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					Journal of Genetics and Genomics (Formerly Acta Genetica Sinica) August 2007, 34(8): 749-755

Research Article

Selection of Maize Inbred Lines with High Regeneration and Susceptibility to Agrobacterium tumifacien
Yu Wang1, 2, Shaohong Fu3, Ying Wen2, Zhiming Zhang1, Yanli Xia1, Yuzhen Liu1, Tingzhao Rong1, Guangtang Pan1,
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1. Institute of Maize Genetics and Breeding, Sichuan Agricultural University, Ya’an 625014, China; 2. Research Institute of Sugar Crops in Sichuan Province, Zizhong 641200, China; 3. The 2nd Agricultural Science Rresearch Institute of Chengdu, Wenjiang 611130, China

Abstract: Ten-maize inbred lines of maize (Zea mays L.) with high-induction rate and proliferation ability of embryonic calli were selected from 70-maize inbred lines by immature embryo culturing. Some of the embryonic calli were transferred onto regeneration medium to examine the ability of regeneration, some were transformed via Agrobacterium tumifaciens C58 carrying intron-β-glucuronidase (gus) gene, and GV3301 carrying the green fluorescent protein (gfp) gene to study the susceptibility of different genotypes in maize to A. tumifaciens. All embryonic calli initiated from 10-maize inbred lines were able to regenerate into plantlets, and the regeneration frequencies of inbred lines 6010, 6038, 6015, 6051, and 6060 were 61.11%, 31.94%, 45%, 33.33%, and 56.94%, respectively, which were higher than that of other lines. Analysis of variance indicated that the susceptibility of the various genotypes in maize to A. tumifacien C58 showed a significant difference among each other, and the inbred lines 6010, 6015, 6051, 6050, 6058, 6060, 6069, 6077 were susceptible to A. tumifacien C58, of which frequency of gus expression were over 70%. Expression of GFP was observed in six-inbred lines (6050, 6015, 6051, 6058, 6069, 6077). The inbred lines 6051, 6010, 6015, 6060, and 6050 had the high regeneration and the susceptibility to A. tumifaciens C58; and the inbred lines 6051, 6015, and 6060 had the high regeneration and the susceptibility to Agrobacterium tumifaciens GV3301. Keywords: maize inbred lines; embryogenic calli; Agrobacterium tumifacien; gfp; gus

Genetic engineering of the important cereal crops such as barley (Hordeum vulgare L.), maize (Zea mays L.), oat (Avena sativa L.), rice (Oryza sativa L.), sorghum (authorization), and wheat (Triticum aestivum L.) have been achieved using major antibiotic and herbicide resistance genes as selectable markers [1−3]. Novel selected markers have been developed to increase the efficiency and flexibility of

cereal transformation and to overcome drawbacks sometimes associated with the use of certain makers. Transgenes encoding glucuronidase (GUS) has been the most widely used visual reporter in cereal transformation systems. Recently, green fluorescent protein (gfp) gene, isolated from the jellyfish, was used as a useful reporter gene for analysis of gene expression and for visual screening of transgenic cells. Ge-

Received: 2006−09−26; Accepted: 2006−12−03 This work was supported by the National Natural Sciences Foundation of China (No. 30370889), the Program for Changjiang Scholars and Innovative Research Team in University of China (No. IRT0453), Beijing Agricultural Innovative Platform-Beijing Natural Science Fund Program, the National High-tech R&D Program of China (No. 2006 AA100103), the National Key Technologies R&D Program (No. 2006 BAD01A03), and the Program of the National Ministry of Agriculture (No. 2003-Q03). ① Corresponding author. E-mail: pangt@ sicau.edu.cn; Tel: +86-835-288 2714 www.jgenetgenomics.org

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netic transformation of plants by the gram-negative soil bacterium A. tumifaciens is the only known example of inter-kingdom genetics exchange. Although great advances have been made over the past decade in increasing the number of plant species that can be transformed and regenerated using Agrobacterium by altering the bacterium, many important species or inbred lines remain highly recalcitrant to Agrobacterium-mediated transformation because of changing culture condition of recipient and optimizing system of transformation. The objectives of this research were to identify maize inbred lines with high-plant regeneration ability and susceptibility to A. tumifacien and to research the application of gus and gfp as visual reporters in screening and monitoring of maize transformants.

system. This vector carries genes htp and nptII conferring hygromycin resistance (HR) and kanamycin resistance (KR), respectively. Agrobacterium C58 (pkyl71) was grown in agitated (200 r/min) liquid YEB medium supplemented with 50 mg/L rifampicin, and 100 mg/L kanamycin for 14 h at 28℃. Agrobacterium GV3301 (pCambia1302) was grown in agitated (200 r/min) liquid YEB medium supplemented with 50 mg/L rifampicin, 100 mg/L kanamycin, and 50 mg/L gentamycin for 14 h at 28℃ The OD600 was measured and adjusted to a final optical density of 0.5–0.7 (approximately 0.5109–1.2109 cfu/mL). 1.3 Plant transformation

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1.1

Materials and Methods
Plant materials

Seventy-maize inbred lines maintained at the Maize Research Institute of Sichuan Agricultural University were used in this study. Immature zygotic embryos of maize inbred lines measuring 1.8–2.2 mm in length were aseptically dissected from field-grown ears harvested 11–13 days post pollination, which were cultured embryo-axis side down on N6-based medium at 28℃ in darkness. Friable and embryogenic calli were isolated and subcultured onto fresh maintenance medium at an interval of 21 days. To ensure the reliability of results, over 200 immature zygotic embryos of each inbred line were inoculated. Embryonic calli were transferred onto MS regeneration medium to produce plantlets after the third subculture. 1.2 Bacterial strains and vectors The C58 strain of A. tumifaciens consists of plasmid pkyl71 with gus-cct1 (the C termini Arabidopsis cry1 gene), nptⅡgene conferring resistance to kanamycin, and a β-glucuronidase gene bearing a portable intron. The strain GV3301 carrying plasmid pCambia1302 and 35S-gfp were used as the vector

Friable and embryogenic calli were placed in a vacuum chamber with the suspension of Agrobacterium medium containing 100 μmol/L acetyl syringone. Vacuum (–80 Pa) was applied for 10 min, and then released slowly, finally, calli were blotted dry on sterile filter paper and incubated on cocultivation medium. 1.4 Microscopic observation of GUS assay and GFP activity

After a 3-day cocultivation at 24℃ in dark, calli were washed thrice with sterile distilled water for 20 min and blotted dry on sterile filter paper, and then were used to assay GUS histochemically as described by Jefferson (reference). In brief, GUS staining was carried out by immersing embryonic calli or plantlets in the GUS staining solution containing 1 mmol/L of 5-bromo-4-chloro-3-indoyl glucuronide (X-Gluc) (Duchefa, Haarlem, the Netherlands), 0.1% Triton X-100, 0.5 mmol/L K3Fe(CN)6, 0.5 mmol/L K4Fe (CN)6, and 100 mmol/L sodium phosphate buffer pH 7.0. Samples were incubated overnight at 37℃. The mixture on which GUS assay was carried out was then removed, and tissues were rinsed twice with 70% ethanol. Tissues were examined under a binocular microscope. GFP activity was directly observed by Nikon fluorescence microscope, after a 3-day cocultivation period at 24℃ in dark, the embryonic calli
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were assayed by gfp transient expression through the Nikon-80i fluorescence microscope in the wavelength 330–380 nm scope.

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2.1

Results
Induction of embryonic calli

In plant genetic engineering, the highly efficient regeneration system is an important condition for successful transformation. Numerous studies on genetic transformation of maize proved that embryonic calli induced from immature embryo are perfect recipient. To select maize inbred lines that could induce embryonic calli, immature zygotic embryos of seventy-maize inbred lines were cultured on N6-based medium. Ten-maize inbred lines that produced embryonic calli were finally selected, and the inductionTable 1 Embryonic calli induction and plant regeneration
Efficiency of embryonic calli induction (%) 33.08 (260) 90.59 (170) 9.52 (210) 85.64 (390) 62.86 (280) 64.62 (260) 44 (200) 16.86 (350) 71.48 (270) 27.31 (260)

rate of embryonic calli was calculated approximately 40 days after culture initiation. The ten-maize inbred lines produced embryonic calli, and the induction rate of embryonic calli of maize inbred lines 6015, 6038, 6050, 6051, and 6069 were over 50%, and the proliferation ability of embryonic calli of the inbred lines 6015, 6038, 6050, 6051, 6069, and 6077 were very strong. Although induction rate of embryonic calli of the inbred line 6077 was low (27.31%), the proliferation ability was high (Table 1). After two months of culture, embryonic calli were transferred onto regeneration medium to generate plantlets. The results of plant regeneration indicated that the ten-maize inbred lines could regenerate into plantlets (Fig. 1), and the regeneration frequency of the inbred lines 6010, 6060, 6038, 6051, and 6015 were relatively higher than other lines.

Genotype of explants 6010 6015 6034 6038 6050 6051 6058 6060 6069 6077

Proliferation ability of embryonic calli (tube) 1 3 0.5 3 2 4 1 1 3 2

Regeneration rate (%) 61.11 (72) 31.94 (72) 16.67 (72) 45.00 (60) 27.78 (72) 33.33 (72) 16.67 (72) 56.94 (72) 8.33 (72) 8.33 (72)

Embryonic calli induction efficiency = (No. of embryonic calli/No. of embryo inoculated) × 100%. Proliferation ability of embryonic calli: the tube number of embryonic calli obtained from a tube embryonic calli after one subculture. No. of regeneration plantlets: the number of the regenerating plantlets which were above 5 cm in height. Total number of calli tested is given in parentheses.

Fig. 1

Induction and differentiation of embryonic (typeⅡ) calli

1: Embryonic (typeⅡ) calli; 2: Regenerating calli; 3: Regeneration plantlets.

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2.2

Genotypes with susceptibility to A. tumifacien C58

showed a high expression in calli and buds differentiated from calli (Fig. 2, 2−4). There was no blue stain in the control calli. The degree of GUS expression was not uniform in different lines, some showed strong expression and others weak expression. 2.3 Genotypes with susceptibility to A. tumifacien GV3301

The expression of the GUS-intron gene as detected by GUS color staining is a reliable indicator of plant transformation, as the GUS-intron gene can express efficiently in plant cells but not in Agrobacterium. A histochemical GUS assay was carried out after a period of 3-day cocultivation at 24℃ in dark. The frequency of the gus expression in the different genotypes indicated that the susceptibility of different genotypes in maize to A. tumifacien C58 varied. Variance analysis indicated that the gus expression varied significantly in different genotypes (Table 2). The gus expression frequency of the inbred lines 6034 and 6038 (8.62% and 11.11%, respectively) were significantly lower than other lines, indicating that the inbred lines 6034 and 6038 had no susceptibility to A. tumifacien C58. The frequency of gus expression in other genotypes were over 70%. Maize inbred lines 6051, 6010, 6015, 6060, and 6050 were considered to be gene acceptor materials, because they have stronger reproducibility and were susceptible to A. tumifacien C58. There was no blue stain in calli from the maize inbred lines 6034 and 6038, but the blue stain could be observed in the other 8 genotypes of calli (Fig. 2). After two subcultures, the inbred lines in which a high expression of the GUS gene was detected in calli still
Table 2

When observed under fluorescence microscope, gfp-expressing cells could be easily distinguished from nontransformed, control cells, because of the presence of green stripe or spot on expressing cells of calli. To determine if these fluorescence expression was stable, gfp-expressing cells that produced green spot were isolated and placed onto the subculture medium for culture to proceed. The result showed that gfp expression could be detected on calli under the fluorescence microscope after a 2–3 weeks subculture period (Fig. 3). Different gfp expression rate in the 10-inbred lines indicated that the susceptibility of different genotypes in maize to A. tumifacien GV3301 varied (Table 3). Expression of GFP were detected in lines 6051, 6015, 6060, 6069, 6058, and 6077, but not in lines 6038, 6034, 6050, and 6010. This showed that the inbred lines 6038, 6034, 6050, and 6010 were susceptible to A. tumifacien GV3301. Maize inbred lines 6051, 6015, and 6060 were considered as gene acceptor material, because they had high regeneration and were susceptible to A. tumifacien GV3301.

Sensitivity of different genotypes in maize embryonic calli to A. tumifacien C58
Experiment 1 22(30) 30(40) 2(38) 2(25) 20(30) 30(34) 21(32) 15(25) 20(30) 26(30) No. of calli with GUS activity Experiment 2 28(32) 34(36) 3(38) 3(25) 23(35) 40(44) 30(34) 20(25) 26(32) 24(32) Experiment 3 30(34) 36(40) 5(40) 3(22) 28(35) 32(34) 24(30) 17(20) 29(38) 24(30) Mean GUS activity as % of calli tested 84.21a 86.20a 8.62b 11.11b 71a 91.07a 78.13a 74.28a 75a 80.43a

Genotype of explants 6010 6015 6034 6038 6050 6051 6058 6060 6069 6077

Mean GUS expression frequency in the table indicates the average GUS transient expression frequency of three experiments. Means followed by the same letter were not significantly different at 1% level. Total number of calli tested is given in parentheses. www.jgenetgenomics.org

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Fig. 2 Histochemical GUS assay 1: GUS expression in different genotypes transformed calli tissue; 2: GUS expression in untransformed control calli tissue; 3: gus transient expression in transformed calli tissue; 4: gus transient expression in transformed calli tissue and bud.

Fig. 3 GFP transient expression 1 and 2: The embryonic calli transformed was assayed by GFP transient expression; The green spot was observed on the embryonic calli transformed by fluorescence microscope.

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Table 3

Fluorescence assay of embryonic calli transformed by A. tumifacien GV3301
No. of calli tested 45 40 34 56 42 45 45 48 44 40 No. of calli with GFP activity 18 0 0 15 9 0 13 0 7 11 Frequency of gfp transient expression 40 0 0 26.79 21.43 0 28.89 0 15.51 27.50 6051 6038 6034 6015 6060 6050 6069 6010 6058 6077

Genotype of explants

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Discussion

Since the isolation of jellyfish gfp gene, this gene has been widely used in plants, microorganism, and animals for the research in molecular biology. Conventionally, to analyze expression of gene, such as GUS gene, NPT gene, additional substrate must be added to the tissue to be analyzed or extract the enzymes encoded by the genes from the tissues or transformants. These treatments will damage the tissues or transformants. However, if gfp gene was used as a selectable marker in primary selection of transformed tissue, transgenic tissue can be efficiently obtained without the use of an antibiotic resistance gene, and the expression of gene can be observed directly in living cells without damaging the tissues or transformants, and the putatively transformant can be isolated and further selected. Application of this selection system may improve transformation efficiency, reduce the time of selection, and diminish the concerns often raised in relation to the use of antibiotic or herbicide resistance marker genes in transgenic plants. In this experiment, we found that certain genotypes of maize inbred lines were exactly highly recalcitrant to Agrobacterium-mediated transformation and to regeneration. In this study, 10-maize inbred lines that could induce embryonic calli were selected and regenerated into plantlets from 70-maize inbred lines. Finally, inbred lines 6051, 6010, 6015, 6060, and 6050 that were susceptible to Agrobacterium C58 with high-regeneration potential, and 6051, 6015, and 6060 that were susceptible to Agrobacterium GV3301

with high-regeneration ability were selected. The degree of GUS expression was not uniform in different lines, some showed strong expression and others showed weak expression. This might be due to individual inbred line having a different number of transgenic copies [4]. Prols and Meyer et al. [5−7] also reported that different expression intensities could be due to methylation of the chromosomal integration region. Differences in cell size, metabolic activity, and penetration of staining substrate have also been reported to result in the differences in color intensity [8]. So in this study, only the embryonic calli with blue stain but not intensities of expression were considered. It was concluded that plant regeneration ability and genotype are major limiting factors in Agrobacterium-mediated transformation. To ensure the successful transformation, recipient genotypes prior to genetic transformation must be selected. In future, it is very important to extend the host range of plants for transformation, and the success of the transformations may be attributed to the manipulation of the plants. Recently, several groups have started to identify plant genes and protein products involved in the process of transformation. Recent studies identified that several plant genes involved Agrobacterium-mediated transformation. For example, mutation of the histone H2A-1 gene HTA1 results in decreased T-DNA intergration, over-expression of HTA1 gene in mutant plants could generally “sensitize” plant cells to Agrobacterium-mediated transformation to restore transwww.jgenetgenomics.org

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formation proficiency [9−11]. In future, the efficiency of transformation by Agrobacterium can be improved by extending the host range of plants. The identification and manipulation of these genes, is which may eventually lead to their manipulation to improve transformation during the Agrobacterium-mediated transformation process, still remains a challenge to the scientific biotechnology community. References
1 Gordon-Kamm WI, Spencer TM, O’Brien JV, Star WG, Daines RJ. Transformation of maize using microprojectile bombardment: an update and perspective. In Vitro Cell Dev, 1991, 27: 21−27. Casas AM, Kononowicz AK, Zehr UB, Tomes DT, Axtell JD, Butler LG, Bressan RA, Hasegawa PM. Transgenic sorghum plants via microprojectile bombardment. Proc Natl Acad Sci USA, 1993, 90: 11212−11216. Torbot KA, Rines HW, Somers DA. Use of paromomycin as a selective agent for oat transformation. Plant Cell Rep, 1995, 14: 635−640. Hobbs SLA, Warkentin TD, Delong CMO. Transgene copy number can be positively or negatively associated with trans-

gene expression. Plant Mol Biol, 1993, 21: 17−26. 5 Prols F, Meyer P. The methylation patterns of chromosomal integration regions influence gene activity of transferred DNA in Petunia hybrida. Plant J, 1992, 2: 465−475. 6 7 Meyer P. Understanding and controlling transgene expression. Trends Biol Technology, 1995, 13: 332−337. Meyer P, Niederhof I, ten Lohuis M. Evidence for cytosine methylation of non-symmetrical sequences in transgenic Petunia hybrida. EMBO J, 1994, 13: 2084−2088. 8 Martin T, Wohner RV, Hummel S, Willmitzer L, Frommer WB. The GUS reporter gene system as a tool to study plant gene expression. In: Gallagher SR, ed. GUS protocols: The GUS gene as a reporter of gene expression. Academic Press, San Diego, 1992, 23−43. 9 Mysore KS, Nam J, Gelvin SB. An Arabidopsis histone H2A mutant is deficient in Agrobacterium T-DNA integration. Proc Natl Acad Sci USA, 2000, 97: 948−953. 10 Narasimhulu SB, Deng XB, Sarria R, Gelvin SB. Early transcription of Agrobacterium T-DNA genes in tobacco and maize. Plant Cell, 1996, 8: 873−886. 11 Mysore KS, Bassune B, Deng XB, Darbinian NS, Motchoulski A, Ream W, Gelvin SB. Role of the Agrobacterium bacterium VirD2 protein in T-DNA transfer and integration. Mol Plant-Microbe Interact, 1998, 11: 668−683.

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具有高再生性且对农杆菌敏感玉米自交系的筛选
王 钰1, 2, 付绍红3, 文 颖2, 张志明1, 夏燕莉1, 刘玉贞1, 荣廷昭1, 潘光堂1
1. 四川农业大学玉米研究所,雅安 625014; 2. 四川省制糖糖料工业研究所,资中 641200; 3. 成都市第二农业科学研究所,温江 611130 摘 要:以 70 个高代玉米自交系的幼胚为材料在 NB 培养基上进行离体培养,通过多次继代选择,从中筛选出了胚性愈伤 组织诱导率高、克隆能力强的 10 个自交系,将这 10 个自交系的一部分胚性愈伤组织用作绿苗再分化,研究其再生能力; 另一部分用农杆菌 C58 和 GV3301 转化,利用植物载体中携带的 gus 和 gfp 报告基因的瞬时表达为指标研究玉米基因型对 农杆菌的敏感性。绿苗再分化结果表明,10 个自交系都具有绿苗再分化能力,其中自交系 6010、6038、6015、6051 和 6060 的绿苗再分化力相对较高,分别为 61.11%、31.94%、45%、33.33%和 56.94%。gus 瞬时表达率方差分析结果表明:玉米基 因型对农杆菌 C58 的敏感性存在极显著差异,在自交系 6034、6038 的胚性愈伤组织上没有检测到 gus 瞬时表达,即这两 个基因型对农杆菌 C58 不敏感,不能被其转化;其他 7 个基因型的平均 gus 瞬时表达率均大于 70%,说明这 7 个基因型对 农杆菌 C58 敏感。GFP 荧光检测结果表明,在自交系 6034、6038、6069 和 6010 的胚性愈伤组织上没有检测到 gfp 瞬时表 达,在其他 6 个自交系上检测到绿色荧光蛋白的表达。因此,认为自交系 6015、6051 和 6060 是对农杆菌 GV3301 敏感且 具有高再生能力的玉米转基因受体材料;自交系 6051、6010、6015、6060 和 6050 是再生能力强且对农杆菌 C58 敏感的玉 米转基因受体材料。 关键词:玉米自交系;胚性愈伤组织;农杆菌;gus 报告基因;gfp 报告基因 作者简介:王钰(1981− ),女,四川乐山人,硕士,研究方向:植物基因工程。E-mail: yuer0119_cn@sina.com.cn www.jgenetgenomics.org


				
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Description: selection of maize inbred lines with high regeneration and