGT

TGATTCAGCTAGTGC-3

Tf/overexpression construct pFK-PciI-F Forward 5′-ACATGTCATCAACATCATCATC-3′pFK-XhoI-R Reverse 5′-CTCGAGTCAAGTGGCTCCTACTCCAAG-3′

q, quantitative; sq, semiquantitative.

Table S3. Auxin applications

Leaflet missing onapplication site No reaction Total

Control 0 20 2010 mM IAA 21 35 56

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