Cre and lox-reporter gene constructs
To test the efficacy of gene activation via Cre/lox-mediated excision in tobacco and cowpea, we first constructed two cassettes AtRps5apro:Cre:phaseolinterm (AtRps5aproCre), and AtUbq3pro:lox-PINIIterm-lox:ZsGreen:NOSterm (AtUbq3prolox). In the cassette AtRps5aproCre (Fig. 1a), the Cre gene was controlled by the promoter (1684 bp) from Arabidopsis thaliana ribosomal protein subunit 5a (AtRps5a, AT3G11940; Weijers et al. 2001) and the phaseolin 3’ terminator. In the cassette AtUbq3prolox (Fig. 1b), the ZsGreen gene was controlled by the promoter (1721 bp) from A. thaliana polyubiquitin 3 (AtUbq3, AT5G03240, including its 5′ UTR and leading intron) and the nopaline synthase (NOS) terminator. A potato Proteinase Inhibitor II (PINII) terminator (Keil et al. 1986) flanked by two modified lox sites was inserted between the AtUbq3pro and ZsGreen coding sequence (CDS). The cassette AtRps5aproCre, together with a fluorescent-marker cassette GmEF1apro:DsRed:NOSterm (GmEF1aproDsRed) and a selectable-marker cassette, was transferred into the T-DNA region of a binary vector pPZP201BK (Covert et al. 2001) (Table S1). In the cassette GmEF1aproDsRed, a DsRed gene was controlled by the promoter (2166 bp) from soybean (Glycine max) elongation factor 1a (GmEF1a, Glyma.17G186600, including its 5′ UTR and leading intron; Li et al. 2015) and the NOS terminator. In the selectable-marker cassette, the selectable marker gene was controlled by the promoter (930 bp) from potato (Solanum tuberosum) polyubiquitin (StUbq, GenBank: L22576; Garbarino and Belknap 1994) and the NOS terminator. Flanked by the same promoter and terminator, three selectable-marker CDS, neomycin phosphotransferase II (nptII), hygromycin phosphotransferase (hpt), and Bialaphos resistance (bar), were evaluated side by side for their effect on the efficiency of cowpea transformation. The cassette AtUbq3prolox was transferred into the T-DNA region of the binary vector pORE (Coutu et al. 2007), together with a kanamycin resistance cassette (S1pronptII) wherein the nptII gene containing a Ricinus communis catalase 1 (CAT1) intron was controlled by the S1 promoter and S3 terminator from subterranean clover stunt virus (SCSV) (Schünmann et al. 2003) (Table S1). The cassettes AtUbq3prolox and S1pronptII were also transferred into the T-DNA region of the binary vector pPZP201BK, together with the cassette GmEF1aproDsRed as a fluorescent marker (Table S1).
To further evaluate Cre/lox-mediated gene activation in cowpea, two additional cassettes were constructed as AtDD45pro:Cre:OCSterm (AtDD45proCre) and AtUbq10pro:loxP-GmUbq3pro:tdTomatoER:OCS-loxP:ZsGreen:NOSterm (AtUbq10prolox). In the cassette AtDD45proCre (Fig. 1a), the synthetic codon-optimized Cre gene containing a modified CAT1 intron was controlled by the promoter (1002 bp) from A. thaliana DOWN REGULATED IN DETERMINANT INFERTILE (DD) 45/Egg Cell-secreted protein 1.2 (AtDD45/EC1.2, AT2G21740; Steffen et al. 2007; Sprunck et al. 2012) and the octopine synthase (OCS) terminator. In the cassette AtUbq10prolox (Fig. 1c), the synthetic codon-optimized ZsGreen gene was controlled by the promoter (1500 bp) from A. thaliana polyubiquitin 10 (AtUbq10, AT4G05320, including its 5’UTR and leading intron) and the NOS terminator. Between the AtUbq10pro and the ZsGreen CDS, a cassette GmUbq3pro:tdTomatoER:OCSterm (GmUbq3protdTomato) flanked by two loxP (the original lox from bacteriophage P1) sites was inserted in a reverse direction for the AtUbq10 promoter. The tdTomato gene (a variant of DsRed) (Shaner et al. 2004) was controlled by the promoter (919 bp) from soybean (G. max) ubiquitin 3 (GmUbq3, Glyma.20G141600, including its 5’UTR and leading intron) and the OCS terminator (Fig. 1c). The cassettes AtDD45proCre and AtUbq10prolox were transferred respectively into the T-DNA region of the binary vector pAGM4673 (Weber et al. 2011), together with a spectinomycin resistance cassette (Che et al. 2021) (Table S1). Considering that the 35S enhancer in the spectinomycin resistance cassette might interact with the AtDD45pro in the cassette AtDD45proCre and affect its tissue-specificity, a 2 kb transformation booster sequence (TBS) (Singer et al. 2011) was placed between the cassette AtDD45proCre and the spectinomycin resistance cassette (Table S1).
Transient assay by microprojectile bombardment
Mature cowpea seeds were surface sterilized with 10% commercial bleach (6% (w/v) sodium hypochlorite; Clorox, Oakland, CA, USA) for 30 min with agitation at 150 rpm, followed by rinsing with sterilized reverse osmosis deionized (RODI) water at least five times. After sterilization, seeds were immersed in sterilized RODI water overnight. Cotyledons were excised from the imbibed seeds and the embryo axes were used for microprojectile bombardment. With one side touching the medium, ten embryo axes were laid within a circle of 2 cm in diameter at the center of a Petri dish containing 0 MS medium composed of 1× Murashige and Skoog (MS) salts and vitamins, 3% (w/v) sucrose (Research Products International, Mt Prospect, IL, USA), and 8 mg/l agarose (pH 5.7). Microprojectile bombardment was conducted using a PDS-1000/HeTM system (Bio-Rad Laboratories, Hercules, CA, USA) under 1550 psi helium in a vacuum of 23 in Hg, with the Petri dish carrying embryo axes positioned on the sample platform 5 cm below the launch assembly. Each bombardment delivered approximately 156 ng DNA composed of an equal molar amount of the Cre cassette and lox-reporter gene cassette and 50 µg gold microcarriers 0.6 µm in diameter (Bio-Rad Laboratories). Each sample was bombarded twice. Upon evaluating the cassettes AtRps5aproCre and AtUbq3prolox harbored in the plasmid pBlueScript KS(+), a trace amount of the cassette GmEF1aproDsRed harbored in the plasmid pBlueScript KS(+) also was added so that we could check the quality of each bombardment. With the lox-flanked GmUbq3protdTomato in the lox-reporter cassette, the cassette GmEF1aproDsRed was omitted upon evaluating the cassette AtDD45proCre in vector RC2677 and the cassette AtUbq10prolox in vector RC2717 (Table S1). Cowpea embryo axes were observed for ZsGreen expression 24 h after bombardment.
Agrobacterium inoculum and plant transformation
All recombinant binary vectors (Table S1) were introduced into competent cells of Agrobacterium strain AGL1 using the freeze-thaw method (Chen et al. 1994). Agrobacterium carrying the recombinant vectors was stored in 15% glycerol at −80 °C. For tobacco transformation and cowpea transformation using Method 1 and 2 (see below), Agrobacterium inoculum was prepared as follows: Agrobacterium from the glycerol stocks was cultured in 2-ml liquid Lysogeny broth (LB) medium (ThermoFisher Scientific, Waltham, MA, USA) containing 100 mg/l kanamycin, 50 mg/l rifampicin, and 50 mg/l carbenicillin, with incubation at 150 rpm and 28 °C for 24 h. The bacterial culture was diluted 1:100 with liquid LB medium containing the same antibiotics and incubated overnight at 150 rpm, 28 °C. The culture was centrifuged for 10 min at 6000 rpm using a J2-21M centrifuge (Beckman Coulter, Brea, CA, USA), and re-suspended in liquid co-culture medium, with OD600 adjusted to 0.5–0.6. For cowpea transformation using Method 3, Agrobacterium inoculum was prepared according to Che et al. (2021).
The AtRps5aproCre and AtUbq3prolox were introduced into tobacco PI 552484 (seeds purchased from Lehle Seeds, Round Rock, TX, USA) following the protocol of Clemente (2006) with some modifications (Zhang et al. 2020). Transgenic plants containing either the cassette AtRps5aproCre or AtUbq3prolox were recovered with selection on either 20 mg/l hygromycin or 200 mg/l kanamycin, according to the selectable marker in the plasmids used for transformation (Table S1). Plants derived from different leaf disc explants were independent, while those from the same explants were potentially the same lines.
Different Cre or lox-reporter cassettes were introduced into cowpea IT86D-1010 following three distinct methods. The cassettes AtRps5aproCre and AtUbq3prolox were introduced by either Method 1 described by Popelka et al. (2006) with some modification or Method 2 wherein explants were cotyledonary nodes from seedlings pre-conditioned with 5 mg/l 6-benzylaminopurine (BA). Both methods started with mature cowpea seeds surface sterilized as previously described (Sect. “Transient assay by microprojectile bombardment”). In Method 1, sterilized seeds were immersed in sterilized RODI water overnight. The cotyledonary nodes from the imbibed seeds were used as explants for Agrobacterium-mediated transformation after removing the seed coat, shoot tips, cotyledons, and radicals. Every ten explants were immersed in 2-ml inoculum consisting of Agrobacterium suspended in a liquid co-culture medium (CCM, Table S2) supplemented with 0.02% (v/v) Silwet-77 (Lehle Seeds, Round Rock, TX, USA) in a 10-ml borosilicate glass test tube (Cat# 14-961-27, ThermoFisher Scientific, Waltham, MA, USA), then sonicated for 20 s using an FS30H sonicator (ThermoFisher Scientific). After 30 min incubation at room temperature, explants were blotted dry with sterile filter paper and transferred onto CCM (Table S2) with a piece of filter paper on top of the medium to prevent direct contact between explants and the medium, 20–25 explants each plate. In Method 2, sterilized mature seeds were cultured on a germination medium (GM, Table S2) for 4 d. Seedlings with a greening cotyledonary node were selected, followed by excision of cotyledons, shoot tips, and roots. The remaining cotyledonary nodes were used as explants for transformation. Every five explants were immersed in 2-ml inoculum consisting of Agrobacterium resuspended in a liquid modified co-culture medium (CCM’, Table S2) supplemented with 0.02% (v/v) Silwet-77 in a 10-ml borosilicate glass test tube then sonicated for 4 min. After 30-min incubation at room temperature, explants were blotted dry and placed on CCM’ plates (Table S2), 12 explants each, with a piece of filter paper between explants and the medium. In both methods, after a 5-day co-culture, explants were washed in a liquid shoot induction medium (SIM, Table S2) then blotted dry. Explants were transferred to SIM (Table S2) supplemented with 20 mg/l hygromycin, 5 mg/l phosphinothricin (PPT), or 200 mg/l kanamycin according to the selectable marker gene used in the vectors (Table S1). Explants forming shoots were transferred to fresh SIM every other week and monitored for DsRed expression. Explants with transgenic shoots were transferred to a shoot elongation medium (SEM, Table S2) supplemented with the same selective agent used in SIM for shoot development and rooting.
Transgenic shoots that failed to form roots were recovered by in vitro grafting adapted from a method for sunflower (Zhang and Finer 2016). Sterilized mature seeds were germinated under a sterilized folded paper towel saturated with sterile RODI water in MagentaTM GA7 vessels (Sigma-Aldrich, St Louis, MO, USA), with ten seeds in each box. Rootstocks were made from 5-day-old seedlings by removing the shoot tips and cotyledons. As a scion, a developed transgenic shoot (> 0.5 cm) with its base shaped into a wedge was inserted into the longitudinal incision (0.5–1 cm) made in the side of the rootstock’s hypocotyl. The hypocotyl tissue from both sides held the scion in place. The grafted plants were grown in GA7 vessels containing 0MS medium supplemented with 30 mg/l meropenem (ABBLIS Chemicals, Houston, TX, USA) for 2–3 weeks. Elongation of the scions indicated graft success. The successfully rooted or grafted plants were transferred to soil, acclimatized in plastic containers with the lids gradually opened over a 3-week period. Plants were transferred to the greenhouse to set seeds when they were sufficiently hardened and growing vigorously.
The cassettes AtDD45proCre and AtUbq10prolox were introduced into cowpea using Method 3 (Che et al. 2021) given its higher efficiency in recovering transgenic plants. In brief, mature seeds were surface-sterilized overnight using chlorine gas, then soaked overnight in a bean germination medium (Che et al. 2021). Embryo axes were isolated by removing seed coats, cotyledons, and plumules without damaging the meristematic dome and collected in sterile RODI water. After removing water, embryo axes (100–200 pieces) were infected with 15-ml Agrobacterium inoculum plus 50-μl sterile Poloxamer 188 10% solution in a 100 × 25 mm Petri dish (ThermoFisher Scientific). After sonication for 3 s, 10 ml of inoculum was added to each Petri dish and incubated at 60 rpm, room temperature, for 1.5 h in the dark. Explants were removed from inoculum and transferred to filter paper (VWR Cat# 28320-020) wetted with 700 μl of infection medium (Che et al. 2021) in a 100 × 25 mm Petri dish, with every 30 explants in a pile. After 2-day co-culture at 21 °C under dim light with a 16/8 h light cycle, embryo axes were inserted into SIM (Table S2) supplemented with 50 mg/l spectinomycin with shoot apex and cotyledonary node above the medium. The shoot apex was removed after 5-day incubation on SIM to facilitate the formation of axillary shoots. After 4 weeks, transgenic shoots were either transferred to a rooting medium (Che et al. 2021) for rooting or a shoot elongation medium (Che et al. 2021) for further growth before transfer to the rooting medium. Plantlets derived from different cotyledonary-node explants were independent, while those from the same explants were considered as potentially the same lines.
All plant tissues were incubated at 25 °C, with a 16/8 h light cycle, except for the cowpea transformation using Method 3 wherein plant tissues were incubated at 25 °C and 24-h light after co-culture. MS salts, MS vitamins, and acetosyringone (AS) were from PhytoTechnology Laboratories (Lenexa, KS, USA). Unless otherwise noted, all chemicals were from Millipore Sigma (St. Louis, MO, USA).
Genomic DNA of tobacco and cowpea was extracted from leaf tissue using the cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle 1990). The presence of the transgene was verified by polymerase chain reaction (PCR) using corresponding primer combinations (Table S3). We conducted PCR using PrimeStar GXL DNA polymerase (Takara Bio, Kusatsu, Japan) in a 25 µl reaction for amplicons larger than 1 kb and using GoTaq® Master Mix (M7123, Promega, Madison, WI, USA) in a 20 µl reaction for amplicons smaller than 1 kb. The PCR setup followed the manufacturers' instructions with 20–100 ng genomic DNA as a template. PCR products were visualized under UV light after electrophoresis in 1% agarose gels and staining with ethidium bromide.
Evaluation of transgene segregation in transgenic lines
In tobacco, after surface sterilization according to Zhang et al. (2020), T1 seeds of the lines harboring the cassette AtRps5aproCre were germinated on 0MS medium containing either 200 mg/l kanamycin or 20 mg/l hygromycin depending on the selectable marker used in the vectors (Table S1). After 2 weeks, seedlings were observed for DsRed expression and counted. Surface-sterilized T1 seeds of tobacco lines harboring the cassette AtUbq3prolox (from vector pZZ017, Table S1) were germinated on 0MS medium containing 600 mg/l kanamycin for a stricter selection to avoid possible false positives. After 2 weeks, the green seedlings were counted as transgenic while the seedlings showing chlorosis at cotyledons or the shoot apex were counted as non-transgenic.
In cowpea, mature T1 seeds of the lines harboring the cassette AtRps5aproCre, AtUbq3prolox (from vector pZZ031B, Table S1), or AtUbq10prolox were observed for DsRed/tdTomato expression and counted. If fluorescence was not evident in dry seeds, a proportion of T1 seeds from those lines was germinated between sheets of filter paper saturated with sterile RODI water and observed for DsRed/tdTomato fluorescence after 5 days. For the cowpea lines harboring the cassette AtDD45proCre, the presence or absence of the transgene in T1 progeny was determined by PCR using genomic DNA extracted from leaf tissues of 2-week-old T1 seedlings grown in the greenhouse. The segregation ratios of the transgene in tobacco and cowpea transgenic lines were calculated and tested by the Chi-square test.
Estimation of copy number by quantitative PCR (qPCR)
Genomic DNA from transgenic plants harboring the cassette AtRps5aproCre or AtUbq3prolox was digested by restriction endonuclease HindIII-HF® (New England Biolabs, Ipswich, MA, USA) while genomic DNA from transgenic plants harboring the cassette AtDD45proCre or AtUbq10prolox was digested by NcoI-HF® (New England Biolabs). After purification with the DNA Clean and Concentration-5 Kit (Zymo Research, Irvine, CA, USA), digested DNAs in 20-fold dilution served as the qPCR templates. The qPCR reactions were conducted with the SYBR green I/HRM dye program in a LightCycler® 480 system (Roche, Basel, Switzerland) following the manufacturer’s protocol of LightCycler 480 SYBR Green I Master V13 (Roche, Mannheim, Germany) in a 20 µl volume. The amplification efficiency of each primer combination was estimated based on qPCR data of a 5-log serial dilution (0.0016, 0.008, 0.04, 0.2, 1×) of a DNA mixture that contained an equal amount of DNA from each tested sample using the absolute quantification method in the LightCycler 480 software (Roche, Release 1.5.0). The qPCR results with two technical replicates for each sample were analyzed using the advanced relative quantification method in the LightCycler 480 software. Primers for qPCR were designed using either Geneious R10 (Auckland, New Zealand) or Realtime PCR Tool from Integrated DNA Technology (San Jose, CA, USA) (Table S4). The tobacco tubulin α-chain gene (NCBI accession # XM_016623993) served as a reference gene (Głowacka et al. 2016) for the assays in tobacco. To identify single-copy genes used as reference genes for cowpea, the Plants Datasets (Embryophyta odb9) from Benchmarking Universal Single-Copy Orthologs (BUSCO) was aligned to the whole-genome shot-gun (WGS) assembly (MATU00000000 at NCBI) of cowpea IT97K-499-35 (Muñoz-Amatriaín et al. 2017) using the BUSCO v3 software (Simão et al. 2015; Waterhouse et al. 2018). The amino acid sequences of those orthologs classified as “complete” BUSCO genes were aligned to the reference protein database at NCBI using the BLASTP program to acquire the annotation of the genes. After removing mitochondrial and chloroplastic genes, the cowpea nucleotide sequences which encode the amino acid sequences were aligned to the cowpea WGS assembly to verify whether the gene sequence was unique in the assembly. The genes without any duplication in the genome were considered as single-copy genes in the cowpea genome and potentially used as reference genes for qPCR. After testing the amplification efficiency and specificity of the first six genes from the list, a predicted cowpea F-box protein (VuFbox, Phytozome12 transcript name Vigun07g146600) was selected and used as the reference gene for the assays in cowpea. The copy numbers of the transgene were calculated based on the ratios of the transgene to the reference gene.
Genomic DNA of 10 µg from the cowpea lines of the cassettes AtRps5aproCre and AtUbq3prolox was digested overnight by SpeI-HF® and MluI-HF® (New England Biolabs), respectively. Restriction fragments were separated by gel electrophoresis and then transferred to GeneScreen Plus® hybridization transfer membrane (Cat# NEF1017001PK, PerkinElmer, Waltham, MA, USA). Hybridization was conducted using a digoxigenin (DIG)-labeled probe targeting either the Cre gene for the cassette AtRps5aproCre or the ZsGreen gene for the cassette AtUbq3prolox and using DIG Easy HybTM as hybridization buffer following the Roche DIG application manual for filter hybridization (Eisel et al. 2008). The probes were generated and labeled by PCR with primers p3769/p3770 and p3700/p3701 (Table S3) for Cre and ZsGreen, respectively using the Roche PCR DIG labeling mix following the manufacturer’s protocol. Detection was conducted using the chromogenic assay with NBT/BCIP according to the Roche DIG application manual for filter hybridization (Eisel et al. 2008). All chemicals used for making buffers, as well as blocking reagent (Cat# 11096176001), anti-DIG-AP (Cat# 11093274910), and NBT/BCIP (Cat# 11681451001), were from Millipore Sigma.
Identification of cowpea plants homozygous for the transgenes
The ideal plant materials for evaluating Cre/lox-mediated gene activation upon crossing were homozygous plants derived from lines where the transgene segregated as a single locus. For the cassette AtRps5aproCre, at least six transgenic T1 seedlings from the single-locus lines were grown in the greenhouse to set seeds, and their T2 seeds were observed for DsRed expression. If all T2 seeds harvested displayed DsRed fluorescence, the T1 plants were homozygous for the transgene. For the cassettes AtUbq3prolox, AtDD45proCre and AtUbq10prolox, the homozygotes were identified using qPCR (Sect. “Estimation of copy number by quantitative PCR
(qPCR)”) from the single-locus lines. If the ratio of the transgene to the reference gene in the T1 progeny doubled compared to the T0 mother plants, the T1 progeny would be homozygous for the transgene. Expression of DsRed/tdTomato in T2 seeds from the homozygous T1 of the AtUbq3prolox and AtUbq10prolox lines also was observed to verify their homozygosity. The homozygous plants were grown in the greenhouse to increase seeds and make crosses.
We also attempted to identify homozygous progeny that inherited a single locus from the AtRps5aproCre or AtUbq3prolox lines with multiple copies of the transgene. The numbers of cowpea transgenic lines for the cassettes AtRps5aproCre and AtUbq3prolox were low due to the low efficiency of the transformation methods applied. To include additional lines harboring those cassettes in the analysis, it was necessary to utilize high copy-number lines. Since the transgenes segregated in the T1 generation, progeny inheriting only one of the transgene loci from those lines could be obtained if enough progeny were screened by qPCR, T1 progeny that likely carried two copies of the transgene were identified and grown in the greenhouse to set seeds. If all the T2 seeds from a T1 displayed DsRed fluorescence, the T1 progeny had become homozygous for the single locus inherited from the T0 parents. If the fluorescence trait segregated among T2 seeds from a T1 progeny, the segregation ratio would indicate the number of transgene loci inherited by the T1 progeny. The T1 progeny with an approximately 3:1 segregation ratio were further advanced to obtain homozygous plants using the same approach as previously described.
Tobacco crossing and F1 embryo isolation
Flowers were emasculated at stage 10 (Koltunow et al. 1990) and covered with a tailored pollination envelope. After 2 days, the emasculated flowers on the AtRps5aproCre lines were pollinated with pollen from the AtUbq3prolox line. Meanwhile, the AtRps5aproCre lines and the AtUbq3prolox line were self-pollinated as controls. The pollinated flowers were tagged and covered with tailored pollination envelopes. Ovules were harvested 6–8 days after pollination (DAP), and embryos were isolated using the method described by Fu et al. (1996) with some modifications (Zhang et al. 2020). Isolated embryos were observed for ZsGreen expression.
Cowpea crossing and F1 progeny analysis
Cowpea flowers were emasculated in the evening before anthesis, followed by covering the peduncles of emasculated flowers with moist tailored pollination envelopes. The next morning, emasculated flowers were pollinated and tagged, and the peduncles of the pollinated flowers were covered by moist tailored pollination envelopes. After harvest, mature F1 seeds were observed for ZsGreen expression. Genomic DNA was extracted from leaf tissues of F1 seedlings. F1 progeny were genotyped by PCR to verify whether they inherited both the Cre and lox-reporter genes. With the use of p3785/p3786 and p4825/p4826, PCR could also confirm the Cre/lox-mediated excision in F1 progeny inheriting both the Cre and lox-reporter genes, as the excision would result in a smaller amplicon due to the removal of the intervening DNA sequence flanked by two lox sites (Fig. 1b, c).
Observation of fluorescence
Expression of fluorescent marker genes (i.e., ZsGreen, DsRed, and tdTomato) in plant tissues was observed using a Stemi SVII stereomicroscope equipped with an HBO illuminator (Zeiss, Thornwood, NY, USA), a FITC filter set (λexcitation = 480 nm, and λemission = 515 nm; Chroma Technology, Bellows Falls, VT, USA), and a DsRED filter (λexcitation = 545/25 nm, dichroic 565LP, λemission = 605/70 nm; Chroma Technology). Expression of ZsGreen in isolated embryos was observed under a Zeiss Axioskop 2 plus fluorescence microscope equipped with an 89 North® PhotoFluor LM-75 illumination system (Chroma Technology) and a FITC filter set. Images were taken using an AxioCam camera (Carl Zeiss, Oberkochen, Germany) and the AxioVision LE64 software.
Nomenclature of transgenic cowpea
We gave a unique code to every T0 transgenic cowpea plant recovered from tissue culture. For the AtRps5aproCre and AtUbq3prolox, each plant was named by a six-digit code. The first two digits were the last two digits of the vector used; the middle two digits indicated the independent explant giving rise to transgenic shoots (i.e., independent lines); and the last two digits indicated the plants recovered from the particular line. In the code “120200”, for example, ‘12’ indicated the use of vector pZZ012; ‘02’ indicated the independent line 2; and ‘00’ indicated the first plant recovered from line 1202. For the AtDD45proCre and AtUbq10prolox, each plant was named by a code starting with “R” followed by six digits. In the code “R771402”, for example, ‘77’ indicated the use of vector RC2677; ‘14’ indicated the independent line 14; and ‘02’ indicated the second plant recovered from line R7714. Regardless of the last two digits, as long as the first four digits were the same, the plants were potentially clones from the same line unless additional evidence showed that they were not.