Stability of inheritance of transgenes in maize (Zea mays L.) lines produced using different transformation methods

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1 Euphytica (2005) 144: DOI: /s C Springer 2005 Stability of inheritance of transgenes in maize (Zea mays L.) lines produced using different transformation methods Yongsheng Zhang, Xiaoyan Yin, Aifang Yang, Guosheng Li & Juren Zhang School of Life Science, Shandong University, Jinan , P.R. China ( author for correspondence; jrzhang@sdu.edu.cn) Received 30 April 2004; accepted 8 October 2004 Key words: maize, genetic transformation, transgene stability, comparison of transformation methods Summary The inheritance and stability of the acetolactate synthase (als) transgene were compared in transgenic maize plants, generated using the pollen-tube pathway, particle bombardment, or Agrobacterium-mediated methods of transformation. Progeny populations generated by successive selfing or backcrossing of primary transformants were analyzed over three generations, using PCR and herbicide screening, to examine segregation and als activity, respectively, and transgenic homozygous plants were selected. The pollen-tube method resulted in a higher rate of primary normal transgenic plants and a less-stable transmission of the als locus than did the other two methods. When transferred by the particle bombardment and Agrobacterium-mediated methods, the als gene was in a much higher proportion of Mendelian transmission than transferred by the pollen-tube method. Compared to the Agrobacterium-mediated transformation, the particle bombardment method tends to create multiple copies and insert sites of the als gene in maize genome, which delaying the homogenization of the als locus with advancing generations. Agrobacteriummediated transformation resulted in a greater proportion of stable, low copy number (in general 1 2) transgenic events, facilitating the stable inheritance of the als gene, and producing multiple desirable transgenic plants. Introduction Several transformation methods are currently available for the genetic transformation of crops. The principal methods used are particle bombardment and Agrobacterium-mediated transformation; these methods have been used to produce the majority of recently transformed crop lines (Komari et al., 1998). However, these techniques have certain disadvantages, especially with regard to the obligatory processes of in vitro culture and plant regeneration, which are time-consuming, complicated, and often result in phenotypic abnormalities and reduced fertility of the resulting transgenic plants. Transformation with a lesser-known method, the pollen-tube pathway, bypasses the regeneration of plants from callus and the problems associated with this step. First reported by Zhou et al. (1983) for cotton, and still controversial, this method has proven successful in crops such as cotton (Zhou et al., 1988; Huang et al., 1999), rice (Luo and Wu, 1988), wheat (Zeng et al., 1994; Chong et al., 1998), maize (Otha, 1986; Wang et al., 2001), and soybean (Hu & Wang, 1999). Optimized versions of this method would offer a system characterized by simplicity, reduced costs, and a low equipment requirement, and could be used for the transformation of plant varieties for which the regeneration of plants from callus is difficult. The success of plant genetic manipulation requires not only the ability to deliver functional DNA into the cell, but also that of producing multiple transgenic plants that stably inherit and express the transgenes. Many studies have analyzed the progeny of the primary transformants (Fromm et al., 1990; Register et al., 1994; Peng et al., 1995; Duan et al., 1996; Scott et al., 1998; Wu et al., 2002), which revealed that the inheritance and stability of the transgenes varied in the offspring. However, most of them concerned the particle bombardment and recently

2 12 Agrobacterium-mediated methods of transformation. There have been few similar reports on the pollen-tube method. One of the typical studies is that of Zeng et al. (1998), who analyzed the transgenic wheat obtained by the pollen-tube method over three generations. In most studies of other plant species that were treated using this method, only the T0 or T1 generation was examined. To best use transformation technologies in breeding programs, the differences in the results obtained using these methods must be examined, as well as the time required and the frequency of desirable transgenic plants produced. However, it is lack of experiments to deal with all these systems, especially for keeping other factors like target plant varieties and interest genes constant. In the project described herein, we examined successive generations of transgenic lines, produced using the three methods, to determine the long-term stability and heritability of transgenes in maize. A mutant herbicide-resistance acetolactate synthase gene (als), derived from Arabidopsis thaliana,was transferred into the maize inbred line, Qi319, using the pollen-tube method, particle bombardment, and Agrobacteriummediated transformation. The inheritance and stability of the als gene were compared through the T3 progeny of lines derived from the three methods, and their appropriateness for use in breeding programs was evaluated. The als gene was used as both a selectable marker and a target gene. Materials and methods Plant material, plasmids, and bacterial strain The maize elite inbred line, Qi319, was used for transformation. The plasmids used were p35s-als (7.7 kb) and CAMBIA1300-als (14 kb), which both contained the chlorsulfuron resistance gene als derived from a mutant A. thaliana line. The als gene was fused between the CaMV 35S promoter and the CaMV terminator. Purified plasmid DNA of p35s-als was used for transformation with the pollen-tube method and particle bombardment. A strain of A. tumefaciens LBA4404 harboring the mini-ti plasmid pcambia1300-als was used for Agrobacterium-mediated transformation. Maize transformation via the pollen-tube pathway Seeds of the maize Qi319 line were sown in early May. The ears of the plants were bagged before the silks appeared. Each morning, ears judged to be receptive to pollination that day were marked and prepared to be treated with an exogenous DNA solution. The styles were severed about 2 cm from the ear axis using sterile scissors, and 200 µl of plasmid DNA solution (100 µg ml 1 ; TE) were applied to the severed surfaces of the styles using a microsyringe. The treatments followed either procedure A, in which the DNA was applied just prior to self-pollination, or procedure B, in which the DNA was applied after 8 10 h of selfpollination. Several ears with severed styles were allowed to self-pollinate as controls. Mature seeds were harvested and sown in pots. Seedlings at the three-leaf stage were screened by spraying a 20 mg l 1 solution of the herbicide Luhuanglong (25% chlorsulfuron by weight; Shenyang Agricultural Chemical Company, Shenyang, China; Li et al., 2001), a concentration sufficient to eliminate non-transformants. Survivors were transplanted into the field and allowed to grow to maturity. Genomic DNA from leaves was examined by PCR and Southern blotting to confirm the plants as transgenic. Maize transformation via particle bombardment Maize immature embryos were aseptically isolated from the kernels of inbred line Qi319 and then cultured on modified MS medium plus 2 mg l 1 2,4-D to produce embryonic calli. The calli were then transferred onto fresh medium with unchanging compositions every 2 weeks for subculture (Li et al., 1990). The procedure of maize transformation was as described by Li et al. (2001). The parameters used for bombardment were as follows: the helium pressure was kpa; the vacuum was kpa; the gold powder size was 1.0 mm; the amounts of particles were 100 mg gold (167 ng DNA correspondingly) per shot; the number of calli per shot was 120 or so. The calli cultured for one generation after bombardment were selected for three subcultures on medium supplemented with 1 mg l 1 Luhuanglong. Then the herbicide-resistant calli were placed on hormone-free medium with the same concentration of herbicide for regeneration. Green shoots were transferred to root-boosting medium, and the plantlets with three to four leaves were transplanted to flowerpots. Maize transformation via the Agrobacterium-mediated method The embryogenic calli of inbred line Qi319 were produced as used in bombardment transformation. The

3 13 maize transformation procedure was as described by Quan et al. (2004). Maize calli inoculated with bacteria were cultured on MS medium plus 100 µmol l 1 acetosyringone in the dark for 3 days, and then rinsed three times with sterile water, cultured on modified MS medium for 15 days in darkness, and transferred to modified MS medium plus 1 mg l 1 Luhuanglong for three-generation selection. The herbicide-resistant calli was then transferred to hormone-free medium plus 1 mg l 1 Luhuanglong and regenerated into plantlet in the same way as described in bombardment transformation. Confirmation of transgenic plants by PCR and Southern blotting Total DNA was extracted from leaves using the CTAB method. The sequences of PCR primers for the als gene were as follows: P1, 5 -ACAGGAC- AAGTCTCTGGTCG-3 ; P2, 5 -GGGTTAGCAACA- GACGCT-3. The reaction conditions were as reported by Li et al. (2001). The PCR primers did not amplify maize endogenous als. Maize genomic DNA digested with EcoRI, which cleaves the plasmid DNA inside of the als gene and produces a 2.5-kb fragment, or with PstI, which cleaves the plasmid DNA at one site outside of the als gene, were used in the Southern blot analysis. The hybridization protocols were implemented according to the manufacturer s instructions of the DIG High Prime Labeling and Detection Starter Kit (Roche) as previously reported by Li et al. (2001). Analysis of progeny T0 transgenic plants produced using the three methods were self-pollinated or cross-pollinated with untransformed plants to construct families. Only the herbicideresistant and PCR-positive plants in one generation were chosen to produce lines analyzed in next generation. Multiple lines with sufficient individuals were tested using PCR and herbicide screening in each generation. Transmission of the als gene was confirmed using the PCR assay. The ratios of positive to negative plants in each line were compared to the expected Mendelian inheritance ratio using Chi-square analysis. Expression of the als gene was tested using an ALS activity assay, a foliar application of 20 mg l 1 Luhuanglong to three-leaf plantlets. Individuals were rated as resistant or susceptible after 14 days, and the data were analyzed using the Chi-square test. Results Stability of inheritance of the als gene in progeny of transgenic maize produced via the pollen-tube pathway In preliminary studies to develop the pollen-tube protocol, we optimized some parameters of maize transformation using this method, including purifying the plasmid DNA with the PEG protocol, applying 20 µg DNA per treated ear and treating the style after 8 10 h of self-pollination in procedure B, etc. The method proven to be successful in prior transformation was performed in this experiment. A total of 44 maize Qi319 ears were treated, half with procedure A and half with procedure B. Twenty-eight of the treated ears set seeds, but the number of seeds set was lower than that of normal, probably because of the higher humidity in the bag when dropping the solution on style. Over 1700 seeds of normal phenotype were obtained, and 1200 of these seeds were sown in pots for screening of the seedlings. Morphological variations, such as dwarf, were observed in some plants, but the variations did not occur in the subsequent generation. Foliar application of herbicide to three-leaf plantlets and subsequent analysis of survivors using PCR and Southern blotting identified 17 plants as transgenic. As seen in Figures 1 and 2, the band patterns correlating to transgenic plants using the PCR (lanes 1, 2, 6 8, Figure 1) and Southern blotting (lanes 4 7, Figure 2) were identical with that of plasmid DNA (lane 4, Figure 1; lane 2, Figure 2), with no signal from the non-transgenic plant (lane 5, Figure 1; lane 3, Figure 2). Sixteen of the 17 transgenic plants were transformed with procedure A and one from procedure B. All 17 of the transgenic plants were fertile, and the plants were carefully self-pollinated to obtain progeny. Figure 1. PCR analysis of T0 plants transformed using the pollentube method. Lanes 1, 2, 6 8, positive results of transgenic plants; Lane 3, λ/hindiii marker; Lane 4, result of plasmid DNA; Lane 5, negative result of non-transgenic plant.

4 14 Figure 2. Southern analysis of T0 plants transformed using the pollen-tube method. Lane 1, λ/hindiii marker; Lane 2, result of plasmid DNA/EcoRI; Lane 3, negative result of non-transgenic plant; Lanes 4 7, results of transgenic plants genomic DNA/EcoRI. The seeds of 16 T0 transgenic plants from procedure A were grown for examination of the T1 generation plants. As seen in Table 1, 12 of 16 lines inherited the als gene, but none of them segregated as Mendelian fashion. By PCR analysis, the proportions of the positive plants in each line varied from 30.2% (positive/negative = 13/30) in line QP4 to 6.7% (positive/negative = 6/83) in line QP6 (Table 2). Four lines, QP3, QP7, QP10 and QP14, have lost the als gene. After herbicide screening, about 14.3% of the T1 plants were resistant, in which the majority were positive in PCR assay. In T1 generation, a few resistant plants were confirmed as negative by PCR reaction (Table 2). It is presumed that some of these plants were escape events from the herbicide screening, despite the severe screening process used in the experiments. In the T2 generation, a total of 65 lines from 12 primary transformants were tested (Table 1). The PCR assay revealed that 47 lines carried the als gene, with the highest frequency of positive plants in line QP8-3 (64.4%; positive/negative = 38/21) and the lowest in line QP12-2 (10.9%; positive/negative = 6/49) (Table 2). A greater proportion of the QP8 and QP5 populations were transgenic, as compared to the other lines. A Chi-square test showed that the segregation of the als gene in line QP8-3 was in a 3:1 Mendelian ratio (Table 2), confirmed by herbicide screening, indicating that line QP8-3 is probably a stable transgenic line. After herbicide treatment, about 27% of the T2 plants were herbicide resistant. All of the plants from 15 lines died after herbicide screening. Plants of T3 generation from 115 lines were examined as described earlier (Table 1). Out of 115 lines screened, 88 lines, mainly derived from QP4, QP5, QP8, and QP9, carried the als gene. A Chi-square test showed that in the QP4 line, the als gene segregated at a 3:1 Mendelian ratio in lines QP4-3-1, QP4-3-3 and QP4-4-1 (Table 2). Seven T3 lines of QB8-3, for example lines QP8-3-3 and QP8-3-4 (Table 2), showed uniformly positive in PCR assay, which was confirmed by herbicide screening. This result confirmed that line QP8-3 is a stable transgenic line, in which several transgenic homozygous plants were obtained. In three lines of the QP5 line, more than 50% of the individuals carried the als gene, but none had simple segregation ratios (line QP5-5-2 as example, Table 2). Similarly, more than 40% of the plants, in five lines derived from the QP9 line, contained the als gene, but in none did the gene segregate according to the expected ratio (line OP9-1-2 as example, Table 2). By herbicide screening, the proportion of resistant plants averaged 51% in T3 generation. Based on the earlier analysis, 20 transgenic lines in T4 generation, derived from QP8-3 and QP4-3, were developed to evaluate the herbicide tolerance. The three-leaf plantlets were sprayed with varying concentrations of herbicide. The result of this assay demonstrated that the herbicide tolerance of transgenic plantlets varied with the concentration of herbicide (data not shown). With 20 mg l 1 of herbicide, for example, less than 70% of the plants in three lines were herbicide resistant, seven lines were uniformly resistant, and resistant individuals were all confirmed to contain the als gene by Southern analysis. However, with a treatment of 40 mg l 1 of herbicide, plants from only four lines showed resistance. Stability of inheritance of the als gene in progeny of transgenic maize generated by particle bombardment A total of 135 regenerated plants were obtained using particle bombardment in this experiment, and 21 were confirmed to be transgenic using PCR and Southern blotting. Figure 3 shows the result of PCR assay, in which the expected bands of transgenic plants appeared in lanes 2 7. The plants that survived transplantation were generally unhealthy and displayed reduced fertility. Some of the plants were significantly shorter, flowered later, and were partially sterile, as compared to non-transgenic controls. Morphologically abnormal flowers that failed to set seeds were also observed on several plants. The inheritance and stability of the als gene delivered using this method were analyzed in the progeny of five primary transgenic plants:

5 15 Table 1. Comparison of three transformation methods by segregation analysis of the als gene up to three generations a Pollen-tube pathway Particle bombardment Agrobacterium-mediated transformation LA LM LA LM LA LM NL No. % No. % NL No. % No. % NL No. % No. % T1 generation PCR analysis Herbicide screening T2 generation PCR analysis Herbicide screening T3 generation PCR analysis Herbicide screening a NL number of lines screened; LA lines inheriting the als gene; LM lines inheriting the als gene according to Mendelian fashion, including the lines separating as Mendelian fashion and that of uniformly transgenic; No. numbers of corresponding lines; % percent to number of lines screened.

6 16 Table 2. PCR analysis and herbicide screening of some progenies of a few transgenic plants obtained by the pollen-tube transformation method a PCR analysis Herbicide screening Generation Line Positive/negative Expected ratio χ 2 Resistant/susceptible Expected ratio χ 2 T1 QP4 13/30 3: /30 3: QP5 9/39 3: /37 3: QP6 6/83 3: /83 3: QP8 11/31 3: /30 3: QP9 4/24 3: /23 3: T2 QP4-3 26/27 3: /26 3: QP5-5 8/34 3: /33 3: QP8-3 38/21 3: /21 3: QP9-1 16/31 3: /31 3: QP12-2 6/49 3: /49 3: T3 QP /19 3: /18 3: QP /20 3: /18 3: QP /13 3: /13 3: QP /32 3: /31 3: QP /0 39:0 39/0 39:0 QP /0 44:0 44/0 44:0 QP /38 3: /37 3: a With one degree of freedom; χ = 6.64; χ = 3.84; χ = 2.71; χ = Figure 3. PCR analysis of T0 plants transformed using particle bombardment. Lane 1, λ/hindiii marker; Lanes 2 7, positive results of transgenic plants; Lane 8, negative result of non-transgenic plant; Lane 9, result of plasmid DNA. QB29, QB68, QB105, QB10, and QB28. To supply information about the copy number and insertion sites of als in the host genome, Southern blot analysis was performed by digesting genomic DNA of T0 transgenic plants and some T1 progeny with PstI. Here results of the QB29, QB68 lines were shown as example. Five lines in T1 generation, obtained by backcrossing the transgenic T0 plants with non-transgenic Qi319 plants, were examined by PCR and herbicide screening. As shown in Table 1, all five lines inherited the als gene, but only the QB29 T1 plants were indicated segregating as Mendelian fashion by Chi-square test (Table 3). In Southern blots, the banding patterns of the QB29 (lane 2, Figure 4) and T1 plants (lanes 1 5, Figure 5) indicated that the three copies of the als gene are inherited as a single dominant locus. For the QB68 line, Southern analysis of QB68 (lane 3, Figure 4) and T1 plants (lanes 2 7, Figure 6) revealed three als loci in the T0 plant. However, a Chi-square test showed that the T1 plants deviated significantly from the expected ratio of 7:1 (Table 3). For the QB105 line, the T1 plants deviated the expected Mendelian ratio (Table 3). PCR analysis showed that relatively fewer T1 plants of QB10 and QB28 contained the als gene (Table 3). In T2 generation, all lines examined carried the als gene, in which majority were inherited as Mendelian fashion (Table 1). The T2 plants of QB29 line showed following Mendelian segregation fashion (line QB29-11 as example, Table 3). In the QB68 T2 plants, PCR analysis confirmed the inheritance of the als gene as a single dominant locus. However, in line QB68-7 (Table 3), the ratio of herbicide-resistant plants was lower than expected, possibly because of a tandem-repeat als locus

7 17 Table 3. PCR analysis and herbicide screening of some progenies of a few transgenic plants obtained by particle bombardment method a PCR analysis Herbicide screening Generation Line Positive/negative Expected ratio χ 2 Resistant/susceptible Expected ratio χ 2 T1 QB29 10/18 1: /18 1: QB68 29/18 7: /32 7: QB105 25/16 36/39 QB10 21/40 13/48 QB28 9/6 9/6 T2 QB /10 3: /14 3: QB /13 3: /45 3: QB /10 3: /21 3: QB /25 1: /35 1: QB /34 3: /43 3: QB /23 1: /25 1: T3 QB /11 3: /18 3: QB /0 29:0 48/0 48:0 QB /9 3: /12 3: QB /0 25:0 51/0 51:0 QB /0 33:0 48/0 48:0 QB /12 3: /12 3: QB /0 35:0 43/0 43:0 QB /0 29:0 46/0 46:0 a With one degree of freedom; χ = 6.64; χ = 3.84; χ = 2.71; χ = Figure 4. Southern analysis of genomic DNA of QB29, QB68, and QB105 digested with PstI. Lane 1, result of plasmid DNA/PstI; Lane 2, result of QB29; Lane 3, result of QB68; Lane 4, result of QB105. Figure 5. Southern analysis of genomic DNA of T1 plants of line QB29 digested with PstI. Lane 1, result of QB29-14; Lane 2, result of QB29-13; Lane 3, result of QB29-11; Lane 4, result of QB29-6; Lane 5, result of QB29-3.

8 18 Figure 6. Southern analysis of genomic DNA of T1 plants of line Q68 digested with PstI. Lane 1, result of plasmid DNA/PstI; Lane 2, result of QB68-17; Lane 3, result of QB68-12; Lane 4, result of QB68-11; Lane 5, result of QB68-7; Lane 6, result of QB68-3; Lane 7, result of QB68-2. (corresponding to the 7.7-kb fragment, Figure 6). For the QB105 and QB10 lines, the als gene segregated in a Mendelian fashion in T2 lines (lines QB and QB10-2 as example, Table 3). Among the progeny of QB28, for example, line QB28-2 showed no Mendelian segregation pattern (Table 3). But in line QB28-6, the plants containing and lacking the als gene occurred in the expected 1:1 ratio (Table 3). The T3-generation lines were also examined as mentioned earlier, and the majority of them inherited the als gene following the Mendelian fashion (Table 1). In QB29 T3 plants, analysis of line QB gave results similar to those obtained with T2 plants (Table 3). In the QB68 line, the results of T3 generation revealed four corresponding T2 plants as transgenic heterozygous, for example QB (Table 3). Moreover, QB and QB were confirmed as transgenic homozygous plants by analysis of T3 populations (Table 3). In addition, among the QB105 progeny, for example, QB was transgenic heterozygotes, and QB was transgenic homozygotes (Table 3). Similarly, QB and QB were found to be transgenic homozygotes (Table 3). By spraying three-leaf plantlets with the herbicide of 40 mg l 1, most transgenic homozygous plantlets grew normally, but the transgenic heterozygous plants were killed (data not shown). the long tissue-culture periods required for these methods. Plants that appeared healthy after being transplanted into the field were subjected to PCR and Southern blotting analysis. Out of 78 plants, 19 were confirmed as transgenic. The expected patterns of transgenic plants are shown in Figure 7 (lanes 2 8, PCR assay) and in Figure 8 (lanes 4 8, Southern blotting). Mature seeds were harvested from 16 transgenic plants, which were then self-bred or backcrossed with nontransformed plants. In the T1 generation, 16 independently derived transgenic lines were developed, of which 11 lines (QN1 QN11) were self-bred progeny and five (QN12 QN16) were backcrossed progeny. The T1 plants of most lines were more vigorous than the parents, although some plants of line QN5 had an abnormal morphology of leaves with fading stripes, and a few did not produce progeny. As seen in Table 1, 12 lines, Figure 7. PCR analysis of T0 plants transformed using Agrobacterium-mediated method. Lane 1, λ/hindiii marker; Lanes 2 8, positive results of transgenic plants; Lane 9, negative result of non-transgenic plant; Lane 10, result of plasmid DNA. Stability of inheritance of the als gene in progeny of transgenic maize produced via Agrobacterium-mediated transformation Plants regenerated from callus infected with Agrobacterium generally gave results similar to plants produced by particle bombardment, suggesting that phenotypic abnormalities and reduced fertility are mainly due to Figure 8. Southern analysis of T0 plants transformed using Agrobacterium-mediated method. Lane 1, DL15000 marker; Lane 2, plasmid DNA/PstI; Lane 3, negative result of non-transgenic plant; Lanes 4 8, results of transgenic plants genomic DNA/PstI.

9 19 Table 4. PCR analysis and herbicide screening of some progenies of a few transgenic plants obtained by Agrobacterium-mediated transformation a PCR analysis Herbicide screening Generation Line Positive/negative Expected ratio χ 2 Resistant/susceptible Expected ratio χ 2 T1 QN1 5/24 3: /24 3: QN2 27/3 15: /3 15: QN4 20/6 3: /6 3: QN5 17/11 3: /11 3: QN13 16/12 1: /12 1: T2 QN1-1 28/10 3: /10 3: QN2-1 16/13 3: /13 3: QN4-1 25/0 25:0 25/0 25:0 QN8-1 29/0 29:0 29/0 29:0 QN /6 3: /6 3: T3 QN /0 25:0 25/0 25/0 QN /0 19:0 19/0 19/0 QN /7 3: /7 3: QN /0 18:0 18/0 18/0 QN /14 3: /14 3: QN /0 24:0 24/0 24/0 a With one degree of freedom; χ = 6.64; χ = 3.84; χ = 2.71; χ = ofthe 11 self-bred lines and three of the five backcrossed generation lines, carried the als gene. Four T0 transgenic plants had lost the als gene during sexual reproduction, probably because they were transgenic chimerisms. The transgenic lines QN4, QN5, QN13 (Table 4) and four other lines each showed a Mendelian segregation pattern indicative of a single dominant locus, and the segregation pattern of the QN2 line (Table 4) suggested that it contained two unlinked loci. In the rest of the lines, such as line QN1 (Table 4), the als gene did not segregate in a Mendelian fashion. Treatment of three-leaf plantlets with herbicide revealed resistant individuals in all lines, with varying frequencies. Moreover, all PCR-positive plants were herbicide resistant. Several of the T1 transgenic plants with normal appearance were self-bred to obtain mature seeds. In the T2 generation, a total of 34 lines were tested (Table 1), of which 25 were derived from self-bred progeny and nine from backcrossed progeny of the T0 plants. By PCR analysis, plants from 27 lines were confirmed carrying the als gene with varying frequencies, and plants from the other seven lines, such as lines QN4-1 and QN8-1 (Table 4), which were all self-bred offspring of the T0 plants, were confirmed as uniformly transgenic. Sixteen of the 27 segregating lines, such as lines QN1-1 and QN13-1 (Table 4), segregated in a 3:1 Mendelian ratio. After herbicide treatment, all PCR positive plants were confirmed as resistant. Overall, these results with the T2 generation further confirmed the predictions derived from the Mendelian segregation of the corresponding T1 lines, and several transgenic T1 homozygous plants were identified. Thus, in general, a low number of copies of the als gene (1 2) were inserted in the T0 transformants obtained using the Agrobacterium transformation method, which is also shown in the pattern of Southern blot (Figure 8). A portion of T2-generation transgenic plants was selected to undergo progeny analysis in the T3 generation (Table 1). Out of 60 lines, the plants of 24 (40%) lines were all PCR positive, such as lines QN1-1-2, QN4-1-1, QN6-1-1 and QN (Table 4); the plants of 32 (53.3%) lines showed Mendelian segregation of the als gene, such as line QN5-1-2 (Table 4), and the plants of four (6.7%) lines segregated in a pattern that deviated from the Mendelian ratio, such as line QN (Table 4). Thus, about 40% of the T3 plants were transgenic homozygous plants. These plants were treated with different concentrations of the Luhuanglong herbicide to evaluate the herbicide tolerance. The resistance of the transgenic plants from different lines varied, presumably due to differences derived from the original T0 plants (data not shown).

10 20 The result showed that maize inbred lines, tolerant of up to 50 mg l 1 of herbicide, were obtained using Agrobacterium-mediated transformation. Discussion The aim of the present investigation was to evaluate the inheritance and stability of transgenes transferred by the pollen-tube pathway, particle bombardment, and Agrobacterium-mediated transformation. The latter two methods have been reported to work successfully with some maize genotypes (Register et al., 1994; Ishida et al., 1996; Bohorova et al., 1999; Li et al., 2001; Zhao et al., 2001; Frame et al., 2002), but few reports have been published on maize transformation using the pollen-tube pathway. Several pioneer works by Otha (1986) and Wang et al. (2001) have reported that transformation could be achieved using a mixture of pollen and exogenous DNA. However, in successful transformations of rice (Luo & Wu, 1988), wheat (Zeng et al., 1994; Chong et al., 1998), soybean (Hu &Wang, 1999) and cotton (Zhou et al., 1988; Huang et al., 1999), foreign DNA was applied to cut styles shortly after pollination. In the present study, therefore, both methods (procedures A and B) were used. From the results, the als gene was transferred using both procedures, but procedure A created a higher rate of transgenic plants than procedure B. The transgenic plants derived from the pollen-tube method developed normally, flowered, and in most cases were indistinguishable from non-transgenic plants, whereas those produced by particle bombardment or Agrobacteriummediated transformation often showed phenotypic abnormalities and reduced fertility. Therefore, fertile primary transgenic plants are easier to obtain using the pollen-tube method than the other two methods. With all of these transformation methods, the input exogenous DNA is maintained in transgenic plants by integration into the host genome, which could be proved with the observation of Mendelian segregation ratios in subsequent sexual generations. In the present study, the inheritance of the als gene was analyzed over three generations of plants, generated using the three different methods. Both Mendelian and unexpected segregation ratios of the als locus were observed in each population, and transgenic homozygous plants were obtained with varying proportions. In the population derived from the pollen-tube method, only four lines showed a 3:1 Mendelian segregation ratio, including one T2 line (line QP8-3) and three T3 lines (lines QP4-3-1, QP4-3-3 and QP4-4-1). Seven T3 lines from QP8-3 were confirmed as uniformly transgenic. In other lines, the number of transgenic plants was lower than expected. Transgenic homozygous plants were obtained in the T2 line of QP8-3. Zeng et al. (1998) reported no evidence of Mendelian inheritance of marker genes in progeny of transgenic wheat obtained using the pollen-tube method, but the population tested was far smaller than that used in this study. Wang et al. (2001) only examined the T1 progeny of transgenic maize generated via this method, giving no information of the transgene inheritance over generations. In lines generated using the particle bombardment method, one line (line QB29, 1/5 of the T1 lines) exhibited a one-locus Mendelian segregation pattern in the T1 generation. However, after successive selfing or backcrossing, the majority of the T2 lines segregated in the expected ratio. Transgenic homozygous plants were identified in the T2 generation of the QB68, QB10, QB28, and QB105 lines. In the population generated by Agrobacterium-mediated transformation, by comparison, eight (1/2 of the T1 lines) segregated as expected ratio in the T1 generation. About half of the T2 lines inherited the als gene as Mendelian fashion. Seven T1 plants were identified as transgenic homozygous plants by progeny analysis. Therefore, the transmission of als gene introduced via the pollen-tube method did not follow a Mendelian fashion in most cases, which contrast to the most simple transmission of als gene introduced via the other two methods, especially in later generations. Moreover, the phenomena, the poor als gene transmission and disagreement between herbicide and PCR response, were observed in population obtained using the pollen-tube method. So it is presumed that these effects be related to this special approach to some extent. However, a screen of a large population of offspring produced a stable transgenic line of QP8-3, even in the lowest proportion, indicating that the als gene delivered by this method could be inherited as expected once it occurs stably in the host genome. It is thereby necessary to elucidate the underling mechanisms of this novel transformation method to alleviate some of these problems. In the present study, segregation analysis showed that the als copy number was generally lowest (about 1 2) when transgenic plants were generated using the Agrobacterium-mediated method. Comparatively, different plasmid DNA insertion patterns were revealed by Southern blot analysis in lines created using particle bombardment, but there are few als loci. One possible explanation for this effect is that the als locus

11 21 functioned as the selectable marker as well as the target gene. However, the various als insertion patterns yet contributed to the abnormal segregation in several lines, such as in lines QB68, QB105 and QB28-2 (Table 3), and the production of transgenic homozygous plants, which appeared later and in lower numbers than that of Agrobacterium-mediated transformation. Moreover, tandemly repeated transgenes, appeared in T0 plant of QB68 and the progeny, resulted in the inactivation of the als gene in some transgenic plants, such as in line QB68-7 (Table 3). The use of maize transformation in breeding programs requires the production of multiple transgenic homozygous plants that display stable transgene inheritance. As shown in this report, the early steps of the pollen-tube method are simple, but the screening of large populations of offspring was required to obtain sufficient numbers of acceptable transformants. 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