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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The visual marker GUS has been utilized in this study to understand the Arabidopsis thaliana vacuum infiltration transformation process by Agrobacterium tumefaciens. High transformation frequencies of up to 394 transgenic seeds per infiltrated plant were achieved. The results showed that the majority of the transgenic seeds from single infiltrated plants were from independent transformation events based on Southern analysis, progeny segregation, distribution of transgenic seeds throughout the infiltrated plants and the microscopic analysis of GUS expression in ovules of infiltrated plants. GUS expression in mature pollen and anthers was monitored daily from 0 to 12 days post-infiltration. In addition, all ovules from a single infiltrated plant were examined every other day. GUS expression frequencies of up to 1% of pollen were observed 3–5 days post-infiltration, whereas frequencies of up to 6% were detected with ovules of unopened flowers 5–11 days post-infiltration. Most importantly, transgenic seeds were obtained only from genetic crosses using infiltrated plants as the pollen recipient but not the pollen donor, demonstrating Agrobacterium transformation through the ovule pathway.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Over the years, a vast amount of information has been generated in Arabidopsis. One of the vital tools in the study of gene function and regulation is genetic transformation and recovery of transgenic events. Even though Arabidopsis is very amenable for molecular genetic manipulations, Agrobacterium-mediated transformation using root, leaf disc, stem or hypocotyl segment, or seed ( Kemper et al. 1992 ; Koncz et al. 1990 ; Lloyd et al. 1986 ; Sheikholeslam & Weeks 1987; Valvekens et al. 1988 ) has been difficult, mainly because (i) the transformation efficiencies were low; (ii) the process was labor-intensive and time-consuming; and (iii) the recovered transgenic plants exhibited different degrees of somaclonal variation and reduced fertility ( Evans & Sharp 1986; Feldmann & Marks 1987; Larkin & Scowcroft 1986; Scholl et al. 1981 ; Van den Bulk et al. 1990 ). Bechtold et al. (1993) first reported Arabidopsis in planta transformation by vacuum infiltration with Agrobacterium, which eliminated the in vitro steps. Since then, other in planta transformation methods for Arabidopsis ( Chang et al. 1994 ; Katavic et al. 1994 ), or modifications to the original Bechtold transformation method ( Clough & Bent 1999; on the Arabidopsis internet sites) have also been reported. Very recently, the components of the transformation method were assessed, and improvements to the method were reported ( Clough & Bent 1999; Richardson et al. 1998 ). However, the reported transformation frequencies were usually relatively low, and, most importantly, the biological mechanism of transformation remains unknown.

Here we report the results of our studies on the distribution of transgenic seeds on the V0 plants, the molecular analysis of the transgenics for independence of transgenic events, the stages during which transformation happens, and the target cells for infiltration transformation.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Vacuum infiltration transformation

The transformation procedure is based on Bechtold et al. (1993) , and the protocol posted on the Arabidopsis net by Andrew Bent (11.01.94, http://net.bio.net). The procedure is simple with few tissue culture steps involved; is fast so that transgenic seeds can be obtained within 2 months of infiltration; and is high through-put, therefore many constructs can be tested in a short time-frame and a huge number of transformants can be analyzed. Extensive characterization of transformation was carried out using uidA gene as a scorable marker ( Fig. 1). No transient GUS expression was observed in the floral tissues within the first 2 days of vacuum infiltration. Figure 2 shows typical transient GUS expression in floral buds 3–5 days post vacuum infiltration. Whereas no GUS expression was detected in any part of the control plant infiltrated with a control Agrobacterium strain containing no uidA gene ( Fig. 2b), GUS expression was observed in the floral buds of plants infiltrated with Agrobacterium harboring a uidA gene ( Fig. 2a). Typically, little GUS expression was detected in any other parts of the plants. Under the growth conditions (16 h light at the intensity of 150 μΕ at 22°C and 8 h dark at 20°C, with a relative humidity of 75%), transformed seeds were usually recovered within 3 weeks after vacuum infiltration. Seeds developed after 3 weeks usually did not yield any transformants. The transformed seeds were usually scattered in the siliques ( Fig. 2c).

image

Figure 1. Plasmid map of pMON15737.

The vector contains the nptII gene as a selectable marker and the uidA gene as a visual scorable marker. The uidA is under the control of FMV promoter and contains an intron. The nptII gene is driven by the nos promoter. The designation of the genetic elements is as follows: nptII, neomycin phosphotransferase II; 5′ nos, promoter of nopaline synthase; nos 3′, 3′ signal of nopaline synthase; FMV, 35S promoter from the Figwort Mosaic Virus; E9 3′, 3′ signal from pea rbcS E9 gene; CP4, CP4 EPSP synthase from Agrobacterium sp. strain CP4; spc/str, coding region for Tn7 adenylyltransferase conferring resistance to spectinomycin and streptomycin; ori-322, E. coli origin of replication; ori-V, the vegetative origin of replication, functional only when the trfA protein is present in the same cells.

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image

Figure 2. GUS expression in infiltrated Arabidopsis plant 3–5 days post vacuum infiltration transformation, or in immature seeds 3 weeks post-infiltration.

(a) Plant infiltrated with Agrobacterium containing pMON15726 (FMV-uidA).

(b) Plant infiltrated with Agrobacterium containing no uidA gene.

(c) Immature seeds in siliques expressing GUS.

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High transformation frequencies were achieved routinely. Ninety to nearly 400 transgenic seeds per infiltrated plant were produced, with an average of 211 transgenic seeds per plant. Such high transformation frequencies enabled the studies to elucidate the target for vacuum infiltration transformation.

Distribution of transgenic seeds on infiltrated plants

To better understand the timing (sites) and the nature of Arabidopsis vacuum infiltration transformation by Agrobacterium, the distribution of transgenic seeds on single infiltrated plants was investigated. At the time of infiltration, the lengths of existing secondary bolts were measured. Four weeks after infiltration, individual siliques were harvested and their relative positions on the plant were mapped. Each silique was then sterilized and germinated on selective medium separately. The selected plants were also later subjected to Southern analysis to confirm the integration of the transgene, and to determine the independence of transformation events. Figure 3 shows the distribution of transgenic seeds (V1 seeds) on a single infiltrated plant (V0 plants). It is clear that the transgenic seeds were scattered in the siliques and distributed throughout the entire infiltrated plant, which indicates that it is not likely that all the transformants were from a single transformation event. Further comparison of transformation frequencies (number of siliques with GUS-expressing seeds divided by the total number of siliques at the time of harvest) for bolts at different developmental stages from the same plant shows no significant difference at the silique level ( Table 1).

image

Figure 3. Distribution of transgenic seeds on a single plant.

The small insert represents the plant at the time of vacuum infiltration. The secondary bolts were numbered and their lengths were measured and shown in centimetres. The green bars on the plant represent siliques bearing transgenic seeds, whereas the black ones represent siliques bearing no transgenic seeds. The numbers next to the green bars show the number of transgenic seeds recovered from each silique.

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Table 1.  Comparison of transformation frequencies for bolts at different developmental stages from the same plant
Bolt numberaBolt length (cm) bNo. of GUS+ siliques/total cTransformation frequency (%)
  • a

    Bolt length refers to the length of the bolts at the time of vacuum infiltration.

  • b GUS + siliques/total stands for the number of siliques bearing GUS-expressing seeds versus the total number of siliques at the time of harvest.

  • c

    Transformation frequency is expressed as the number of siliques with GUS-expressing seeds divided by the total number of siliques at the time of harvest.

15.019/5336
23.019/4840
31.421/6234
41.08/2532
50.59/2339
6invisible at infiltration6/1540

Genetic and molecular analyses of V2 progenies

Molecular and genetic analyses were performed to determine: (i) whether transformation was due to integration of the transgene into the Arabidopsis genome; (ii) whether the transformants arose from independent transformation events, or from a single or a few transformation events; and (iii) segregation of the transgene in progenies. In our studies, the infiltrated plant was designated as V0 plant. Seeds harvested from the infiltrated plants and the resultant seedlings were designated as V1 seeds and V1 plants, respectively. Seeds collected from the V1 plants and the resultant seedlings were then designated as V2 seeds and V2 plants, respectively, which segregated for resistance or susceptibility when kanamycin was used as the selectable marker. As shown in Table 2, most of the V2 populations segregated at a 3 : 1 ratio, indicating the integration of the transgene in the nuclear genome with one active locus. On the other hand, some V2 progenies from single infiltrated V0 plants segregated at ratios other than 3 : 1, indicating that not all transgenic V1 seeds from the same infiltrated plant arose from the same transformation event.

Table 2.  Segregation of aV2 populations for kanamycin resistance/susceptibility
bV1 plant no.No. of resistant progenyNo. of susceptible progenyApparent segregation ratio
  1. a V 1 plants were designated as the plants obtained from V1 seeds from infiltrated plants (V0). Seeds harvested from the V1 plants and the resultant seedlings were then designated as V2 seeds and V2 plants, which segregated for resistance or susceptibility.

  2. b The number before ‘–’ refers to the original infiltrated plant number (V 0), whereas the number after ‘–’ stands for the V1 plant number. Therefore, V1 plants with the same number before ‘–’ were from the same infiltrated V0 plant.

54–1216723 : 1
54–23581163 : 1
54–3229623 : 1
54–5176473 : 1
54–6250927 : 1
54–7251753 : 1
54–8202494 : 1
54–9181920 : 1
45–1187603 : 1
45–2331428 : 1
45–3243753 : 1
45–4262319 : 1
45–5202634 : 1
1–259163 : 1
1–10178306 : 1
1–113031242 : 1
1–12183683 : 1
1–16272853 : 1
1–172072011 : 1
1–233521173 : 1
1–28219783 : 1
1–29207743 : 1
1–313771377 : 1
1–32261476 : 1

Direct evidence for transgene integration and independent transformation came from Southern analysis of individual transformants obtained from single infiltrated plants. Figure 4 reveals that most transgenic plants from a single infiltrated plant showed distinct hybridization patterns when the genomic DNA was digested with BamHI and probed with the uidA gene fragment ( Fig. 1), suggesting unique integration sites within the Arabidopsis genome. Furthermore, the Southern analysis showed that the majority of the transformants contained a single unique band, indicating that the T-DNA was integrated at a single chromosomal location. It should be noted that the genomic DNA from most of the lanes contained two additional common bands, one of which has the entire Ti plasmid backbone sequence. This result suggests that the left border was partially defected and was often skipped.

image

Figure 4. Southern hybridization of DNA from some individual transgenic plants from a single infiltrated plant.

DNA was digested with BamHI, and the resulting fragments were resolved by gel electrophoresis and transferred to a nylon membrane. The membrane was hybridized with a 32P-labeled uidA gene fragment. In (a), lanes 2–5 contained 0.3, 1, 3, 10 μg DNA from 3 day post-infiltrated Arabidopsis plant, respectively; lanes 6–8 contained 0.02, 0.2 or 2 μl of Agrobacterium DNA. An appropriate amount of wild-type Arabidopsis genomic DNA was added to each lane to make up a total of 10 μg genomic DNA. Panels (b–e) contained genomic DNA from individual transgenic plants from a single infiltrated V0 plant. The designations are using D2-4-74 as an example: D (infiltrated V0 plant D) 2 (bolt #2)-4 (branch #4)-7 (silique 7) 4 (seed #4). The arrows point to the 8.9 kb BamHI fragment containing the entire Ti plasmid backbone sequence. The stars point to the 5.5 kb common band due to head to head T-DNA integration.

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Transformation target as revealed by microscopic analysis

Even though high rates of transformation could be obtained by vacuum infiltration with Agrobacterium, little was known about the mechanism of transformation. Two approaches were taken in this study to elucidate where and when transformation takes place by vacuum infiltration. One of the approaches was to monitor transformation microscopically using uidA as a reporter gene. The second approach was by genetic crosses described in the next section. In the first approach, Arabidopsis plants were infiltrated with Agrobacterium strain ABI harboring pMON15737 (containing uidA and nptII genes). GUS expression in mature pollen was monitored daily from 0 to 11 days post-infiltration from many infiltrated plants. In addition, every ovule of unopened flowers from a single infiltrated plant was examined every other day. Table 3 summarizes the results from this study. GUS expression was observed in pollen 3–5 days post-infiltration at frequencies of up to 1%; nevertheless, ovule transformation frequencies of up to 6% were obtained 5–11 days post-infiltration. Figure 5(a–e) shows GUS expression in pollen grains 3–5 days post-infiltration (a,b), and in ovules 5–11 days post-infiltration (c,d,e). GUS expression was observed in the ovaries of unopened flowers throughout the plants. For a given ovary, GUS expression was usually detected in several ovules scattered in the ovary, consistent with the observation at the seed (silique) level. GUS expression was typically localized at the micropylar area, where the egg cell is located.

Table 3.  GUS expression in pollen grains and ovules over time
PollenOvuleOvary
Time (DPI)bTotal no. observed c% Total no. observed%Total no. observed%
  • a

    DPI stands for days post-infiltration.

  • b

    Total number of pollen grains, ovules or ovaries observed.

  • c

    Percentage stands for transformation frequencies for pollen, ovule or ovary. Pollen transformation frequency is expressed as the number of GUS-expressing pollen divided by the total number of pollen observed. Ovules transformation frequency is presented as the number of GUS-positive ovules (from unopened flowers) divided by the total number of ovules observed. Ovary transformation frequency is defined as the number of ovaries containing GUS-positive ovules divided by the total number of ovaries observed.

118300  
227100  
331860.03840210
478301  
541310.927601.86918.8
663500  
7thousands022806.15736.8
8thousands0  
9thousands075200.91889.6
10thousands0  
11thousands048402.312124
image

Figure 5. GUS expression in pollen grains, ovules and seeds of infiltrated Arabidopsis plants.

(a) An anther containing GUS-expressing pollen grains; (b) pollen expressing GUS 4 days post vacuum infiltration; (c) an ovary containing many GUS-expressing ovules; (d) a close-up of GUS-expressing ovules from an unopened flower. The GUS expression was localized around the micropylar area; (e) a close-up of a more mature, GUS-expressing ovule; (f) seeds with seed coat, showing GUS expression throughout the seeds; (g) seeds with seed coat, showing localized GUS expression; (h) seeds with the seed coat removed, and GUS expression throughout the seeds; and (i) seeds with seed coat removed and the localized GUS expression.

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In the same study, some infiltrated plants were allowed to grow to maturity to collect the seeds. Seed transformation frequencies were determined for correlation with those of pollen or ovule by either germinating on kanamycin-containing medium, or by assaying for GUS expression. In general, seed transformation frequencies correlated very well with those of the ovule, suggesting that the ovule is most likely to be the target for vacuum infiltration transformation. Figure 5(f–i) shows GUS expression in mature seeds. Two different patterns of GUS expression were detected: those that were completely blue (f) and those with more localized expression (g). The embryos showed the same patterns when the seed coat was removed (h,i).

Transformation target as revealed by crosses

Even though the microscopic study provided strong evidence that ovule transformation frequencies correlated well with seed transformation and that the ovule was most likely the target for infiltration transformation, direct evidence was needed to prove that the ovule was indeed the target for infiltration transformation. Sexual crosses were conducted with the infiltrated plants serving as either the male or the female parent. Four sets of treatments were included in the experiment: (1) wild-type controls, where anthers from the recipient plants were removed and the stigma pollinated with pollen from wild-type plants for the assessment of pollination frequencies; (2) the infiltrated plants serving as the pollen recipients, and the wild-type, non-infiltrated plants serving as the pollen donors; (3) the wild-type non-infiltrated plants serving as the pollen recipients, and the infiltrated plants serving as the pollen donors; and (4) the emasculation controls, where anthers from the infiltrated plants were removed and the plants allowed to grow to maturity to see if hand-emasculation was complete, and to rule out the possibility of cross-pollination from other flowers or plants. After crosses were performed, any pre-existing flower buds were removed to avoid confusion. The plants were also monitored daily to remove any new developing flower buds to ensure that any siliques developed were those from crosses. Any transformants obtained from treatment 2 would provide direct evidence for the pathway of ovule transformation; whereas those from treatment 3 would prove the pathway of pollen transformation. Table 4 shows information on the crosses of all four treatments. No siliques were obtained from the emasculation control, showing that the emasculation was effective, and that the possibility of cross-pollination could be eliminated. Therefore, any seeds that developed should be the result of the performed crosses, rather than the result of cross-pollination from other flowers/plants, or the result of possible incomplete removal of anthers. About 40–60% of the crosses executed yielded seeds. Fifteen kanamycin-resistant seedlings were obtained from treatment 2 where the infiltrated plants served as the pollen recipient (female parent). These plants were also assayed for GUS expression and all of them showed GUS activity. No transformants were generated from treatment 3 where the infiltrated plants served as the pollen donor (male parent).

Table 4. a Summary of four different types of crosses performed
bCross No. attemptedNo. successfulc Percentage efficiencyNo. of transgenic seeds
  • a

    Results presented in this table do not reflect transformation.

  • b

    Crosses: Wt × Wt where pollen donors and recipients were both wild-type plants; Inf × Wt where the infiltrated plants served as the pollen recipients and the wild-type plants served as the pollen donors; Wt × Inf where the wild-type plants served as the pollen recipients and the infiltrated plants served as the pollen donors; Emasc. Ctrl where the infiltrated plants were hand emasculated and allowed to grow to maturity.

  • c

    Percentage efficiency was expressed as the number of successful crosses divided by the number of crosses attempted (×100).

Wt × Wt301343.3
Inf × Wt1878746.515
Wt × Inf1388863.70
Emasc. ctrl30000

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Results from two independent approaches clearly demonstrated that the female gamete (ovule) is the target for Arabidopsis vacuum infiltration transformation by Agrobacterium: the microscopic analysis for GUS expression and the genetic crosses. GUS expression was usually localized around the micropylar area where the egg cell is located. Care was taken to distinguish between opened and unopened flowers to eliminate the possibility of detecting GUS expression due to cross-pollination by possible transgenic pollen. Very low frequencies (up to 1%) of GUS expression in pollen were observed 3–5 days post-infiltration. This may be partly due to the fact that the uidA gene is under the control of the FMV promoter in both pMON15737 and 15726, and that the FMV promoter has very low expression in cotton and corn pollen (C. Armstrong, personal communication). However, this should not affect the result of stable seed transformation through the approach of genetic crosses. Failure to generate transformants when infiltrated plants were used as the pollen donor may be due to (i) the inability of Agrobacterium to transform pollen; or (ii) the relatively small scale of crosses performed given the fact that pollen transformation was observed at very low frequencies (up to 1%) in a very short timeframe (3–5 days post-infiltration).

It seems that because Agrobacterium remained in the plants as evidence by overgrowth of Agrobacterium when the harvested seeds were germinated in vitro in the absence of antibiotics in the medium, transformation could occur repeatedly over a period of time. Most of the transformation happened before the flowers were open. However, some might take place after fertilization occurred. This was supported by the observation of localized GUS expression in some seeds and of chimeric GUS expression in some kanamycin-resistant seedlings (data not shown). Some of the skewed segregation data ( Table 2) might also be due to the chimeric nature of the transformants.

Several lines of evidence support that most of the transformants from a single infiltrated plant arose from independent transformation events. The direct evidence was provided by Southern analysis showing the unique hybridization patterns of individual V1 plants from the same infiltrated V0 plant. The detailed map of transgenic seed distribution ( Fig. 3) showed that seeds were randomly distributed throughout the entire infiltrated plant. Furthermore, the transgenic seeds were usually scattered within the siliques rather than aligned linearly. If they were ontogenetically derived from the same tissue, one would have expected them to be clustered. In addition, microscopic observation of GUS expression in different opened and unopened flower buds after infiltration provided visual evidence that transformants were present in many individual flower buds on different branches/bolts throughout the plants over a period of time.

Two classes of GUS-expressing V1 embryos were observed in this study ( Fig. 5): those where the entire embryos showed GUS activity; and those where part of the embryos showed GUS activity. The fully stained embryos can be explained by transformation through the female gamete. The second pattern could only result from transformation of a cell in an early embryo which gave rise to the observed localized GUS expression in the mature embryos.

In this study, we demonstrated the target for infiltration transformation and high transformation efficiencies with Arabidopsis. The system is very efficient, with high numbers of independent transgenic seeds able to be generated within 2 months after infiltration transformation. It provides a high throughput system for gene evaluation and understanding the transformation mechanism should help in adapting the system for other plant species.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Plant growth conditions

Arabidopsis thaliana ecotype Columbia was used for the studies. Seeds were germinated and plants grown in 2.5 inch square pots filled with a soil mixture (two-thirds Redi-earth and one-third perlite) in a growth chamber. The pots were covered with a window mesh. Each pot contained two plants. The plants were subirrigated every other day for 30 min and fertilized bi-weekly with Peters 20–20–20. The growth conditions were 16 h light (150 μΕ) at 22°C/8 h dark at 20°C, with a relative humidity of 75%. After emerging to about 2 cm, the primary bolts were clipped to encourage the growth of secondary or tertiary bolts.

Agrobacterium infiltration transformation

Agrobacterium tumefaciens strain ABI harboring the binary vectors pMON15737 ( Fig. 1) or 15726 were used for transformation. Both vectors contain the nptII gene as a selectable marker and the uidA gene as a visual scorable marker. In both constructs, uidA is under the control of FMV (Figwort Mosaic Virus, Sanger et al. 1990 ) promoter and contains an intron ( Vancanneyt et al. 1990 ). The nptII gene is driven by the FMV promoter in pMON15726 or the nos promoter in pMON15737, respectively. Small scale Agrobacterium cultures were grown in liquid LB medium with appropriate antibiotics at 28°C overnight. The small scale cultures were then diluted 50-fold into LB medium with appropriate antibiotics for large scale overnight cultures. Cells were then harvested by centrifugation at 5000 r.p.m. (about 3000 g) for 15 min, and resuspended in infiltration medium to an OD600 of 0.8. The infiltration medium consists of half-strength MS ( Murashige & Skoog 1962) salts, full-strength B5 vitamins ( Gamborg et al. 1968 ), 0.5 g l–1 2-(N-morpholino)ethanesulfonic acid (MES), 5% sucrose, 0.044 μm benzylaminopurine and 0.02% Silwet L-77 (Osi Specialties, Inc., A Witco Company, Endicott, NY, USA) at pH 5.7. Infiltration was performed using a Rubbermaid square container containing the Agrobacterium suspension, placed inside a vacuum desiccator (Nalgene Nunc International, Milwaukee, WI, USA), and a house vacuum. Four pots with Arabidopsis plants were inverted and placed into the container holding the Agrobacterium suspension. A house vacuum (24–27 inch Hg) was applied for 10 min or as specified, and released quickly. The pots were then removed from the Agrobacterium suspension and placed on their sides in a flat lined with an absorbent pad for drainage. The flat was covered with a clear plastic germination dome to maintain high humidity. The germination dome was removed the next day and the pots were turned upright. The plants were allowed to grow to maturity and seeds were harvested.

Sterilization and in vitro selection of transgenic seeds

The harvested seeds were sterilized using a chlorine gas sterilization method as follows: seeds were placed into 15 ml plastic Corning tubes with the caps loosely tightened. They were then placed inside a vacuum desiccator. A 5.25% sodium hypochlorite solution (200 ml) was added to a glass beaker inside the desiccator. The desiccator lid was immediately put on after 2 ml of concentrated HCl was added to the hypochlorite solution. A house vacuum was applied very briefly to seal the device and the seeds were left in the device overnight. They were then sprinkled onto solidified kanamycin-containing culture plates to select for transformants. The selective medium contained MS salts ( Murashige & Skoog 1962) with Gamborg’s B5 vitamins, 1% glucose, 0.5 g l–1 MES, 0.7% phytagar (GIBCO BRL Life Technologies Inc., Gaithersburg, MD, USA) supplemented with 60 mg l–1 kanamycin, 500 mg l–1 carbenicillin and 250 mg l–1 cefotaxime. Seed germination conditions were 16 h light (150 μΕ) at 22°C/8 h dark at 20°C. After about 2 weeks, kanamycin-resistant seedlings were transplanted into soil, grown to maturity and individually harvested.

GUS assay and microscopic observation

β-Glucuronidase activity was detected histochemically by incubating either the whole seedlings or plants, or different parts of the plants at 37°C overnight in a solution containing 1 m m 5-bromo-4-chloro-3-indolyl-β- d-glucuronide, as described by Jefferson (1987) and Jefferson et al. (1987) .

The expression of GUS activity (as shown in blue) was observed either under a dissecting microscope (Carl Zeiss, Inc., Thornwood, NY, USA), or under a compound microscope (Carl Zeiss, Inc.) for detailed analysis of the localization of GUS expression.

Southern analysis

Genomic DNA was isolated from individual plants using the method of Doyle & Doyle (1990). A roughly equal amount of DNA (10 μg DNA per lane) was digested with the restriction enzymes BamHI, resolved by electrophoresis on a 0.8% agarose gel, transferred to a nylon membrane and probed with a gel-isolated, 32P-labeled uidA gene fragment following the manufacturer’s protocol for the GeneScreen Plus membrane (DuPont, Wilmington, DE, USA).

Crosses

Crosses were performed between 11.00 hours and 13.30 hours 4–10 days post-infiltration. The plants used as females were hand-emasculated and anthers from freshly opened flowers of donor plants were harvested and pollinated by touching the anthers onto the stigmas of the emasculated plants. The pollinated flowers were labeled and any remaining opened or unopened flowers from the same plant were removed to avoid any confusion at harvest.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The authors would like to thank Drs Charles L. Armstrong and J. Austin Burns for their critical review of the manuscript, and Dr Charles Armstrong for his comments and discussion regarding the project. The authors would also like to thank Dr Michael Fromm for his input and critical comments on the project.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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