Evidence for stable transformation of wheat by floral dip in Agrobacterium tumefaciens
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- Zale, J.M., Agarwal, S., Loar, S. et al. Plant Cell Rep (2009) 28: 903. doi:10.1007/s00299-009-0696-0
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Hexaploid wheat, one of the world’s most important staple crops, remains a challenge for genetic transformation. We are developing a floral transformation protocol for wheat that does not require tissue culture. This paper presents three transformants in the hard red germplasm line Crocus that have been characterized thoroughly at the molecular level over three to six generations. Wheat spikes at the early boot stage, i.e. the early, mid or late uninucleate microspore stages, were immersed in an infiltration medium of strain C58C1 harboring pDs(Hyg)35S, or strain AGL1 harboring pBECKSred. pDs(Hyg)35S contains the NPTII and hph selectable markers, and transformants were detected using paromomycin spray at the whole plant level, NPTII ELISAs, or selection on medium with hygromycin. Strain AGL1, harboring pBECKSred, which contains the maize anthocyanin regulators, Lc and C1, and the NPTII gene, was also used to produce a Crocus transformant. T1 and T2 seeds with red embryos were selected; T1 and T2 plants were screened by sequential tests for paromomycin resistance and NPTII ELISAs. The transformants were low copy number and showed Mendelian segregation in the T2. Stable transmission of the transgenes over several generations has been demonstrated using Southern analysis. Gene expression in advanced progeny was shown using Reverse Transcriptase-PCR and ELISA assays for NPTII protein expression. This protocol has the potential to reduce the time and expense required for wheat transformation.
KeywordsAgrobacteriumFloral transformationIn plantaTriticum aestivum (L.)Floral dip
Neomycin phosphotransferase gene
Anthocynanin regulatory genes of maize
Dissociation element of maize
Polymerase chain reaction
Enzyme-linked immunosorbent assay
Genetic transformation is an essential tool for analyzing gene function in plants. Most published protocols for the transformation of hexaploid wheat (Triticum aestivum L.) involve the use of tissue culture, skilled personnel and specialized equipment that may not be available to all researchers, especially those in developing countries. Currently, transgenes are typically introduced using particle bombardment (biolistics) and Agrobacterium-mediated transformation of dissected explants. Particle bombardment has been used to transform embryogenic wheat calli (Vasil et al. 1992; Weeks et al. 1993) and dissected scutella (Nehra et al. 1994), and T-DNA transfer by Agrobacterium has been achieved with embryos, pre-cultured immature embryos, and embryogenic calli (Cheng et al. 1997). These approaches rely on the totipotency of individual plant cells to dedifferentiate into unorganized callus tissue, become embryogenic and regenerate into whole plants through organogenesis. Immature embryos are primarily used because of their greater capacity to regenerate plants (Zale et al. 2004).
Cereal transformation via a tissue culture phase has been successful, but involves several limitations. The use of tissue culture allows selection of single transformed cells which are regenerated in a whole plant and this lessens the production of genetic chimeras. However, the tissue culture approach causes somaclonal variation due to either epigenetic effects or chromosomal rearrangements (Kaeppler et al. 2000; Mohan Jain 2001). For example, many of the two hundred thousand T-DNA lines produced by tissue culture in rice are somaclonal variants (An et al. 2005). In the past, transformation success in wheat has been limited to a relatively few genotypes that regenerate well from tissue culture (Jones 2005; Pellegrineschi et al. 2002). Biolistics can also cause multiple T-DNA insertions and gene silencing in subsequent generations (Taylor and Fauquet 2002).
Attempts at floral transformation of allohexaploid wheat (Triticum aestivum L.) were published prior to the development of the floral dip method in Arabidopsis. The target tissue in these experiments was the pollen, and a basal medium containing Agrobacterium harboring genetic constructs was pipetted into open wheat florets at anthesis. In transformants isolated by Hess et al. (1990), the T-DNA underwent size alterations or appeared to be lost in subsequent generations. Langridge et al. (1992) concluded that floral transformation of wheat, barley and maize at anthesis led to artifacts on gels in the T0 generation possibly due to transformation of an endophytic bacterium.
In planta transformation of the model dicotyledonous species, Arabidopsis thaliana by vacuum infiltration of whole plants (Bechtold et al. 1993) and the floral dip (Clough and Bent 1998) are now routine, and have contributed greatly to the rapid forward and reverse genetics research in this species. Three different laboratories have confirmed that the target of T-DNA transfer in Arabidopsis is primarily the female ovule, and segregation data showed that the T-DNA insertions are hemizygous (Bechtold et al. 2000; Desfeux et al. 2000; Ye et al. 1999). Similar approaches have been developed for the in planta transformation a number of other dicotyledonous species such as Shepard’s purse (Capsella bursa-pastoris), radish, and alfalfa (Medicago sativa) (Bartholmes, et al. 2008; Curtis and Nam 2001; Weeks, et al. 2008).
A number of advances in reducing the dependence on tissue culture have recently been made in wheat transformation. An in planta transformation method in which an Agrobacterium coated needle is used to inoculate a germinating wheat seedling has been developed (Supartana et al. 2006). Another method involves inoculating Agrobacterium on the basal portion of cut seedlings with no intervening callus phase and requires minimal tissue culture (Zhao et al. 2006).
The objective of this research was to determine whether stable transformants of wheat could be obtained by the floral dip if the treatment were performed at an earlier stage of floral development than previously used (Hess et al. 1990; Langridge et al. 1992). This was based on the rationale that the target of transformation may be the ovule, as in Arabidopsis (Bechtold et al. 2000; Desfeux et al. 2000; Ye et al. 1999). An infiltration medium containing a surfactant and acetosyringone was used to produce multiple stable transformants in the ‘Crocus’ genotype, a hard red wheat germplasm line that possesses the double recessive alleles for high crossability with rye (Zale and Scoles 1999). In one version of the protocol, putative transformants were screened with a paromomycin spray at the whole plant level (Cheng et al. 1997) or germinated on hygromycin medium followed by NPTII ELISAs. In another version of the protocol, the embryos of T1 seeds were visually screened for color changes induced by the maize anthocyanin regulatory genes. Southern hybridizations for transgene copy number have shown that the T-DNA has integrated in the wheat genome with stable transmission of the transgenes over three to six generations. Finally, RT-PCR and NPTII ELISA assays demonstrated expression of the transgenes in advanced progenies.
Materials and methods
Plant material and growth conditions
Crocus spring wheat germplasm was obtained from the USDA/ARS National Germplasm Resources Information Network (GRIN http://www.ars-grin.gov/). Crocus is a hard red spring wheat germplasm line adapted to the Northern Great Plains and it also possesses the crossability alleles (Zale and Scoles 1999). The original rationale was that if the crossability alleles of ‘Crocus’ provide a decreased barrier in wide crosses, and then they might also result in a reduced barrier to Agrobacterium in the pistil. For example, these alleles are associated with decreased expression of nucleases in the carpal at anthesis (Zale, unpublished) and might increase the probability of floral transformation. Moreover, a red seeded wheat line was chosen because expression of maize Lc/C1 in white wheat lines is known to be detrimental to plant growth (McCormac et al. 1997). Zuzanna spring wheat is a Czech variety and was a gift from Dr. Ludmila Ohnoutkova, The University of Tennessee, Department of Plant Sciences.
The wheat plants were grown in the greenhouse or in walk-in growth rooms, with day temperatures at 23 ± 4°C with a 16 h light cycle; the night temperatures were 16 ± 4°C with an 8-h dark cycle. The intensity of the lighting never fell below 400 μE/m2 per second.
The Agrobacterium tumefaciens strains C58C1 and AGL1 were used in transformation experiments. The C58C1 strain was chosen because its relative, C58, has been used to transform monocots including wheat (Cheng et al. 1997) and dicots (Long et al. 1997; Wilson et al. 1996). AGL1 is a derivative of C58 containing a deletion in the recA gene that lessens genetic rearrangements of the Ti plasmid in Agrobacterium (Lazo et al. 1991), and is useful in monocot transformation (Pacurar et al. 2007; Wu et al. 2007).
Agrobacterium culture and infiltration medium
Five ml of LB broth was inoculated with the appropriate antibiotics and grown overnight at 22 ± 4°C with shaking at 2×g. The next day, 2.5 ml of this culture was used to inoculate 250 ml of YEP broth containing the antibiotics and 200 μM acetosyringone, and grown at 22 ± 4°C with shaking at 2×g until the OD600 = 0.8–1.0. The YEP/Agrobacterium culture was centrifuged at 6,400×g for 15 min at room temperature and the supernatant discarded. The cells were gently re-suspended in infiltration medium consisting of half-strength Murashige and Skoog (MS) (Murashige and Skoog 1962) medium (Fisher Biotech, Pittsburgh, PA), 5% w/v sucrose, 0.5 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer, pH 5.8 (Bechtold et al. 1993; Clough and Bent 1998), and 200 μM acetosyringone. The wetting agent Silwet L-77, at 0.4% (v/v) was added just prior to dipping.
Preparation of the wheat spikes and floral transformation
Screening of transformants
Screening of putative transformants typically commenced in the T1 generation but never with PCR because of its sensitivity of to bacterial contamination and the preponderance of positive plants. Seeds derived from T0 plants treated with pBECKSred, were visually examined under a stereoscope and those with red embryos (see Fig. S1) were selected for further testing. Seeds were washed in 30–70% commercial bleach for 30 min with shaking, rinsed three times in sterile distilled H2O, 5 min per wash, and planted in 6 in. pots containing potting mix.
A series of sequential screens were then used to identify putatively transformed T1 plants containing pDs(Hyg)35S or pBECKSred. Screening for expression of the NPTII gene at the whole plant level was performed by spraying a 2% paromomycin solution containing 0.02% Tween 20 on seedlings at the two–three leaf stage and scoring for minimal bleaching 7–10 days later (Cheng et al. 1997). Tissue was harvested from paromomycin-screened plants and tests for NPTII protein expression by ELISA assay performed according to manufacturer’s instructions (Agdia, Elkhart, Indiana). Positive plants were saved if ELISA readings were greater than those of wild-type Crocus germplasm.
Segregation ratios and statistical analyses
Segregation of T2 progeny derived from independent transformation events was determined using 2% paromomycin at the whole plant level (Cheng et al. 1997), NPTII ELISAs, growth on hygromycin, or scoring for seed color changes induced by the maize anthocyanin regulatory genes. The segregation ratios were subjected to χ2 Goodness of Fit tests (Mead et al. 2003).
Genomic DNA digestions, probes and Southern blots
Genomic DNA was extracted with either using the DNAzol™ procedure (Invitrogen, Carlsbad, CA) or the CTAB DNA extraction procedure (Kleinhofs et al. 1993). Genomic DNA (25 µg) was digested to completion with restriction enzymes (New England Biolabs, Ipswich, MA), and blotted onto Gene Screen™ membrane (Perkin Elmer NEN, Waltham, MA) or Hybond N+™ (Amersham Pharmacia, Piscataway, NJ) using the alkaline transfer procedure (Sambrook and Russell 2001). The rapid downward Southern blot (Chomczynski 1993) method was employed to improve transfer of the DNA to the membrane. Left border analysis was performed to determine copy number (Cheng et al. 1997), and transgene analysis performed using enzymes that cut on either side of the transgene (Jordan 2000).
The NPTII or hph probes used in Southern hybridizations were PCR amplified. Gene specific primers to the NPTII sequence were used to amplify a 929 bp product from pBECKSred using the NPTII Forward 5′-GCTTGGGTGGAGAGGCTATT-3′ and Reverse 5′-CGAAGAACTCCAGCATGAGA-3′. An 809 bp hph product was amplified from the hygromycin gene of pDs(Hyg)35S using forward primer 5′-GATGTAGGAGGGCGTGGATA-3′ and the reverse primer 5′-ATTTGTGTACGCCCGACAGT-3′. The C1 probe was obtained by digestion of pBECKSred with EcoRI and excision of the 1.1 kb band. All probes were gel purified using the Qiaex II kit (Qiagen, Valencia, CA).
Radioactive hybridizations were performed by denaturing the DNA probes and labeling with Easy Tides α-32P dCTP specific activity 6,000 Ci (Perkin Elmer-General Electric, Waltham, MA) using Megaprime™ random primer labeling kit (Amersham-General Electric, Piscataway, NJ). Unincorporated α-32P dCTP was removed using Nucaway™ spin columns (Ambion-Applied Biosystems, Austin, TX). Labeled probes with at least 1.0 × 106 counts per min per ml of hybridization buffer were used in hybridization. The blots were hybridized at 43–45°C using Ultrahyb™ hybridization buffer (Ambion-Applied Biosystems, Austin, TX) and washed at the recommended temperatures and stringencies. Blots were exposed to Kodak Biomax™ MS film (Fisher Scientific, Pittsburgh, PA) or exposed to BIORAD phosphorimaging screens (Hercules, CA). Phosphor images were scanned on a BIORAD phosphorimager (Hercules, CA) and the scans were analyzed using Quantity One™ software (BIORAD, Hercules, CA). Image J software (National Institute of Health; http://rsb.info.nih.gov/ij/) was used to subtract background, remove noise, and alter contrast from some of the digital images.
Reverse transcriptase PCR (RT-PCR)
RNA was extracted using the Qiagen RNeasy™ extraction kit (Valencia, CA). RNase-free DNase I polymerase was used to degrade plant genomic DNA before the RT reaction. RT-PCR was done in two steps using the Masterscript™ RT-PCR System from 5 Prime (San Francisco, CA) according to the instructions. Mastermix 1 (15 μl) was set-up as advised by the vendor. Mastermix 2 was total RNA (200 ng–1 μg in 5 μl) heated at 65°C and chilled on ice. Both tubes were mixed together and incubated for 90 min at 42°C.
PCR was performed with Taq DNA polymerase (Takara, Clonetech, Mountain View, CA) using primers: C1 Forward: 5′-TCGGACGACTGCAGC TCGGC-3′, C1 Reverse: 5′-CCTCGTGCTTATTGGACA-3′ to generate a product of 213 bp. PCR conditions were: initial denaturation at 94°C for 4 min; 30 cycles of 92°C for 30 s; 55°C for 30 s; 72°C for 30 s; and final extension at 72°C for 5 min. The NPTII primers used were NPTII Forward 5′-TGCTCCTGCCGAGAAAGTAT-3′, NPTII Reverse 5′-AATATCACGGGTAGCCAACG-3′ to generate a 356 bp product or Forward 5′-GTAGCCGGATCAAGCGTATG-3′, NPTII Reverse 5′-GCTCGACGTTGTCACTGAAG-3′ to yield a 150 bp product.
PCR conditions were: initial denaturation at 94°C for 4 min; 25 cycles of 92°C for 30 s; 60°C for 30 s; 72°C for 30 s; and final extension at 72°C for 5 min. The hph primers used were hph Forward 5′-GTGTCACGTTGCAAGACCTG-3′ and hph Reverse 5′-ACATTGTTGGAGCCGAAATC-3′ to generate a 322 bp product using the same thermal cycler program as for NPTII primers with an annealing temperature of 52°C.
Crosses with Zuzanna spring wheat
To unequivocally validate that gene transfer was nuclear, high NPTII ELISA transgenic plants were used as the pollen parents in crosses with Zuzanna, a spring wheat cultivar. Zuzanna plants were emasculated prior to pollen shed and covered in glassine bags for 2 days. Pollen from one spike of transgenic wheat line was dusted on the pistils of Zuzanna, the spikes were bagged, and allowed to set seed. Hybrid seed was collected and tested by PCR for the presence of the NPTII gene.
Dipping of Crocus wheat at various stages of microspore development
Stage of dippinga
Number of florets dipped
Number of T1 seeds
T4, T5, T6
T1, T2, T3
0 (red foci on some endosperm)
Transformation of Crocus with pDs(Hyg)35S
Two experiments using spikes at the uninucleate stage of microspore development were performed using infiltration media containing strain C58C1 carrying pDs(Hyg)35S (Table 1). The infiltration medium was formulated essentially as described for the Arabidopsis floral dip (Bechtold et al. 1993; Clough and Bent 1998) except that acetosyringone was added. Acetosyringone is a phenolic compound that induces the virulence genes in Agrobacterium (Hirooka et al. 1987). The first transformant, 3B2, was generated after dipping six unemasculated mid-late uninucleate stage spikes, with clipped florets, into infiltration media for 2 min. The spikes were bagged for 2 days to maintain humidity, and the plants were left to self-pollinate. Seed set after dipping was reduced (Table 1). The T1 seeds were sterilized with a commercial bleach solution, planted, and the seedlings sprayed with 2% paromomycin at the two to three leaf stage to screen for resistance conferred by the NPTII gene (Cheng et al. 1997). One T1 Crocus plant showed minimal spotting due to the paromomycin spray and was advanced to the T2 for further analysis. One hundred and twenty-one T2 progeny were tested for resistance to paromomycin spray. The T2 plants segregated in a 3:1 ratio of 93 resistant: 28 sensitive (χ2 = 0.187; probability (p) = not significant (NS)) suggesting one T-DNA insertion. To confirm the segregation analysis, genomic DNA was isolated from 14 T2 seedlings and analyzed in PCR with the NPTII primers. Ten out of 14 showed the presence of NPTII suggesting 3:1 segregation (χ2 = 0.095; p = NS; Fig. S2).
The second transformant, 14C1, was obtained after dipping spikes of Crocus with C58C1 harboring pDs(Hyg)35S twice for 2 min on two consecutive mornings. The wheat spikes were at the early to mid uninucleate microspore stage, when the spike was approximately four cm in length. Such double dipping is also practiced in Arabidopsis (Bent 2000). Seed set was severely reduced after the two treatments and only one T1 seed was recovered (Table 1). This T1 seedling showed minimal spotting with 2% paromomycin and was advanced to produce T2 seeds. The T2 seeds were plated on MS medium with hygromycin (60 mg/L) to determine segregation ratios. Segregation in the T2 generation produced 275 hygromycin resistant:25 hygromycin sensitive plants (15:1; χ2 = 2.02, p = NS) suggesting the presence of two T-DNA insertions. Sensitive plants were very chlorotic.
A different Southern analysis strategy was used to analyze the hygromycin markers in 14C1 and 3B2. Genomic DNA was digested with BglII, an enzyme that cuts on either side of the hygromycin transgene and probe in the pDs(Hyg)35S T-DNA (Fig. 1a). The presence of the hygromycin transgene results in an 800 bp band when a DNA blot is hybridized with the hygromycin probe in T6 progeny 3B2, and in the T5 progeny of 14C1, thus confirming transmission of the hygromycin transgene (Fig. 3b).
Finally, to demonstrate unequivocally that the T-DNA had integrated into the nuclear genome of the transgenic plants, 14C1 T2 transformants were used as the pollen parents in crosses with ‘Zuzanna’ wheat as the female parent. Two F1 ‘Zuzanna’/14C1 hybrid seeds were germinated and the seedlings were tested for the presence of the NPTII transgene by PCR. The ‘Zuzanna’ parent tested negative, whereas the F1 hybrids tested positive demonstrating that the NPTII transgene is transmitted in crosses via the pollen (Fig. 3c).
Transformation of Crocus with pBECKSred
In working with the pDs(Hyg)35S transformants, one limitation in identifying transformants was the need to use the paromomycin screen to identify primary transformants. Thus, additional floral dip experiments were performed using pBECKSred, a T-DNA vector containing both the NPTII marker and two maize regulators of anthocyanin synthesis, C1 and Lc, whose expression allowed a visual screen for red pigmentation in transformed seed.
Floral dip experiments with infiltration media containing Agrobacterium AGL1 harboring pBECKSred (McCormac et al. 1997) were performed on Crocus spikes at different stages of microspore development including the late uninucleate, the binucleate, and trinucleate stages (Table 1). No transformation events, as determined by red embryos, screens for paromomycin resistance and NPTII ELISAs, were isolated in 3,000 T1 seeds derived from dipping of binucleate stage spikes or in 4,200 T1 seeds derived from dipping of trinucealate stage spikes (Table 1). It was observed that a small number of seeds from these experiments did show red foci on the endosperm; however, red pigmentation on the endosperm was not sufficient to identify transformants.
Southern analysis was used to show integration of the T-DNA transgene into the wheat genome, to determine transgene copy number, and to detect transgene transmission to the T2 and T3 generation. Left border analysis was performed by digesting T2 and T3 genomic DNA with BamHI, an enzyme that cuts to the right of the NPTII probe (Fig. 1b). Hybridization of genomic DNA blots with the NPTII probe detected a single band of 5.5 kb in 1CR1 T2 (Fig. S4) and T3 plants (Fig. 4b). This indicates that a single copy of the NPTII transgene was stably transmitted over the T2 and T3 generations.
If the T-DNA of pBECKSred is integrated into the Crocus genome, the 2.5 kb C1 transgene should also be present. 1CR1 T3 genomic DNA was digested with HindIII, an enzyme cuts on either side of the C1 probe resulting in a band of ~2.5 kb (Fig. 1). Hybridization of DNA blots with the C1 probe detected the expected 2.5 kb C1 band in 1CR1, but not in Crocus wild-type (Fig. 4c). This confirmed the transformation of 1CR1 with the pBECKSred T-DNA.
Reverse transcriptase (RT)-PCR
This research establishes that the floral dip can be used to isolate stable low-copy number transformants in wheat without the use of tissue culture. Three well-characterized transformants have been isolated in the germplasm line Crocus, and Southern analysis has shown the transgenes to be integrated in the wheat genome and are stable for three–six generations. The transgenes showed the expected segregation ratios for a nuclear insertion event, and this was verified by transmission through pollination. Transgene expression was observed in the T5 and T6 generations.
The developmental stage of the flowers dipped proved to be crucial for successful floral transformation of wheat. In the early attempts at wheat transformation, Agrobacterium infiltration medium harboring various genetic constructs with acetosyringone and Tween 20 was pipetted into uncut florets of wheat at or near the time of anthesis in an effort to transform the pollen (Hess et al. 1990; Langridge et al. 1992). Langridge et al. (1992) failed to demonstrate transmission of the transgene to the next generation. It is possible that such transformation events obtained late in floral development, if obtained at all, may have a tendency toward generating chimeras. In the present study, preliminary research failed to identify a single transformation event in 25,000 putative transformants produced by dipping at stages closer to anthesis (i.e. within 4 days prior to anthesis; J. Zale and C.M. Steber, unpublished). Similarly, no transformants were produced in Arabidopsis if dipping occurred later than 4 days before anthesis (Clough and Bent 1998). Conversely, in wheat, little or no seed set was obtained when florets are dipped too early when the spike was approximately 4 cm long. The ideal stage for dipping appears to be the mid to late uninucleate microspore stage when the spike has not emerged from the sheath. The spike will be approximately 6–7 cm long at this stage and, depending on environmental conditions, approximately 4–7 days before anthesis. This stage coincides with that preferentially chosen for microspore culture (Liu et al. 2002). The target tissue for the T-DNA transfer to wheat has not been determined, but based on the work in Arabidopsis, the ovule is the likely target for T-DNA transfer (Bechtold et al. 2000; Desfeux et al. 2000; Ye et al. 1999).
The transformation efficiency in these experiments was estimated at 0.44% (number of transformants/number of florets dipped). The originally reported transformation efficiency of Arabidopsis by the floral dip ranged between 0.23 and 0.47% in the presence of 0.005% Silwet L-77 (Clough and Bent 1998). If transformation efficiency is reported as the number of transformants/the number of seeds set, then the efficiency is much higher at 6.8% for these three experiments. The latter transformation efficiency was relatively high due to low seed set in the production of 14C1. It appears that dipping at the early uninucleate microspore stage, and/or dipping twice, in an Agrobacterium infiltration medium containing Silwet L-77 significantly decreases seed set in wheat (Table 1). Similarly, any other stress event such as disease, heat, drought, or covering the spikes with heavy glassine bags lowers seed set.
One of the most important considerations in identifying transformed plants is the choice of a selectable marker or reporter genes that are reliable indicators of the transformation event. The maize Lc/C1 anthocyanin regulators in pBECKSred (McCormac et al. 1997) turn tissue red and can be used to identify transformation events by visually scoring for red embryos in T1 seed. However, the stress of dipping can also produce false positives and induce anthocyanin accumulation in the seed, particularly in a red wheat variety. Therefore, using a secondary screen would be appropriate.
The choice and concentration of selection agent are also important considerations when using this protocol. Wheat has a naturally high tolerance to many antibiotics when seeds are plated on nutrient medium (Langridge et al. 1992) and in hydroponics (D. J. Guerra and C. M. Steber, unpublished). However, the concentration of selection agent should not be so high as to be lethal to low copy number T-DNA transformants. We chose not to use geneticin G418 or any other kanamycin analog in nutrient medium because of the prevalence of escapes with NPTII selection (Rakosy-Tican et al. 2007). Screening based on the bleaching induced by the 2% paromomycin spray (Cheng et al. 1997) is, at times, subjective and influenced by environmental conditions. Under higher temperatures, plant tissue will become desiccated and this may confound scoring. One must also consider the strength of the promoter when assessing tolerance or sensitivity and compare the plants tested against wild-type control plants that have also been sprayed.
The large size of the wheat genome at 17,000 Mbp per haploid nucleus (Bennett and Leitch 1996) makes it difficult to detect low copy number T-DNA transfer to wheat. Dirty genomic DNA preparations, degraded DNA, problems with DNA transfer to the membrane, and poor probe labeling can yield negative results in Southern analysis. Conversely, related sequences may be detected due to homology with the probe if the washes are not sufficiently stringent. Langridge et al. (1992), detected bands at 800 bp and 8 kb band on EcoRI and EcoRI/PstI digested Southern blots with the NPTII probe in the T0 generation after treating wheat, barley and maize plants with Agrobacterium, but these bands were not transmitted to the next generation. The authors concluded that these bands might have been due to transformation of an endophytic bacterium. The present study occasionally detected faint 500–800 bp bands in advanced progeny of 3B2 when EcoRI-digested Southern blots were hybridized with the NPTII probe. This band may be due to incomplete transfer of the NPTII transgene at the left border.
Three well-characterized transformations events isolated in the ‘Crocus’ genotype show that the method is reproducible and can produce transformation events that are stable over three–six generations. Transformants have also been produced in Chinese Spring and Bobwhite, however further work is needed to determine whether some genotypes are more amenable than others. A significant obstacle to obtaining transgenic wheat seed using this method is the low seed following dipping. Future work will optimize this protocol so that transformants can consistently be produced while maximizing seed set. These experiments will test different Agrobacterium strains, alternative selectable markers, and different surfactants.
In conclusion, we have produced low copy number T-DNA transformants in Crocus wheat using a floral transformation protocol. The transgenes have integrated into the plant genome, are stable over several generations as determined by Southern analysis, are transmitted in crosses, and are expressed in progeny derived from the primary transformants.
This material is based upon work supported by the National Science Foundation under Grant No. 0638421 (to J.Z.) and the USDA NRI Grant No. 2001-01856 (to J.Z. and C.M.S.). We gratefully acknowledge Dr. L. Ohnoutkova for determining the stage of microspore development and for providing seed of Zuzanna spring wheat.
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