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Published online 7 November 2007
Published in Crop Sci 47:2390-2402 (2007)
© 2007 Crop Science Society of America
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GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY

Genetic Engineering of Maize with the Arabidopsis DREB1A/CBF3 Gene Using Split-Seed Explants

Diaa Al-Abedb, Parani Madasamya, Reddy Tallaa, Stephen Goldmana and Sairam Rudrabhatlac,*

a Plant Science Research Center, Univ. of Toledo, 2801 W. Bancroft, Toledo, OH 43606
b current address: Edenspace Systems Corp., 1500 Hayes Dr., Manhattan, KS 66502
c current address: School of Science, Engineering and Technology, TL 174, Penn State Harrisburg, 777 W. Harrisburg Pike, Middletown, PA 17057-4898

* Corresponding author (svr11{at}psu.edu).


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Transformation of maize (Zea mays L.) split-seed explants from inbred line R23 was performed following particle bombardment with a construct carrying the Arabidopsis transcriptional factor CBF3 under the control of the inducible promoter rd29A and the selectable marker hygromycin phosphotransferase. Overexpressing CBF3 has been shown to enhance cold, drought, and salt tolerance in Arabidopsis, tobacco (Nicotiana tabacum L.), and wheat (Triticum aestivum L.). The CBF3 gene was detected in 18 lines by polymerase chain reaction (PCR), and stable integration of multiple copies of CBF3 was confirmed by Southern blot analysis in three selected lines. Reverse transcription PCR detected expression of CBF3 in the transgenic lines under unstressed conditions despite the use of the stress-inducible rd29A promoter. This constitutive expression was associated with growth retardation and sterility in most of the transgenic lines. Transmission of the gene in a Mendelian fashion to T1 and T2 generations was confirmed in one line by Southern blot analysis. Plants of this line showed stress-inducible expression of the CBF3 gene and hardly detectable expression under unstressed conditions along with significant tolerance to cold, drought, and salinity compared with wild-type plants. These results demonstrate that stress-inducible overexpression of CBF3 has the potential to enhance abiotic stress tolerance in corn.

Abbreviations: DRE/CRT, dehydration responsive element/c-repeat element • GUS, ß-glucuronidase gene • MS, Murashige and Skoog • MSI, multiple shoots induction • PCR, polymerase chain reaction • RT-PCR, reverse transcription polymerase chain reaction

Genetic Engineering of Maize with the Arabidopsis DREB1A/CBF3 Gene Using Split-Seed Explants

Diaa Al-Abedb, Parani Madasamya, Reddy Tallaa, Stephen Goldmana and Sairam Rudrabhatlac,*

a Plant Science Research Center, Univ. of Toledo, 2801 W. Bancroft, Toledo, OH 43606
b current address: Edenspace Systems Corp., 1500 Hayes Dr., Manhattan, KS 66502
c current address: School of Science, Engineering and Technology, TL 174, Penn State Harrisburg, 777 W. Harrisburg Pike, Middletown, PA 17057-4898

* Corresponding author (svr11{at}psu.edu).

Transformation of maize (Zea mays L.) split-seed explants from inbred line R23 was performed following particle bombardment with a construct carrying the Arabidopsis transcriptional factor CBF3 under the control of the inducible promoter rd29A and the selectable marker hygromycin phosphotransferase. Overexpressing CBF3 has been shown to enhance cold, drought, and salt tolerance in Arabidopsis, tobacco (Nicotiana tabacum L.), and wheat (Triticum aestivum L.). The CBF3 gene was detected in 18 lines by polymerase chain reaction (PCR), and stable integration of multiple copies of CBF3 was confirmed by Southern blot analysis in three selected lines. Reverse transcription PCR detected expression of CBF3 in the transgenic lines under unstressed conditions despite the use of the stress-inducible rd29A promoter. This constitutive expression was associated with growth retardation and sterility in most of the transgenic lines. Transmission of the gene in a Mendelian fashion to T1 and T2 generations was confirmed in one line by Southern blot analysis. Plants of this line showed stress-inducible expression of the CBF3 gene and hardly detectable expression under unstressed conditions along with significant tolerance to cold, drought, and salinity compared with wild-type plants. These results demonstrate that stress-inducible overexpression of CBF3 has the potential to enhance abiotic stress tolerance in corn.

Abbreviations: DRE/CRT, dehydration responsive element/c-repeat element • GUS, ß-glucuronidase gene • MS, Murashige and Skoog • MSI, multiple shoots induction • PCR, polymerase chain reaction • RT-PCR, reverse transcription polymerase chain reaction


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
MAIZE IS ONE of the most important commercial crops in the world and is valued for its food, fiber, oil, and other byproducts. Because grain demand could outpace supply, particularly during dry or cold seasons, producing maize cultivars that are drought, cold, and salt tolerant will help ensure grain capacity and price stability. Furthermore, it may allow an expansion in maize acreage, providing farmers with more planting options.

Classical plant breeding protocols have been successfully used to develop lines adapted to survive under or at least mitigate against abiotic and biotic challenges. This technology has a number of disadvantages, most particularly the time needed to select and expand the foundation seed parent(s) (Holmberg and Bülow, 1998). To further enhance productivity by bringing cultivars to market, the emerging technologies of gene transfer are being used as a complement to traditional breeding practices.

Recently, it was reported that the Arabidopsis CBF transcriptional factors family interact with the dehydration responsive element/c-repeat element (DRE/CRT). The DRE/CRT is a cis-acting promoter element that regulates gene expression in response to drought, salt, and cold stress in Arabidopsis (Yamaguchi-Shinozaki and Shinozaki, 1994). Furthermore, expression of this gene enhances drought, salt, and cold tolerance in a number of plant species (Yamaguchi-Shinozaki and Shinozaki, 1994; Stockinger et al., 1997; Kasuga et al., 1999; Gilmour et al., 2000; Pellegrineschi et al., 2004). The main function of the CBF regulon is to protect the plant cells from freezing and water stress (Thomashow, 2001). The CBF regulatory proteins work as switches that activate multiple genes of the cold acclimation and water stress responses (Thomashow, 2001). A number of genes were identified in Arabidopsis to contain the DRE/CRT element, including: KIN1, COR6.6/KIN2, COR15a, COR47/RD17, COR78/RD29a, and ERD10 (Kasuga et al., 1999). These target genes encode for proteins that work on stabilizing cell membranes when subjected to different stress types. In addition, overexpression of CBF3 in Arabidopsis led to increased proline and total sugar levels and, in turn, enhanced freezing tolerance (Gilmour et al., 2000). The DRE in the rd29A promoter also functions in gene expression in response to stress in tobacco plants (Yamaguchi-Shinozaki and Shinozaki, 1994), which suggests that the rd29A promoter regulates the expression of DREB in tobacco as in Arabidopsis. These results indicate that the stress-inducible rd29A promoter is quite useful to overexpress CBF3 for improving drought, salt, and freezing stress tolerance not only in transgenic Arabidopsis but also in other kinds of transgenic plants such as tobacco (Kasuga et al., 2004).

The application of transgenic technologies to maximum effect, however, requires overcoming certain limitations. These include restrictions with respect to genotype (Tomes and Smith, 1985), speed (Vasil et al., 1984; Bhaskaran and Smith, 1990), number of regenerants (Huang and Wei, 2004), and somaclonal variation and albinism (Armstrong and Phillips, 1988). Given these limitations, it becomes mandatory to link a DNA transfer technology to the chosen tissue culture protocol such that transgenics are recovered in sufficient number to warrant the choice of a particular regeneration technology (Christou, 1996).

Embryogenic type II calli derived from immature embryos has been the most used tissue for the regeneration and transformation of maize (Frame et al., 2000; Gordon-Kamm et al., 1990). Recently, interest has been focused on using explants from mature seeds of cereals (Dai et al., 2001). For example, maize shoot tips and shoot meristematic cultures derived from mature seeds have been used as a target for particle bombardment transformation (Zhang et al., 2002; Zhong et al., 2003).

Most recently, fertile maize plants have been regenerated using a new explant "split-seed" (Al-Abed et al., 2006). The split-seed technology is genotype independent and labor and space friendly, as no greenhouses are required to supply constant explant material. Hence the objectives in the present study were: (i) to utilize the split-seed regeneration technology and couple it with a transformation protocol to produce transgenic maize plants segregating for the CBF3 gene, and (ii) to assess the expression of CBF3 with respect to its ability to confer cold, drought, and salinity tolerance.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Explant Source
Seeds of the R23 inbred were obtained from Pioneer Hi-Bred (Johnston, IA). The seeds were washed with antibacterial soap (Bac-down), surface sterilized with 70% ethanol for 1 min, rinsed four times with water, and soaked in 0.1% HgCl2 for 7 min. The seeds were finally rinsed five times with sterile water and soaked in sterile water for 24 h. The seeds were then germinated on MS (Murashige and Skoog, 1962) basal salts and vitamin B5 (Gamborg et al., 1968) and supplemented with 9 µmol L–1 2,4-D (2,4-dichlorophenoxyacetic acid, Sigma, St. Louis, MO) for 3 to 4 d.

Constructs
The plasmid pC1301 containing an intron ß-glucuronuridase (GUS) was obtained from pCambia (Black Mountain, ACT, Australia) (Fig. 1A ). The GUS intron was used to monitor transient gene expression and to define the conditions that lead to maximal gene expression in the scutellum, the coleoptilar ring, and the shoot apical meristems.


Figure 1
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  		Figure 1. Maps of the constructs used for particle bombardment transformation: (A) pC1301 contains the GUS gene under 35S promoter; (B) pPDSG89 contains the CBF3 gene under the control of rd29A.

 
The CBF3 open reading frame (ORF) was amplified from Arabidopsis (ecotype Columbia) genomic DNA by polymerase chain reaction (PCR) with a Bam HI site added to the forward primer, 5'-GGATCCTCTAGATGAACTCATTTTCTGCTTTTTCTG-3', and a Kpn I site added to the reverse primer, 5'-GGTACCTTTTAATAACTCCATAACGATACGTC-3'. The PCR product was cloned into pBluescript SK(–) and confirmed by sequencing. A Hind III and BamHI fragment of rd29A was cloned into a pCambia vector and, subsequently, the CBF3 ORF was released by digesting with BamHI and KpnI, and subcloned into the same vector as described by Kasuga et al. (1999). This construct is referred as pPDSG89 (Fig. 1B).

Explant Pretreatment and Particle Bombardment
After splitting the seeds, the explants were cultured for 24 h on split-seed multiple shoot induction medium (MSI), which consisted of MS basal salts supplemented with vitamin B5, 17.6 µmol L–1 BAP (6-benzylaminopurine, Sigma, St. Louis, MO), and 9.2 µmol L–1 kinetin (6-furfurylaminopurine, Sigma, St. Louis, MO) in addition to: 1 mg L–1 glycine, 400 mg L–1 casein hydrolysate, 30 g L–1 sucrose, and solidified with 8 g L–1 agar. The pH was adjusted to 5.8 before adding the agar. Following this, six split-seed explants per plate were arranged on the same medium with the cut side facing upward and subjected to particle bombardment. All of the plates that were used for particle bombardment were left uncovered in the laminar flow hood for 3 h to air dry the split-seeds before shooting.

Plasmid DNA was isolated using a HiSpeed Plasmid Midi Kit (Qiagen Sciences, Valencia, CA) and the DNA concentration was adjusted to 900 ng µL–1. Gold particles of 0.6 µm in diameter (Bio-Rad Laboratories, Hercules, CA) were coated with 10 µL of plasmid DNA. The coated gold particles were then mixed with 50 µL of 2.5 mol L–1 CaCl2 and 20 µL of 0.1 mol L–1 spermidine and were vortexed for 20 min at 4°C. Subsequently these were washed three times with 200 µL of absolute ethanol and centrifuged for 1 min after each wash. Finally, the particles were resuspended in 35 µL of absolute ethanol and kept on ice. Eight to nine microliters of the suspension was spread on each macrocarrier and allowed to dry before bombardment.

A total of 29 experiments was performed, testing approximately 3000 split-seed explants. The different parameters tested were: He pressure (7.58, 9.31, and 10.69 MPa), target distance (6 and 9 cm), number of shots (one or two). All experiments had a GUS control and two negative controls: one was shot with uncoated gold particles and the other was not shot.

Histochemical ß-Glucuronidase Assay
Transient GUS expression in bombarded split-seed explants was visualized histochemically using X-Gluc (5-bromo-4-chloro-3-indolyl-ß-D-glucuronic acid, PhytoTechnology Laboratories, Shawnee Mission, KS) solution 48 h after bombardment, as described by Jefferson (1987). Bombarded split-seed explants testing different parameters were incubated in GUS solution, 10 explants per tube, for 24 h at 37°C. Transient GUS expression was then monitored under a stereomicroscope (Olympus SZX 12).

Selection and Regeneration
Twenty-four hours after bombardment, split-seed explants were transferred to MSI medium for 4 d in 16/8 h light/dark at 26°C as a recovery period. The explants were then transferred to a selection (MSI) medium supplemented with 25 mg L–1 hygromycin (Bioworld, Dublin, OH). The cultures were then subcultured biweekly on fresh medium three times before root induction. Putative transformants were separated and further cultured in rooting medium consisting of MS salts containing 3.2 µmol L–1 NAA (1-naphthaleneacetic acid, Sigma, St. Louis, MO) and supplemented with 10 mg L–1 hygromycin to ensure stringent selection before transferring to soil.

Molecular Analysis and Inheritance of CBF3 Gene in Progeny
Genomic DNA was extracted from 300 mg of leaf tissues of putative transformants as well as from wild-type controls using a modified cetyl trimethylammonium bromide method as described by Rogers and Bendich (1985). For Southern blot analysis, 10 µg of genomic DNA from transformed and wild-type plants as well as 50 pg of plasmid DNA were digested with either Hind III or KpnI (cut at a single site within the plasmid) and the copy number was estimated by the number of hybridized bands in Southern blot analysis. Digested DNA was separated by electrophoresis in 0.8% agarose gel, and transferred into a Hybond-N+ nylon membrane (Amersham Biosciences, Little Chalfont, UK) according to Sambrook et al. (1989). A 392 base pair (bp) PCR fragment containing the CBF3 coding region of pPDSG89 was amplified using oligonucleotide primers: forward 5'GAAACCGGCGGGTCGTAAGAAGTTT-3' and reverse 5'-TTTCGCTCTGTTCCGCCGTGTAAA-3'. The DNA amplification conditions were as follows: one cycle at 94°C for 3 min; 30 cycles at 94°C (45 s), 60°C (45 s), 72°C (2 min); and a final extension at 72°C (5 min). The DNA was then purified, labeled with {alpha}[32P]-dCTP using a random prime DNA labeling kit (Amersham Biosciences), and used for hybridization. Hybridized membranes were exposed to Kodak x-ray films at –80°C for 48 h.

To confirm the inheritance of the transgene to subsequent generations, a T0 plant was backcrossed to inbred line R23. The seeds obtained from the T0 plants grown in the greenhouse were germinated on MS medium supplemented with 25 mg L–1 hygromycin. The T1 plants were tested further to confirm the inheritance of the CBF3 gene following Southern blot analysis. The T1 plants were self-pollinated to produce the seeds for the T2 generation, which were further analyzed using hygromycin selection and Southern blot analysis.

Reverse Transcription Polymerase Chain Reaction Analysis
Total RNA was isolated from CBF3 transgenic lines 7, 8, and 28 as well as from a wild-type plant growing at 26°C in a growth chamber, using Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA). The RNA was treated with DNase to eliminate false positives from DNA contamination of the RNA. Two hundred nanograms of total RNA was used for cDNA synthesis with a one-shot reverse transcription polymerase chain reaction (RT-PCR) kit (Qiagen, Valencia, CA). The PCR program was as follows: 50°C for 30 min; 94°C for 15 min; 30 cycles consisting of 94°C (45 s), 58°C (45 s), and 72°C (2 min); and a final extension of 72°C for 5 min. Specific primers flanking a 638-bp for the CBF3 transcripts were: forward 5'-AACTCATTTTCTGCTTTTTCTGAA-3' and reverse 5'-TTAATAACTCCATAACGATACGTC-3'. As an internal control, primers for the coding region of the maize Actin were: forward 5'- TACAACGAGCTCCGTGTTTC-3' and reverse 5'- CTTTCTGACCCAATGGTGATG-3'. The products were separated by electrophoresis on a 1.0% agarose gel.

Northern Blot Analysis
Leaf tissues from transgenic plants and wild-type control plants that were treated with cold, drought, and salinity were frozen in liquid N2. Total RNA was isolated using Trizol reagent. The RNA was treated with DNase to eliminate false positives from DNA contamination of the RNA. Fifteen micrograms of total RNA was loaded into a 1% formaldehyde gel and transferred onto a nylon membrane (Hybond-XL, Amersham Biosciences) according to a modified protocol from Sambrook et al. (1989). The blots were then hybridized with {alpha}[32P]-dCTP-labeled probes at 65°C overnight and washed at high-stringency temperature (60°C). The CBF3 probe was prepared using specific primers flanking a 638-bp for the CBF3 transcripts: forward 5'-AACTCATTTTCTGCTTTTTCTGAA-3' and reverse 5'-TTAATAACTCCATAACGATACGTC-3'. A ZmCAT3 (Zea mays catalase 3; accession no. L05934) probe was prepared using specific primes flanking a 558-bp from the coding region: forward 5'-GCAACAACTTCCCCGTCTTCT-3' and reverse 5'-GCTGCTCGTTCTCGTTGAAGA-3'.

The blots were stripped by boiling in 0.1% sodium dodecyl sulfate and rehybridized with a maize Actin probe prepared by using primers for the coding region of the maize Actin, which were: forward 5'- TACAACGAGCTCCGTGTTTC-3' and reverse 5'- CTTTCTGACCCAATGGTGATG-3'. Hybridized membranes were exposed to Kodak x-ray films at –80°C for 24 h.

Stress Tolerance Studies
Constant 10°C Experiments
The T2 and wild-type seeds were sowed in Premier Pro-mix (Premier Horticulture, Quakertown, PA) in trays that hold 36 seeds per tray. A total of 236 T2 and wild-type seeds (118 each) were germinated in a growth chamber under 16/8 h light/dark at 26°C. The T2 generation represents a population of transgenic and segregated nontransgenic seeds. Five-day-old seedlings were challenged at 10°C day and night in a growth chamber also under 16/8 h light/dark. The seedlings were given an equal amount of water and fertilizer. Growth and morphology were monitored on a weekly basis for four weeks and the height of the plants was scored. The seedlings were then transferred to normal growth conditions for two weeks as a recovery period. The survival rate was calculated by dividing the number of surviving plants by the total number of seeds initially planted. Thirty T2 plants and the wild-type plants that survived were randomly selected for continued growth to maturity. The rate of fertility was scored as the number of partially or fully fertile plants divided by the number of selected plants.

Cold and Freezing Tolerance (4, 0, and –2°C)
The T2 seeds were germinated on MS medium containing 25 mg L–1 hygromycin to select for transgenic seeds. After 5 d, germinated seedlings were transferred to soil in trays and placed in a growth chamber under 16/8 h light/dark at 26°C. Wild-type seeds were germinated at the same time on MS medium without hygromycin and transferred to the same type of trays containing soil. A total of 236 2-week-old transgenic and wild-type plants were used for each treatment—4, 0, and –2°C for 2 d, under 16/8 h light/dark—and were then returned to 26°C for two weeks. The number of surviving plants was counted and divided by the initial number of plants.

Drought and Salinity Tolerance
Four-week-old transgenic and wild-type plants were grown in 16.5-cm (6.5-inch) plastic pots (Kord Products, Toronto, ON, Canada) containing Premier Pro-mix (Premier Horticulture, Quakertown, PA) and kept in the greenhouse under 16/8 h light/dark at 26°C and 65% relative humidity. Drought stress was induced by withholding water for 7, 14, or 21 d. A total of 40 transgenic T2 and 40 wild-type plants were used for this study. The plants were monitored on a weekly basis for their phenotypes. The survival rate was scored from each water-withholding period after watering the treated plants and allowing a recovery period of two weeks.

For the salinity stress studies, two-week-old transgenic and wild-type plants grown in trays were subjected to 100, 200, and 400 mmol L–1 NaCl. A consistent volume of NaCl solution, irrespective of molarity, was added to the trays for one week. The plants were then given a two-week recovery period by watering regularly. The survival rate for each treatment was scored by dividing the number of surviving plants by the number of plants initially treated.

Electrolyte Leakage
The determination of electrolyte leakage of plants treated with low and freezing temperatures and drought was conducted as described by Szalai et al. (1996), with some modifications. In the case of drought treatment, the ion leakage was estimated from 3, 7, and 14 d of withholding water, since none of the wild-type plants survived 21 d. Two-centimeter segments from the uppermost part of the leaf of stress-challenged plants were excised directly after each treatment, kept at room temperature for 24 h, and immersed in 20 mL of distilled water in flasks. The flasks were sealed and vortexed for 1 min, followed by 3 h of shaking at 75 rpm at room temperature. To test the level of electrolyte leakage due to low and freezing temperatures and drought, the conductivity (C1) of the solution was measured using an electrical conductivity meter (Model HI 98312, Hanna Instruments, Ann Arbor, MI). The same leaf segments previously used to measure the initial electrolyte leakage (C1) were frozen at –80°C for 2 h, thawed for 1 h, and then immersed in the same solution to estimate the total potential ion leakage (C2). The relative percentage of injury due to stress was determined by dividing the mean ion leakage (C1) by the total leakage from frozen-killed samples (C2).

Statistical Analysis
For GUS expression analysis, the experimental unit was considered to be an explant with three replicates for each experiment. The mean number of GUS spots per explant was compared between each pressure point (covered vs. uncovered) and the number of shots (one or two). The data were analyzed by t-test at P = 0.05, using the SAS statistical analysis software, Version 9.1 (SAS Institute, Cary, NC).

For segregation analysis, the chi-square test at P = 0.05 was used to determine if deviations from expected ratios were within the acceptable range of variation using SAS, Version 9.1.

To determine the difference in the stress tolerance between transgenic and wild-type plants, 118 transgenic and the same number of wild-type plants, from three replicates of 36, were used for each stress treatment. All percentage data from the survival rates and electrolyte leakage were transformed using arcsine transformation before statistical analysis. The data were analyzed by t-test at P = 0.05 to compare the means of transgenic and wild-type plants for each treatment, using SAS, Version 9.1.


   RESULTS AND DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Optimization of Particle Bombardment Parameters for Split-Seed Explants
Since different explants require different parameters for particle bombardment, we started by manipulating different parameters to optimize particle bombardment transformation protocols for split-seed explants. Split-seed explants were used for bombardment with gold particles coated with pC1301 (Fig. 1A), which contained a GUS gene to monitor the expression in bombarded regions of the split-seed explant. In the same experiment, the pPDSG89 plasmid was also used for CBF3 gene transformation (Fig. 1B).

The explants were cultured on MSI medium for 24 h before bombardment, followed by an additional period of culturing after particle bombardment. The data for transgenic plants obtained from all of the experiments are summarized in Table 1 . Since cytokinins were identified as key mediators in plant cell division and cell cycles (Miller et al., 1955; Rediga et al., 1996), the incubation process may have altered the proliferation of targeted cells and made them more receptive to DNA uptake. Our results are consistent with those of Songstad et al. (1996), who found that immature maize embryos cultured for 2 to 4 d before bombardment were successfully transformed and regenerated into transgenic plants. Brettschneider et al. (1997) also reported that an increase in the transformation frequency was observed when immature embryos were cultured on callus induction medium before and after bombardment.


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  		Table 1. Summary of the different parameters used for particle bombardment of split-seed explants that were cultured for 24 h on multiple-shoot induction medium and dried for 3 h before bombardment.

 
Osmotic pretreatment, or partial drying of the target cells before bombardment, has been proven to increase the frequency of successful transformation (Chen et al., 1998; Finer and McMullen, 1990). In many cases, mannitol or sorbitol is added to the medium as an osmotic inducer. Finer et al. (1999) reported that the osmotic treatment step prevented cell death, which occurs from wounding of the cell wall during microparticle penetration. In our experiments at 9-cm distance, we observed a significant difference in the average number of GUS spots from the cultures that were left uncovered in the laminar flow hood for 3 h (osmotic treatment) before bombardment and the covered cultures (P < 0.0001) with one shot using 7.58 and 9.31 MPa. There was no significant difference (P = 0.350), however, when 10.69 MPa was used in this treatment. When cultures were bombarded twice, there was a significant difference in the average number of GUS spots (P < 0.0001) at 7.58 MPa and no significant difference (P = 0.536 and P = 0.810) was observed at 9.31 or 10.69 MPa (Fig. 2 ). A decrease in target distance to 6 cm while keeping the pressure unchanged caused no changes in GUS expression. From these data, it may be concluded that the incidence of transient GUS expression is mainly influenced by pressure and the number of shots.


Figure 2
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  		Figure 2. Effect of drying split-seed explants before bombardment on transient GUS expression. Error bars represent the standard deviation. The means of GUS spots from each pressure point were analyzed by t-test (P = 0.05).

 
Selection and Regeneration of Transformants
We determined that the optimum lethal dosage of hygromycin for untransformed split-seed explants was 25 mg L–1; therefore, this concentration was used to select for putative transformants (Fig. 3A and 3B). After bombardment, explants were given a recovery period of four days on MSI medium before transferring to selection medium for a period of four to six weeks. The primary putative transformed shoots (Fig. 3C) that were regenerated during the first stage of selection were further subjected to a second stage of selection (Fig. 3D). In some cases, the putative shoots that needed further elongation before rooting were subjected to another round of selection for one week (Fig. 3E). A total of 21 antibiotic-resistant transformants were obtained by the end of all of the experiments, and 18 of the initial putative shoots were PCR positives for the CBF3 gene. Hence, the transformation frequency was estimated at 5%. Not all putative shoots elongated at the same rate and 15 plantlets whose growth was retarded were eliminated.


Figure 3
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  		Figure 3. Production of transgenic maize plants using split-seed explant: (A) split-seed explants arranged for particle bombardment; (B) bombarded explants on selection medium; (C) shoot regeneration through first round of selection; (D) shoots after a second round of selection; (E) rooting of a CBF3 putative transformant; and (F) CBF3 transgenic plants grown in the greenhouse.

 
Analysis of T0, T1, and T2 Plants
Leaves from T0 plantlets that were hygromycin resistant were tested for the integration of the CBF3 gene. Three lines that initially showed normal growth were confirmed positive for CBF3 by Southern blot analysis (Fig. 4A ). Southern blot analysis showed that Lines 7 and 8 had an estimated two to three copies of the CBF3 gene and they were sterile. In contrast, Line 28 had five copies but was fertile and went into further analyses.


Figure 4
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  		Figure 4. Southern blot analysis of T0, T1, and T2 CBF3 plants: (A) Southern blot analysis of T0 plants, showing Lane 1—kb ladder; Lane 2—untransformed plant digested with KpnI; Lanes 3, 5, and 7—Lines 7, 8, and 28 digested with Hind III; Lanes 4, 6, and 8—Lines 7, 8, and 28 digested with KpnI; and Lane 10—plasmid positive control digested with KpnI; (B) T1 plants that originated from Line 28, showing Lane 1—kb ladder, Lane 2—untransformed plant; Lanes 3 to 9—plants no. 1, 3, 4, 7, 11, 13, and 14 digested with KpnI; and Lane 11—plasmid positive control digested with KpnI; (C) T2 plants digested with KpnI, showing Lane 1—kb ladder; Lane 2—untransformed plant; Lanes 3 to 20—T2 plants no. 1 to 18; and Lane 21—plasmid positive control.

 
Line 28 was outcrossed to R23 to produce T1 progeny. Segregation analysis of both the selectable marker hpt (hygromycin phosphotransferase) and the CBF3 gene in T1 plants was performed by germinating the seeds on MS medium supplemented with 25 mg L–1 hygromycin (Table 2 ). As expected and despite the fact that the Southern blot analysis unambiguously showed five copies of CBF3, the resulting 91 T1 plants segregated CBF3 in a 1:1 ratio (P = 0.905). This argues that it is probable that the five genes inserted at a single chromosomal locus. In addition, these plants showed normal growth morphology (Fig. 3F) and were further confirmed for the transmission of CBF3 by Southern blot analysis (Fig. 4B). Southern blot analysis of Line 28 and the tested T1 progeny plants showed the same integration pattern with multiple hybridization bands (Fig. 4A and 4B). Dai et al. (2001) also showed that most of the fertile transgenic plants from particle bombardment segregated GUS and hpt genes in a Mendelian fashion.


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  		Table 2. Segregation analysis of T1 and T2 plants for the CBF3 gene. Segregation ratios 1:1 or 3:1 depend on crosses (P = 0.05).

 
Transgenic T1 plants were self-pollinated to produce T2 progeny and the segregation analysis of marker hpt and CBF3 in the T2 plants gave a 3:1 ratio (P = 0.871) also for the presence vs. absence of both genes (Table 2). The inheritance of CBF3 in T2 plants was also confirmed by Southern blot analysis (Fig. 4C).

CBF3 Expression
The expression level of CBF3 was detected in transgenic lines 7 and 8, even under normal growth conditions (26°C), and the plants showed a slight stunted growth and were sterile. In contrast, the expression level in Line 28 was hardly detected in RT-PCR analysis at normal growth temperature and resulted in a fully fertile plant with normal phenotype (Fig. 5 ). Overall, no differences in morphology or growth were observed between the stable transgenic plants expressing the CBF3 gene and wild-type plants under normal growth conditions. Pellegrineschi et al. (2004) observed nonuniform germination in rd29A:CBF3 transgenic wheat seeds from normal and stressed conditions but the plants recovered and showed normal growth and morphology in later stages.


Figure 5
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  		Figure 5. Reverse transcription polymerase chain reaction analysis of CBF3 gene expression in T0 Lines 7, 8, and 28 grown at 26°C; C is a wild-type plant, maize actin was used as a loading control.

 
A high expression level of targeted stress-inducible genes was observed in transgenic plants when CBF3 was driven by the CaMV 35S promoter and those plants showed significant tolerance to drought, freezing, and high-salt stresses (Gilmour et al., 2000; Liu et al., 1998). Transgenic plants with CBF3 under CaMV 35S, however, showed growth retardation under normal growth conditions (Kasuga et al., 2004). Use of the stress-inducible rd29A promoter instead of the constitutive CaMV 35S promoter for the overexpression of CBF3 minimized the negative effects on plant growth in transgenic Arabidopsis (Kasuga et al., 1999). To monitor the expression profile of CBF3 transcripts under stress conditions, plants were grown at 26°C and were then first subjected to cold temperatures gradually, with a duration of 3 h for each period. Northern blot analysis indicated that CBF3 transcripts were first detected at 20°C and the amount of mRNA corresponding to CBF3 was increased to reach the highest level at 4, 0, and –2°C (Fig. 6 ). Zarka et al. (2003) reported that the transcript levels of the CBF genes were detected at 14°C when Arabidopsis plants were subjected to gradually lower temperatures.


Figure 6
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  		Figure 6. Northern blot analysis of CBF3 transcripts after T2 plants derived from Line 28 were treated with different low temperatures. Plants were grown at 26°C and then were gradually exposed to low temperatures (20, 16, 12, 10, 4, 0, and –2°C), 3 h for each temperature. Full length of the coding region of CBF3 was used as a probe and actin was used as a loading control.

 
Analyses of CBF3 Plants
Cold and Freezing Tolerance
We conducted different studies to examine whether CBF3 transgenic plants will be more tolerant to low and freezing temperatures than the wild-type plants. Five-day-old seedlings from T2 and wild-type seeds that were grown in trays at 26°C were transferred to a constant 10°C growth chamber for four weeks under 16/8 h light/dark (Fig. 7A ). After one week, wild-type seedlings began to show injury symptoms and turned yellow, and growth had stopped. In comparison, transgenic seedlings continued to grow at 10°C and remained green (Fig. 7B). By the end of four weeks, 80% of the wild-type seedlings were dead, but transgenic seedlings were more tolerant and survived the 10°C four-week period (Fig. 7C and 7D). After all of the plants were moved to the greenhouse and kept at 26°C for a two-week recovery period, <20% of the wild-type seedlings survived while 83% of the transgenic seedlings survived and continued to grow (Fig. 7E). In addition, 77% of the transgenic plants from this treatment were fertile and set seeds, compared with 0.07% of the wild-type plants (Fig. 7E).


Figure 7
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  		Figure 7. Comparison of growth characteristics, survival rate, and fertility rate between transgenic T2 seedlings derived from Line 28 (left tray) vs. wild-type seedlings (right tray) when kept at 10°C: (A) 5-d-old seedlings grown at 26°C (normal condition); (B) 1-wk-old seedlings; and (C) 4-wk-old seedlings. (D) Mean heights of transgenic and wild-type seedlings at different time periods, with error bars representing the standard deviation; and (E) survival and fertility rates of transgenic and wild-type plants after a 2-wk recovery period from 10°C in the greenhouse, during which surviving plants were grown to maturity.

 
The expression of CBF3 was also monitored at lower temperatures, and the transcript levels were analyzed by Northern blot analysis. When transgenic plants were subjected to cold shock at 4°C, the plants were moved directly from 26°C into 4°C growth chambers for a 24-h period; transcripts of the CBF3 gene were detected by Northern blot analysis after plants were exposed to 4°C for 1 h and the expression level increased after 3 h of exposure and was gradually reduced after 5, 7, 12, and 24 h (Fig. 8A ). In contrast, the transcripts were observed within 15 min of exposure to 0°C and continued to be expressed for the 24-h period (Fig. 8B). To determine whether the CBF3 transgenic plants are resistant to lower (4°C) and perhaps freezing temperatures (0 and –2°C), two-week-old transgenic and wild-type plants grown in trays at 26°C were transferred to 4, 0, and –2°C conditions for 48 h and returned to the greenhouse at 26°C. Directly following the treatments, we observed that all of the wild-type plants wilted, whereas transgenic plants maintained their healthy morphology (Fig. 9A, 9B, and 9C ). We then performed the electrolyte leakage test to evaluate the damage that could have occurred to the cell membrane of the transgenic and wild-type plants as a result of exposure to cold and freezing temperatures. Two-centimeter segments from the upper leaf tip of treated plants were used for the electrolyte leakage test. For the wild-type plants, the ion leakages were 42.8, 78, and 83.9% at 4, 0, and –2°C, respectively, compared with transgenic plants at 11.4, 24.8, and 25.9%, respectively (Fig. 9G). These results clearly demonstrate that the ion leakage of the plants segregating CBF3 was significantly reduced after cold and freezing treatments when compared with control plants (P < 0.0001). To further evaluate the tolerance of transgenic and wild-type plants to the 48-h treatments at 4, 0, and –2°C, the survival rate was also calculated after a two-week recovery period in the greenhouse (Fig. 9D, 9E, and 9F). While 57.4% of the wild-type plants survived the 4°C treatment, 89.2% of the transgenic plants endured. The survival rate percentage of the wild-type plants continued to drop significantly from 0 and –2°C treatments (P < 0.0001), an estimated 40 and 15.7%, respectively, compared with the transgenic plants survival rates of 79.1 and 73.4%, respectively (Fig. 9H).


Figure 8
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  		Figure 8. Northern blot analysis of T2 plants derived from Line 28 that were exposed to 4 and 0°C for different periods of time: (A) accumulation of CBF3 transcripts in response to exposure to 4°C for 1, 3, 5, 7, 12, and 24 h (C is a wild-type plant and TN is a transgenic (T2) plant derived from Line 28 grown at 26°C); (B) T2 plants derived from Line 28 that were exposed to 0°C for 15 min, 30 min, and 1, 3, 6, 12, and 24 h (C is a wild-type plant); actin was used as a loading control.

 

Figure 9
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  		Figure 9. Comparison of cold and freezing tolerance between transgenic T2 plants derived from Line 28 (left tray) and wild-type (right tray) plants: (A, B, and C) plants after exposure to 4, 0, and –2°C, respectively, for 48 h; (D, E, and F) after a 2-wk recovery period under greenhouse conditions (26°C). (G) Electrolyte leakage of transgenic and control plants that were grown at 26°C and were then exposed to 0, 4, and –2°C for 48 h; and (H) comparison of survival rates after cold and freezing treatments of transgenic and wild-type plants after 2 wk of recovery from 0, 4, and –2°C exposure for 48 h.

 
Reports on CBF genes have shown that overexpression induces the expression of COR (cold-responsive) genes in Arabidopsis and in turn leads to water stress tolerance (Jaglo-Ottosen et al., 1998; Thomashow, 2001). Using BLAST search of the GenBank database, we were unable to identify known maize COR genes that are homologous to the Arabidopsis COR genes; however, we identified binding site GGCCCGAC in the promoter region of maize ZmCAT3 (accession no. L05934), which is similar to the DRE/CRT binding site sequence TGGCCGAC in the promoter region of the Arabidopsis COR15a gene. Because chilling tolerance in maize has been shown to be correlated with the upregulation of the ZmCAT3 gene (Prasad, 1997), we wanted to see if the expression of CBF3 would influence ZmCAT3 expression. According to Northern blot analysis, we observed that indeed the level of ZmCAT3 transcripts were higher in transgenic plants than the wild type when grown at normal temperature (26°C) and the level of expression was further increased in transgenic plants when subjected to 4°C for two days (Fig. 10 ). This suggests that CBF3 possibly binds to other stress-related genes in maize and confers tolerance to low and freezing temperatures. Hsieh et al. (2002) reported that neither CBF1 transgenic nor wild-type tomato (Lycopersicon esculentum Mill.) plants survived –2°C treatment for 2 d, but the transgenic plants were more tolerant to chilling treatment than the wild-type plants. It was also observed that the tomato CAT1 transcripts were higher in transgenic CBF1 plants than the wild-type plants. Another report from Lee et al. (2004) showed that rice (Oryza sativa L.) plants expressing CBF1 were not significantly more tolerant to cold treatment than the wild type. Unlike these results, maize CBF3 transgenics express both cold and freezing tolerance paralleling what has been reported in Arabidopsis. Its application to the seed industry is likely to be profound as farmers will enjoy the opportunity of not only earlier planting but also new open markets.


Figure 10
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  		Figure 10. Northern blot analysis of maize CAT3 transcripts in transgenic T2 plants and wild-type plants grown at 26°C or exposed to 4°C for 2 d; C is wild-type plants, T is transgenic plants; actin was used as a loading control.

 
Dehydration and High Salt Tolerance
To investigate the role of the CBF3 gene in maize under drought conditions, transgenic and wild-type plants grown in pods at 26°C were not watered for 21 days. The CBF3 transcripts were observed after 3 d of water deficit and increased during the next four days (Fig. 11 ). To examine the tolerance of transgenic and wild-type plants, the survival rate was scored following 7-, 14-, and 21-day treatments. After one week of dehydration, wild-type plants started to show signs of stress and wilted, while transgenic plants exhibited no stress symptoms (Fig. 12A ). Almost all the wild-type plants died after 14 and 21 days of withholding water, while transgenic plants continued to grow (Fig. 12B and 12C). When plants were watered regularly and monitored for two weeks following a one week drought, 90.1% of the wild-type plants survived, while 99.3% of the transgenic plants survived. Most importantly, 80.2 and 62.4% of transgenic plants survived the 14- and 21-d dehydration periods, whereas 22.6 and 0.3% of the wild-type plants survived this drought stress (Fig. 12D). When comparing the survival rate of transgenic and wild-type plants, transgenic plants were significantly more tolerant to drought conditions than the wild type (P < 0.0001). Moreover, the results of the electrolyte leakage test indicated that the drought tolerance of transgenic plants was significantly greater than the wild type. While 64.5 and 87.7% of the ions leaked from the wild-type plants after seven and 14 days of dehydration, only 23.2 and 26.1% of the ions leaked from transgenic plants after these water-stress periods (Fig. 12E). Hence, the plants expressing the CBF3 gene were significantly more resistant to water stress than wild-type plants (P < 0.0001). Similarly, Pellegrineschi et al. (2004) also reported that wheat plants expressing the CBF3 gene under the control of rd29A were substantially more resistant to water stress than wild-type plants under greenhouse conditions.


Figure 11
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  		Figure 11. Accumulation of CBF3 transcripts in T2 plants derived from Line 28 in response to water deprivation for 3 and 7 d. The numbers 1 to 3 represent three different transgenic plants from the T2 generation; C is a wild-type plant; and actin was used as a loading control.

 

Figure 12
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  		Figure 12. Transgenic maize T2 plants derived from Line 28 (left pots) show more tolerance to drought than wild-type plants (right pots) when not watered for (A) 7 d, (B) 14 d, and (C) 21 d. (D) Comparison of survival rates after drought treatments at the indicated periods and after a 2-wk recovery period in the greenhouse; and (E) electrolyte leakage of transgenic and wild-type plants that were grown at 26°C and were then exposed to dehydration stress for the time indicated.

 
To test whether the expression of CBF3 in transgenic plants will enhance tolerance to salt stress, transgenic and wild-type plants were challenged with 100, 200, and 400 mmol L–1 solutions of NaCl for 7 d. The CBF3 expression was observed after three hours, three days, and seven days of treatment and it was higher in plants treated with 400 rather than 100 or 200 mmol L–1 of NaCl (Fig. 13 ). In the 100 and 200 mmol L–1 NaCl treatments, no differences in morphology were observed between transgenic and wild-type plants after 3 d; however, all of the wild-type plants collapsed from 400 mmol L–1 while transgenic plants looked normal (Fig. 14A , 14B, and 14C). After seven days, no obvious differences between transgenic and wild-type plants were observed following the 100 mmol L–1 treatment, but necrotic symptoms were observed in wild-type plants from the 200 mmol L–1 treatment while transgenic plants looked healthy (Fig. 14D, 14E, and 14F). When plants were given two weeks of recovery by removing any salt residues with regular watering, a significant difference in the survival rate was observed between transgenic and wild-type plants subjected to high salt stress (P < 0.0001). Only 2.1% of the wild-type plants survived the 200 mmol L–1 treatment and none survived the 400 mmol L–1 treatment. This compares with 82.3 and 75.4% of transgenic plants, respectively (Fig. 14G and 14H). These results are in complete agreement with those obtained by Kasuga et al. (1999), who reported that Arabidopsis plants overexpressing the CBF3 gene were significantly more tolerant to a 600 mmol L–1 NaCl treatment (78.6% survived) than wild-type plants (17.9% survived).


Figure 13
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  		Figure 13. Northern blot analysis of the CBF3 gene in T2 plants derived from Line 28 that were exposed to different concentrations of NaCl solution: 100, 200, and 400 mmol L–1 for the periods indicated; C is a wild-type plant and actin was used as a loading control.

 

Figure 14
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  		Figure 14. Transgenic T2 plants derived from Line 28 (left trays) are more tolerant to high salt (NaCl) concentrations than wild-type plants (right trays): (A, B, and C) 100, 200, and 400 mmol L–1 NaCl treatments for 3 d; (D, E, and F) treated plants after 7 d; and (G) treated transgenic and wild-type plants after 21 d in the greenhouse. (H) Survival rate comparison between transgenic and wild-type plants after a 2-wk recovery period.

 
In conclusion, overexpressing the CBF3 gene has been shown to enhance freezing, drought, and salt stress in Arabidopsis (Kasuga et al., 1999). Similarly, according to the above results, maize plants expressing the Arabidopsis CBF3 gene showed higher levels of resistance to cold, drought, and salinity in comparison to the wild type. In maize, this gene is induced following cold, drought, or salinity and, hence, maize split-seeds may serve as the explant of choice for rapidly producing large numbers of transgenic hybrids and inbreds buffered against both abiotic and biotic stresses. Moreover, this protocol can significantly shorten the time for obtaining transgenic maize plants, especially for commercial product development.


   ACKNOWLEDGMENTS
 
We would like to acknowledge primarily funding from the Ohio Plant Biotechnology Consortium, Penn State Harrisburg School of Science, Engineering and Technology, and the U.S. Department of Agriculture, Agricultural Research Service Cooperative Agreement no. 3607-21000-008-01S. Diaa Al-Abed would also like to thank Professor Trevor Thorpe (Univ. of Calgary, Calgary, AB, Canada) for overseeing this work, Ms. Lisa Delp for administrative assistance, and Ms. Dorothy Rimmelin for technical assistance, and is also grateful to the Department of Earth, Ecological and Environmental Sciences at the Univ. of Toledo for providing a graduate stipend. The authors are also grateful to Mr. Bruce Ferguson, chairman and president of Edenspace Systems, for assisting with production costs.


   NOTES
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 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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Received for publication November 16, 2006.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 





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