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Regeneration and transformation of Egyptian maize inbred lines via immature embryo culture and a biolistic particle delivery system

Posted on: Saturday, 22 November 2003, 06:00 CST

SUMMARY

A regeneration system was developed for elite Egyptian maize inbred lines using immature embryos as explants. This system proved to be highly genotype-dependent. Line Gz 643 was identified as the best line, revealing the highest regeneration frequency (42.2%). Addition of L-proline and silver nitrate to culture media greatly enhanced the formation of embryogenic type II callus and the regenerability of some of the tested lines. Transformation of the scutellar tissue of immature embryos from inbred line Gz 643 was performed with the particle delivery system using a single plasmid carrying both the GUS and Bar genes (pAB-6) or by co-transformation with two plasmids, pAct1-F (GUS) and pTW-a (Bar). Different transformation parameters were evaluated, i.e. osmotic treatment, acceleration pressure, and number of shots. Osmotic treatment (0.25 M sorbitol + 0.25 M mannitol) along with the use of either acceleration pressure 1300 psi and one shot per plate (for co- transformation with pAB-6) or 1100 psi and two shots per plate (for transformation with pAct1-F and pTW-a) gave the best results, as expressed by the number of blue spots in the [beta]-glucuronidase (GUS) assay. Stable transformation was confirmed in Ro transformed plants by means of histochemical GUS assay and herbicide application. PCR and Southern blot analysis proved the integration of the full-length genes in some of the transgenics.

Key words: maize; immature embryos; embryogenic callus; plant regeneration; transformation.

INTRODUCTION

The genetic improvement of cereals has been a major focus of plant breeding efforts during the past 50 yr, resulting in remarkable increases in the yield and improvement in the quality of this important group of food crops. However, modern plant biotechnology has provided novel means for crop improvement through the integration and expression of defined foreign genes into plant cells, which can then be grown in vitro to regenerate whole plants. The efficient regeneration of normal and fertile plants from single cells, a basic prerequisite for the production of genetically transformed plants, proved to be rather difficult for gramineous species because of their extreme recalcitrance to manipulation in vitro (Zhang et al., 2002).

The regeneration ability of any plant is influenced by different factors. The type of explant is considered one of the main factors that attracted the concern of many investigators. Immature embryos have been the most widely used explant in many cereals, including maize (Green and Phillips, 1975; Armstrong and Green, 1985; Hodges et al., 1986; Lee and Phillips, 1987; Shillito et al., 1989). Tissue culture of maize immature embryos is capable of producing two different types of embryogenic callus; Type I, a compact organized and slow-growing callus; and Type II, a soft, friable and fast- growing one characterized by its high regeneration capacity. The production of Type II callus arises at low frequency and only for specific genotypes (Armstrong and Green, 1985). Also, regenerability is influenced, to a great extent, by the media composition (Armstrong and Green, 1985; Vain et al., 1989a, b; Songstad et al., 1991; Bohorova et al., 1995; Carvalho et al., 1997).

Another important factor is the genetic background or the genotype of the explants which was found to influence the regeneration potentiality. Bohorova et al. (1995) discussed the effect of the genotype on somatic embryogenesis. Reports by Tomes and Smith (1985) and Hodges et al. (1986) indicated that the regeneration was genetically controlled by nuclear genes in maize. Moreover, studies by Willman et al. (1989) suggested that at least one gene or a block of genes controlled the expression of somatic embryogenesis of maize tissue cultures.

The transfer of defined genes is theoretically the most straightforward method for improvement of crop plants. The microprojectile bombardment system proved to be a powerful technique for genetic modification of maize lines by delivering foreign DNA into scutellar tissues of immature embryos (Koziel et al., 1993; Wan et al., 1995; Songstad et al., 1996; Zhang et al., 1996; Brettschneider et al., 1997; Bohorova et al., 1999; Frame et al., 2000; O'Kennedy et al., 2001; O'Connor-Sanchez et al., 2002).

Thus, the main objectives of the present investigation were: (1) to establish an efficient regeneration system for elite Egyptian maize inbred lines, (2) to investigate the effect of media composition on the type of callus formed by immature embryos, and (3) to optimize transformation conditions of immature embryos using the biolistic particle delivery system, with the GUS reporter gene and the Bar gene as a selectable marker.

MATERIALS AND METHODS

Plant material. Five elite Egyptian maize (Zea mays L.) inbred lines with different or similar genetic background were used in this investigation, viz., Sd 62, G 221D, Sd 7, Gz 643, and Gz 624. Seeds were provided by the Maize Department, Field Crops Research Institute, ARC, Giza, Egypt.

Culture initiation and maintenance. Callus cultures were initiated from immature zygotic embryos produced on self-pollinated ears of field-grown maize plants. Three hundred immature embryos from each genotype (1-2 mm) were aseptically excised according to Green and Phillips (1975) and placed on the solid culture medium with the rounded scutellar side exposed and the flat plumule- radicle axis side in contact with the medium. Two N6-based media CI- 1 (Chu et al., 1975) and CI-2 (Armstrong et al., 1991) were used. Medium CI-1 was supplemented with 2.0 mg l^sup -1^ (9.0 [mu]M) 2,4- dichlorophenoxyacetic acid (2,4-D) and 3% sucrose, while medium CI- 2 was supplemented with 1.0 mg l^sup -1^ (4.5 [mu]M) 2,4-D, 2% sucrose, 2.88 g l^sup -1^ L-proline, and 1.7 mg l^sup -1^ silver nitrate. Cultures were maintained for 2 mo. in the dark at 25[degrees]C by subculturing every 2 wk onto fresh medium of the same composition.

Regeneration and acclimatization. Embryogenic calluses were transferred to MS-based (Murashige and Skoog, 1962) regeneration media as follows: (1) calluses were subcultured on regeneration medium (RM1): MS medium + 1.0 g l^sup -1^ myo-inositol + 1.0 mg l^sup -1^ NAA + 6% sucrose for 2 wk in darkness, (2) subcultured on RM2: MS medium + 1.0 g l^sup -1^ myo-inositol + 3% sucrose for 2-3 wk at 25[degrees]C, 16/8 h photoperiod, and (3) germinated somatic embryos having 1 cm shoots were transferred to the rooting medium (RM3): MS medium + 500 mg l^sup -1^ myo-inositol + 3% sucrose in magenta boxes at 25[degrees]C with 16/8 h photoperiod.

The regenerated plantlets were acclimatized by transferring them to an aquarium containing a modified Hoagland solution (Johnson et al., 1957). When an extensive root system had formed, plantlels were transferred to 15-cm pots containing a 1:1 mixture of sterile peatmoss:soil and placed in the containment greenhouse adjusted to 28[degrees]C with a 16 h photoperiod and 90% humidity. Pots were watered thoroughly with half-strength Hoagland solution. When four to six true leaves were formed, the plants were transferred to larger pots (40 cm in diameter).

Plasmid DNAs. The plasmids pAct1-F and pTW-a were obtained from Prof. Ray Wu (Cornell University) while pAB-6 was kindly provided by Prof. Ahmed Bahei eldein (Ain Shams University). pACT1-F (McElroy et al., 1990) included the GUS coding region controlled by the 1.3-kb 5' region of the rice actin 1 gene (Act1). pTW-a contained the selectable marker gene bar (phosphinothricin acetyl transferase) following the CaMV-35S promoter and rice actin-1 (Act1) 5' intron and terminated with the pin 2 3' region. pAB-6 contained the Bar gene driven by the CaMV 35S promoter and the GUS gene driven by the rice Act1 promoter.

Particle bombardment, osmotic treatment, selection of transformants, and regeneration. Plasmid DNA was precipitated onto tungsten particles (1.1 [mu]m in diameter) (M17, Bio-Rad) following a modification of the original protocol for the Bio-Rad Biolistic PDS-1000/He Particle Delivery System (Zhong et al., 1996). Immature maize embryos (1.0-2.0 mm) of inbred line Gz 643 were bombarded after 4 d incubation on callus induction CI-2 medium in darkness at 25[degrees]C. Sixteen experiments were carried out with a total of 2240 immature embryos. In these experiments, the effect of the different bombardment parameters, i.e. pressure (1100 or 1300 psi), plasmids (pAB-6 or pTW-a: pAct1-F (1:1), number of shots (one or two), and osmotic treatment of the explant, were compared. The osmotic treatment was performed as described by Vain et al. (1993) by placing the embryos on callus induction medium CI-2 supplemented with 0.25 M sorbitol and 0.25 M mannitol. Embryos were subjected to osmotic treatment 4 h prior to bombardment and continued for 16 h after bombardment. Then, the bombarded embryos were transferred to callus induction medium CI-2 free from sorbitol and mannitol.

Calluses produced by bombarded immature embryos were selected for herbicide resistance using PPT (glufosinate ammonium (ammonium-DL- homoalanin-4-methylphosphinate); Riedel-de Haen, Germany) or the herbicide Bialaphos (Meija Seika Kaishi, Ltd., Yokohama, Japan). Selection of PPT- or Bialaphos-resistant callus was initiated 10 d after bom\bardment (Songstad et al., 1996), by placing the bombarded immature embryos on medium CI-2 containing 1 mg l^sup -1^ PPT or Bialaphos. The callus that developed was subcultured every 10-14 d onto fresh selection medium for about 2 mo.

For regeneration, the embryogenic callus that survived was placed on regeneration medium (RM1) supplemented with 1 mg l^sup -1^ PPT or Bialaphos in the dark for 2 wk and then subcultured on regeneration medium (RM2) supplemented with 1 mg l^sup -1^ PPT or Bialaphos under light (16 h) and then the green shoots were placed on rooting medium (RM3) containing 1 mg l^sup -1^ PPT or Bialaphos under light (16 h). All selection and regeneration steps were carried out at 25[degrees]C.

Histochemical [beta]-glucuronidase (GUS) assay. Histochemical localization of the GUS activity in the bombarded tissues was performed 48 h after bombardment according to the method described previously (Zhong et al., 1996) on five immature embryos selected at random from each bombarded plate. In addition, samples (roots or leaves) from regenerated transformed and untransformed control plants were taken from the greenhouse-grown plants and incubated under the same conditions with the GUS-substrate mixture. Green tissues were decolorized by incubation in 75% ethanol for at least 1- 2 h prior to photography. Blue spots were visualized and counted under a binocular stereomicroscope.

PAT activity assay. To determine the activity of PAT, the Bar gene product, it was assayed indirectly by the resistance of transgenic plants to herbicide application. The herbicide BASTA(R) (Hoechst, Germany) containing 200 g l^sup -1^ glufosinate ammonium, the active ingredient, was used for leaf painting of the putalively transgenic plants. Two applications of BASTA were performed by painting approximately 5-10 cm leaf sectors near the tip of the youngest fully-extended leaf at the four- and eight-leaf stages with a 1% solution of the herbicide containing 0.1% (v/v) Tween 20.

Molecular analysis of transgenes. Genomic DNA was isolated from leaf tissues of each putatively transformed plant as well as from untransformed plants of line Gz 643 (control) using the CTAB method (Rogers and Bendich, 1985).

PCR was carried out in a volume of 50 [mu]l containing 50 ng of genomic DNA template, 2 [mu]M primer, 200 [mu]M each of dATP, dCTP, dGTP, and dTTP, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 0.2 mM MgCl^sub 2^, 0.001% gelatin and five units of Taq polymerase enzyme (Promega). Two sets of primers were used to detect the GUS and Bar genes by PCR analysis. One set was GUS-1 (5'- CTCGACGGCCTGTGGGCATTCAGTC-3') and GUS-2 (5'- TAGATATCACACTCTGTCTGGCTTTTGG-3'), while another set was Bar-1 (5'- TGCCACCGAGGCGGACATGCCGGC-3') and Bar-2 (5'-CCTGAAGTCGGACGGCCATGGCGG- 3'). DNA amplification was performed in a Perkin Elmer thermal cycler 2400 programmed as follows: an initial strand separation at 94[degrees]C (5 min), followed by 33 cycles with the following temperature profile: 95[degrees]C (1 min), 55[degrees]C (2 min), 72[degrees]C (2 min), and a final extension at 72[degrees]C (8 min). The PCR products were separated by electrophoresis on 1% agarose gel.

For Southern blot analysis, DNA (10 [mu]g) isolated from leaves of transformed maize plants was digested with the appropriate restriction enzyme(s) in order to detect the integration of the Bar and GUS genes in the regenerated transformed plants according to the protocol described previously (Kreike et al., 1990) with some modifications. For the detection of the GUS gene, DNA was digested with endonecleases BamHI and SacI, while DNA was digested with EcoRI for the Bar gene detection. The digested DNA was electrophoretically separated on a 0.7% agarose gel, transferred to a positively charged nylon membrane (Boehringer Mannheim, Germany) and then cross-linked to the membrane by UV irradiation.

The hybridization probes were prepared by digesting pTW-a with SmaI and pAct1-F with BamHI and SacI to liberate the Bar and GUS DNA, respectively. After separation by electrophoresis, the desired inserts were eluted from the gels, and labeled using a random priming DNA labeling and detection kit (Boehringer Mannheim). The labeled probes were incubated with the membranes at 65[degrees]C for 16-18 h and the hybridization signals were detected by exposure of the membrane to X-ray films.

Statistical analysis. Statistical analysis was performed according to Steel and Torrie (1980) using the SAS computer software (version 5) with associated least significant differences (LSD) function. Experiments were designed as factorial experiments in completely randomized design.

RESULTS AND DISCUSSION

Initiation of callus and formation of somatic embryos. In the present study, immature embryos of five Egyptian maize inbred lines (Gz 643, Gz 624, Sd 62, Sd 7, and G 221D) were screened on two culture media (CI-1 and CI-2) to evaluate genotype culturability and regeneration capacity of these lines.

For each genotype, a total of 300 immature embryos in 10 replicates were used as explants for experiments of callus induction and regeneration. In general, the average number of calluses formed per plate after 2 wk of culture was higher on medium CI-2 as compared to medium CI-1 for all genotypes. However, only two lines, Gz 643 and Sd 62, revealed a significant difference in the average number of calluses produced on both media, with Gz 643 having the highest induction ability.

Tables 1 and 2 show that callus tissue was obtained from the scutellum of immature embryos of all genotypes tested but not all the callus formed during callus initiation was embryogenic. The highest average number of embryogenic calluses was produced by Gz 643 cultured on medium CI-2, indicating that different genotypes possess different potentiality to produce embryogenic callus. Moreover, the components of medium CI-2 tend to enhance the formation of embryogenic callus in lines Sd 62, Gz 643, and Gz 624. Although the concentration of sucrose and 2,4-D was reduced in medium CI-2 as compared to medium CI-1, the addition of proline (25 mM) and AgNO^sub 3^ (1.7 mg l^sup -1^) greatly influenced the quality and quantity of embryogenic callus formed. The callus obtained on medium CI-1 could be classified as Type I, with Type II callus not formed on this medium, whereas a large number of immature embryos of line Gz 643 grown on medium CI-2 formed friable embryogenic callus (Type II) with small somatic embryos on the surface (Fig. 1A). These results indicate that the presence of L- proline (25 mM) and AgNO^sub 3^ (1.7 mg l^sup -1^) in N6 medium are beneficial for the initiation of friable embryogenic (Type II) callus from Gz 643 embryos. The promoting effect of proline has also been reported by Armstrong and Green (1985).

TABLE 1

MEAN NUMBER OF EMBRYOGENIC CALLUSES FORMED AFTER 2 MO., AND THE REGENERATION FREQUENCY (%) OF CULTURE FROM IMMATURE EMBRYOS OF THE FIVE MAIZE INBREDS MAINTAINED ON CALLUS INDUCTION MEDIA (CI-1) AND (CI-2)

TABLE 2

ANALYSIS OF VARIANCE FOR THE NUMBER OF CALLUSES FORMED AFTER 2 WK, THE NUMBER OF EMBRYOGENIC CALLUSES FORMED, REGENERATED PLANTS, AND REGENERATION FREQUENCY

Silver nitrate has also been shown to increase Type II callus production as discussed by Vain et al. (1989a, b). The role of AgNO^sub 3^ as suggested by Beyer (1976, 1979) was that Ag^sup +^ ion inhibited the ethylene action by interfering with C^sub 2^H^sub 4^ incorporation at its receptor sites. Also, McCain and Hodges (1986) observed that AgNO^sub 3^, as an ethylene controlling compound, influenced Type I and Type II calluses differently, indicating a distinct sensitivity of the two types of tissue for ethylene during their initiation. The importance of proline and its connection with ethylene-inhibiting compounds such as AgNO^sub 3^ has been explained by Vain et al. (1989a) as that either proline protected the tissue cultures from a toxic effect of C^sub 2^H^sub 4^ inhibitors or it interacted with C^sub 2^H^sub 4^.

FIG. 1. A, Type II callus showing small somatic embryos on the surface. B, Callus formation on regeneration media. C, Regenerated maize plantlet.

Regeneration of plantlets from somatic embryos. Plant regeneration was induced by transferring 300-500 embryogenic callus pieces to the plant regeneration media. Table 1 shows that the lowest regeneration frequency was exhibited by line Sd 7 (5.62%) while the highest regeneration frequency was obtained in line Gz 643 (42.43%). The regenerability of inbreds Sd 62, G221 D, and Sd 7 was low either on medium CI-1 or CI-2, and the mean number of regenerated plantlets was not significantly different among these lines. It also shows that exogenous application of L-proline and AgNO^sub 3^ only slightly enhanced the regeneration ability of these genotypes. Concerning the inbred lines Gz 643 and Gz 624, the regeneration potentiality of these genotypes significantly increased by changing the callus induction media composition by supplementing the media with L-proline and AgNO^sub 3^ (Fig. 1B, C). The present results indicated that the capacity of plantlet regeneration is correlated with the ability to form embryogenic callus, but not all embryogenic calluses can regenerate, plants. Therefore, the classification of callus as embryogenic does not necessarily imply regenerability. Similar findings have also been reported by Bohorova et al. (1995). In addition, the present study also shows that a large number of normal, fertile plants can be regenerated from Type I as well as Type II embryogenic cultures, with very few abnormalities in regenerated plants. This is consistent with the findings of Bohorova et al. (1999), who reported that regeneration could be obtained from either Type I or Type II callus.

Transient GUS expression in transformed immature embryos. Sixteen independent transformation and co-transformation experiments were carried out using different transformation parameters (i.e. plasmids, osmotic t\reatment, pressure, and number of shots per plate), as shown in Table 2. Inbred line Gz 643 was chosen as a source of immature embryos because it showed the highest regeneration frequency among the lines. The scutellar tissues of immature embryos (1.0-2.0mm long) were bombarded 4d alter culture on callus induction medium using the Bio-Rad He-PDS/1000 Particle Delivery System. The total number of bombarded immature embryos was 2240 with ~ 140 embryos per experiment.

In all transformation experiments, almost every embryo showed GUS- expressing foci, suggesting that the tungsten particles were dispersed to all embryos. The number of foci per embryo varied from several to more than 100. The osmoticum used consisted of a mixture of equimolar mannitol and sorbitol, which was previously reported as the best osmoticum treatment for transformation of microorganisms (Armaleo et al., 1990; Shark et al., 1991).

From the results shown in Table 3, it can be concluded that, in the co-transformation experiments, the use of osmotic pre- and post- treatment with acceleration pressure of 1100 psi and double shots per plate is most efficient, judging from the transient GUS expression. In transformation experiments using pAB-6, the use of an acceleration pressure of 1300 psi, osmotic pre- and post-treatment, and one shot per plate gave the highest value of the transient GUS expression. Moreover, the osmotic treatment of embryogenic maize cells for 4 h before and 16 h after bombardment enhanced the transient expression of the GUS gene, ranging from 1.4- to 2.6- fold, depending on the pressure and number of shots applied per plate during the bombardment process (Fig. 2A). These results are in agreement with the findings of Vain et al. (1993) and Brettschneider et al. (1997).

Analysis of transformants. Histochemical localization of Act1- GUS activity in transgenic R^sub 0^ plants was carried out on roots and leaves of plantlets regenerated from in vitro transformed and untransformed immature embryos. The GUS gene was expressed as blue coloration in the roots of most of the transformed plantlets. The strength of GUS signals in the roots varied between different transformation events (Fig. 2B). Differential GUS expression in different plant parts has been previously reported by Lowe et al. (1995) and Songstad et al. (1996). These authors showed a strong GUS activity was detected in roots and leaves of plantlets, stems, and kernels of F^sub 1^ seeds but GUS was poorly expressed in pollen.

TABLE, 3

TRANSIENT EXPRESSION (NUMBER OF BLUE SPOTS) OF THE GUS GENE IN BOMBARDED IMMATURE EMBRYOS WITH DIFFERENT TRANSFORMATION PARAMETERS

The functional activity of PAT was assessed by localized application of BASTA(TM). About 200 transformed plants of inbred line Gz643 (Fig. 2C) were tested twice for their response to BASTA(TM), as described by Gordon-Kamm et al. (1990). About 10 T^sub 0^ plants showed high resistance to 1% BASTA(TM), and no necrosis was observed on the leaves of these transformed plants up to 30 d after application. Other plants showed varying degrees of resistance to BASTA(TM), and few plants (less than 10%) were susceptible; while all R^sub 0^ untransformed plants were susceptible and showed necrosis in 2 d and the painted leaves died 4 d after BASTA(TM) application (Fig. 2D).

FIG. 2. A, Transient GUS gene expression in transformed immature embryos with osmotic treatment. B, GUS expression in roots of transformed plants (T) and nontransformed control plants (C). C, Regenerated maize plants. D, Leaves of transformed plant (T) and nontransformed control plant (C) painted with BASTA(TM).

In the present study, there was no apparent difference in the selection efficiency of maize tissues transformed with pAB-6 or pTW- a. This can be due to the fact that the Bar gene in the two chimeric plasmids is driven by the same 35S promoter.

Gene integration analysis. Total genomic DNA from leaf samples of putatively transgenic plants resulting from transformation and co- transformation experiments were analyzed by PCR using primers specific to the coding region of the GUS and Bar genes. PCR analysis was performed on 29 putatively transgenic plants resulting from immature embryo transformation and co-transformation experiments. The results revealed that 14 positive events were obtained using the plasmid pAB-6, while 13 positive events were obtained using the pTW- a: pAct1-F. Among the tested samples, two of the transformed plants did not show the GUS DNA fragment, indicating that the gene has been deleted. Similar results have been reported previously by Spencer et al. (1990), who showed that 50% of the Bialaphos-resistant plants tested expressed the nonselected gene encoding GUS. Register III et al. (1994) reported that a transgenc expression cassette was more likely to be rearranged if expression of that gene was not selected for during callus growth. Furthermore, not all plants regenerated from callus representing single transformation events expressed the transgenes, and a nonselectable gene (GUS) was expressed in fewer plants than was the selectable transgene. Figure 3 shows the presence of the GUS DNA fragment (750 bp) at the expected molecular weight in some of the putatively transgenic plants, indicating that these plants are transformed.

To confirm the integration of the GUS gene, genomic DNA from leaves of some of the putatively transformed plants and from the untransformed control plants were digested with BamHI and SacI and hybridized with the GUS probe. The number of inserted transgene copies was estimated by comparing the intensity of the band with that of one and 10 copies of the positive control. As illustrated in Fig. 44, the genomic DNA of the three samples 5, 6, and 7 contained a 1.87-kb GUS-hybridizing band that comigrated with the 1.87-kb fragment, containing the GUS from BamHI, SacI digestion of pAct1-F, while in samples 4 and 8 the GUS signal appeared at a lower molecular weight. Samples 3 and 9 had no GUS-hybridizing signals. These results indicate that the GUS gene is integrated into the genome of the five GUS-positive samples. Samples with the lower molecular weight fragments may result from rearranged copies of the GUS gene. The GUS gene was deleted in the GUS negative samples and absent in the control sample. Similar results have also been reported by Spencer et al. (1990) and Register III et al. (1994). The copy number of the transgenes in the different transformation events was estimated to be two to five copies.

FIG. 3. PCR-amplified DNA from pAct1-F (P), nontransformed (C), and putatively transformed maize plants (1-13); M, DNA molecular weight size marker.

FIG. 4. DNA hybridization analysis of putatively transgenic maize plants. A, Lanes 1 and 2, pAct1-F DNA representing 10 and one copies, respectively. Lanes 3 and 9, GUS-negative plants. Lanes 4- 8, GUS-positive plants. C, untransformed plants. B, Lanes 1 and 2, pTW-a DNA representing 10 and one copies, respectively. Lanes 3 and 9, Bar-negative plants. Lanes 4-8, Bar-positive plants. C, untransformed plants; M, DNA molecular weight size marker.

To confirm the integration of the Bar gene, genomic DNA from leaf samples (of the same transformed plants tested for the GUS gene integration) and from the untransformed control plant were digested with EcoRI. DNA hybridization analysis using the Bar probe revealed that the genomic DNA of five samples (4-8) gave a 0.9-kb Bar- hybridizing band that comigrated with the 0.9-kb fragment containing the Bar from EcoRI digestion of pTW-a (Fig. 4B). Samples 3 and 9 did not show the Bar-hybridizing signal. These results indicate that the Bar gene is integrated into the genome of the five Bar-positive samples, whereas the Bar gene is not integrated into the Bar- negative samples and was absent in the control sample.

Southern blot analysis has been commonly used to confirm the integration of the transgenes at the molecular level. Gordon-Kamm et al. (1990) recovered eight out of 11 PAT-positive events that contained intact copies of a Bar expression unit and the estimated copy number varied between one and two. Koziel et al. (1993) recovered one transformation event per 100 bombarded immature maize embryos. Songstad et al. (1996) bombarded 2.5 d cultured Hill immature embryos and the transformation frequency was 2%. Brettschneider et al. (1997) reported that all plants surviving the selection were positive and contained one or more copies of the PAT gene, and the transformation frequency ranged from 2 to 4% depending on the osmotic pretreatment and the applied bombardment parameters.

Results of this study show that a reproducible regeneration system for Egyptian maize inbred lines was successfully developed using the immature embryo explants. Among the lines studied, Gz 643 possesses the highest regeneration potentiality. Moreover, the conditions for maize transformation have been optimized. This may facilitate the production of novel transgenic maize plants by introduction of foreign genes conferring resistance to insect, fungal diseases, environmental stresses, and other commercially important traits into inbred lines.

ACKNOWLEDGMENTS

This work was supported by the Commercialization and Utilization of Biotechnology (CUB) collaborative research grant executed by the Agricultural Genetic Engineering Research Institute (AGERI) and the Agricultural Biotechnology Support Project (ABSP), Michigan State University (MSU), under the Agricultural Technology and Utilization Transfer (ATUT) Project (USAID grant no. 263-0240-G-00-6014-00).

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HANAIYA A. EL-ITRIBY1*, SHIREEN K. ASSEM1, EBTISSAM H. A. HUSSEIN1,2, FATHY M. ABDEL-GALIL3, AND MAGDY A. MADKOUR1

1 Agricultural Genetic Engineering Research Institute (AGERI), ARC, Giza, Egypt

2 Department of Genetics, Faculty of Agriculture, Cairo University, Giza, Egypt

3 Department of Biochemistry, Faculty of Science, Cairo University, Egypt

(Received 18 March 2002; accepted 5 March 2003; editor L. Herrera- Estrella)

* Author to whom correspondence should be addressed: Email hitriby@ ageri-sci.eg

Copyright Society for In Vitro Biology Sep/Oct 2003

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