Agricultural Sciences in China

2009, 8(6): 658-663

June 2009

A Simplified Seed Transformation Method for Obtaining Transgenic Brassica napus Plants
SONG Li1, 2, ZHAO De-gang1, 2, 3, WU Yong-jun3 and TIAN Xiao-e3
1 2

Guizhou Key Laboratory of Agricultural Bioengineering, Guizhou University, Guiyang 550025, P.R.China Key Laboratory of Green Pesticide and Agricultural Biological Engineering, Ministry of Education/Guizhou University, Guiyang 550025, P.R.China College of Life Sciences, Guizhou University, Guiyang 550025, P.R.China

Abstract
We report here a seed transformation of sonication-assisted, no-tissue culture to rapidly produce transgenic Brassica napus plants. This method comprises the steps of treating seeds by ultrasonic wave, inoculating Agrobacterium tumefaciens with a recombinant ChIFN- gene and germinating directly of treatment seed on wet filter papers. The obtained transformants were verified by GUS histochemical assay and nested PCR amplification. It suggests that seed transformation has a potential use in genetic transformation of rape. Key words: Brassica napus, seed transformation, sonication

INTRODUCTION
Plant transgenic technique is a powerful tool to introduce the desired gene(s) into receptor in the plant bioreactor, which focuses easily on producing large amounts of recombinant protein in a short time. Oilseed rape (Brassica napus L.) is a potential bioreactor of pharmacological products for large area planting and easily separating recombinant protein. Transgenic plants were so far produced primarily from tissue culture of rape cotyledon petioles (Moloney et al. 1989; Damgaard et al. 1997; Zhang et al. 2006) and hypocotyl segments (Cardoza and Stewart 2003; Ramzan Khan et al. 2003; Peng et al. 2006; Zhang et al. 2006) by single Agrobacterium-mediated transformation, which was in general labour-intensive, time-consuming and relatively expensive. Using single Agrobacterium-mediated transformation, a series of tissue culture including coReceived 5 December, 2008 Accepted 2 February, 2009

cultivation, callus induction, shoot initiation, and root inducing is required to culture tissue cell and often 2-5 months are required to obtain complete transgenic plantlets in canola (Ponstein et al. 2002; Das et al. 2006). Tissue culture procedures also have some other adverse effects, such as somatic mutations (RakoczyTrojanowska 2002), plant chimera, losing plants in transplanting and complicated culture medium. Therefore, it is desired for developing a gene transfer of no-tissue culture to build up oilseed rape bioreactor. The direct seed transformation is an alternative procedure as that transgene can go without tissue culture steps and generate large numbers of transgenic plants rapidly. The microscopic injuries caused by ultrasound etching on the surface and internal layers of the targeted tissue provide a channel for the DNA transfer from bacterium to plant in the infection process (Joersbo and Brunstedt 1992; Santarm et al. 1998; Beranov et al. 2008). The successful produc-

SONG Li, Ph D candidate, Tel: +86-851-3865027, Fax: +86-851-3863615, E-mail: lpsssl@126.com; Correspondence ZHAO De-gang, Ph D, Professor, +86-8513865027, Fax: +86-851-3863615, E-mail: dgzhao@gzu.edu.cn
2009, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(08)60261-8

A Simplified Seed Transformation Method for Obtaining Transgenic Brassica napus Plants

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tion of transgenic plants via sonication-assisted Agrobacterium-mediated transformation (SAAT) has been reported in different plant species such as flax (Beranov et al. 2008), soybean (Santarm et al. 1998) and Chenopodium rubrum (Flores Sols et al. 2003) with hypocotyl, cotyledon, and seedling explants. But the SAAT method using seed explant has not been described for rape. Chicken a lpha interferon (ChIFN-) possess powerful, and wide-range of antiviral properties, antiproliferatives, and immunoregulatory functions in birds (Schultz et al. 1995; Plach et al. 1999; Jarosinski et al. 2001; Ruttanapumma et al. 2005). However, use of ChIFN- was limited due to the difficulties in mass production. In order to establish an effi cie nt pl a nt s ys tem to produc e i nt erfer on economically, we have previously shown the bioactive recombinant ChIFN- in lettuce plant (Song et al. 2008). We report herein a successful seed transformation by a SAAT method for the further study of oilseed rape bioreactor. In this work, transgenic B. napus were generated only in 45-60 days after germinating directly from seed explants treated with ultrasonic, which didnt need tissue culture and regeneration processes.

Oil Crops Institute, Guizhou Academy of Agricultural Sciences, Guiyang, China. Healthy seeds of uniform dimension were used in the experiment.

Transformation procedure
B. napus seeds were initially dipped in 75% ethanol for 1 min, and then disinfected with 0.1% (v/v) mercuric chloride for 15 min. After being rinsed thoroughly five times with sterile distilled water to remove the mercuric chloride, the seeds were put in a 50 mL of sterile centrifuge tube containing 20 mL of sterilized water placed in an ice bath, and were sonicated repetitively with 10 min on and 10 min off for a period of time in an MSE Soniprep150 sonifier (Sanyo, Sussex, UK). 5 and 10 m of amplitudes and 0.5, 2.0, and 2.5 h of ultrasonic time were performed respectively in the experiments. The seeds produced were kept in the 25 mL final OD600 0.6-0.8 liquid culture of A. tumefaciens EHA105 bearing plasmid pSFIFN- for ChIFN- gene expression in an XMTD digital homoeothermic water bath (Dongfang, Yuyao, China) at 28C for 1 or 20 h. The period of 0 m of amplitude and 0 h of infection with Agrobacterium (no sonication and sonication without Agrobacterium inoculation applied) were used as a control variant in the experiments.

MATERIALS AND METHODS

Expression of plasmid and bacterial strains
We constructed the plant expression vector pSFIFN- cont a i ni ng Ch IFN- gene, r epor te r ge ne glucoronidase (GUS), and selectable neomycin phosphotransferase (NPT) gene for kanamycin resistance in our previous work (Song et al. 2008). These genes were under the control of Cauliflower mosaic virus (CaMV) 35S promoter and poly A terminator in the T-DNA region. The plasmid pSFIFN- vector was mobilized into Agrobacterium tumefaciens strain EHA105 for the eventual transformation.

Plant growth and selection

After inoculating with Agrobacterium solution for 1 or 20 h, excess A. tumefaciens on the treated seeds were removed by blotting on a sterile filter paper. The seeds were subsequently sown on sterilized moistened wet filter paper in Petri dishes for 2 days co-cultivation in the dark at 27C. Then they were washed with tap water and grown at 27C in a 16 h light/8 h dark photoperiod. When the plumules were around 3-5 cm, they were directly transferred to 8-cm pot s c on t a i ni ng a c omme r c i a l s oi l mi x t ur e (Klasmann, Germany). Due to the presence of NPT gene in the plasmid pSFIFN-, 15 mg L-1 of kanamycin was used to smear on the leaf for selecting transgenic B. napus each day. After 35 days, the plants survived were transferred to the greenhouse for full development.

Plant materials
Mature seed of oilseed rape (Brassica napus L.) line Youyan 10, a local bushy cultivar, was obtained from

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Histochemical detection of GUS expression

45-60 days (no intrinsic GUS activity was detected in any of the non-transformed rapeseed analysed in this study) of kanamycin-resistant lines were used to screen the putative transformed plant by X-gluc histochemical assay in which GUS reporter gene was a powerful tool for analyzing xogenous gene transformation. The B. napus tissues were stained and decolored by a modified method as described for GUS observation (Song et al. 2008).

stage PCR product under the same reaction system and conditions, with the exception that the annealing temperatures of PCR were 54 and 56C, as well as 20 mmol L-1 dNTP of the inner primers (5-GCTG TTCCAGCTTCTCCAC-3 for the forward direction, and 5-CCTGGTGTTTCCGGTAAGG-3for the reverse direction) was used. PCR products were separated on a 1.0 % (w/v) agarose gel.

RESULTS
Seed transformation by sonication-assisted method was performed in the experiment. The procedures of ultrosound treatment and Agrobacterium infection were shown in Fig.1-A, B. Datas of this method were presented in Table. The main stages of transgenic B. napus production from seed explant were presented in Fig.1C, D. Table presented the transgenic plants from GUS and PCR analysis. Seed transformation efficiencies with different treatments were also compared in T0 plants (Table). In this study, higher intensity ultrasonic and longer period of sonication and infection showed that the treatment seeds were liable to death when more A. tumefaciens appearing on the seeds and resulted in a negative impact on seed germination and growth (data not shown). The transformation rate was also affected by the doses of ultrasound and the period of inoculation with bacterium. As shown in Table, SAAT conditions of 5 m per 0.5 h for 20 h inoculation had a better seed transformation rate of 16%. However, the most lines survived were produced by 5 m per 0.5 h of sonication and 20 h infection. GUS gene expression

Nested PCR analysis of transformed plants

The presence of ChIFN- gene in GUS-positive T0 plants was analyzed by nested PCR assay since it is a more sensitive method than Southern analysis (Kojima et al. 2004). Total DNA was extracted from leaf tissues of transgenic plant and control plant following the procedures of Doyle J J and Doyle J I (1990). The first stage of PCR was carried out in a gene thermal cycler (Bio-Rad, Hercules, USA), the reaction mixture (20 L) contained 200 ng of genomic DNA, 1 U Taq polymerase (TaKaRa, Dalian), 4.0 mmol L-1 dNTP, and 20 mol L -1 of the outer primers (5-GTTCTAG AATGGCTGTTCCAGCTTCTC-3 for the forward direction, and 5-GGGGTACCCTATTACTAGGT CCTGGTG-3 for the reverse direction). The PCR cycling conditions were once 94C for 5 min; 5 cycles at 94C for 40 s, 57C for 40 s, and 72C for 40 s; 25 cycles at 94C for 40 s, 59C for 40 s, and 72C for 40 s; and one cycle at 72C for 8 min. The second stage of PCR was performed by amplifying 2 L the first-

Table The summary of parameters tested and analyzed of the transformation results of seed transformation in T0 B. napus 1)
No. of treated seeds2) 50 50 50 50
1) 2)

No. of survived resistant plants3) 2 2 15 44

Amplitude/Sonication time (m h-1)4) 5/2.0 5/2.5 5/0.5 10/0.5

Time of inoculation with Agrobacterium (h) 5) 1 1 20 20

No. of GUS positive plants6) 2 1 11 4

No. of PCR positive plants7) 2 1 8 2

Seed transformation efficiency (%)8) 4 2 16 4

All the transformation lines from the controls were not by sonication, or by sonication without inoculation treatment. Total number of rape seeds for sonication and infection treatment in the SAAT experiments. 3) Number of survived kanamycin resistant plants from the germination seeds treated by sonication. 4) Amplitude and time of ultrasonic treatment in the experi ments. 5) The time of A. tumefaciens infection. 6) Number of GUS positive lines by histochemical assay. 7) Number of transgenic plants recovered from nested PCR assay. 8) Transformation efficiency was determined as the ratio between the number of PCR-positive independent lines and the total number of test seeds in each group.

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(blue inclusions) was observed in 18 transformed lines but no-sonication and no-infection controls (seeds that were not by sonication treatment, and by sonication but no inoculation with Agrobacterium) were negative. The evident blue inclusions were observed in leaf, stem or root tissues in 14 transformed lines (Fig. 1-E, F, G), but only a few blue spots occurred in the leaf tissues from the other plants. The amplified DNA fragment of 573 bp from T0 plants gave a positive result to lines tested (Fig.2). The no-sonication and no-infection controls were uniformly PCR negative.

DISCUSSION
Transgenic B. napus plants were produced by a simple seed transformation method combined sonication treatment, Agrobacterium-mediated, using seed (embryo) as starting materials. The success transformation had occurred for that GUS activity was evident in the leaf, stem, or root tissues of sample plants. PCR further confirmed the presence of ChIFN-a gene in plant genome with an expected band of 573 bp fragment. The same result was also obtained with plasmid DNA replacing the A. tumefaciens strain (data not shown). Results show that seed transformation is efficient and seed (embryo) can used as a suitable target tissue for genetic transformation of rapeseed via SAAT mediatedDNA transfer. The seed germination and transgene efficiency depend on the intensity and length of sonication or inoculation time with A. tumefacie ns in the present experiment. These are the same form as previous resea rch results ( J oersbo a nd Brunstedt 1990; Ananthakrishnan et al. 2007; Beranova et al. 2008). The severer plant tissue damage which resulted in failed seed germination was observed with higher doses of ultrasonic and longer time of inoculation treatment in the present study (data not shown). The degree of plant injury was critical for seed transformation method. It is required to have the SAAT conditions for not only forming sufficient perforations, but also avoiding the transformed cells death, or avoiding else DNA transfering to seed (embryo) cells. The sonication treatment of 5 m per 0.5 h for 20 h infection may be suitable for obtaining the highest seed transformation of 16% among all groups according to the GUS and PCR assays. This rate of this transformation is much higher than that of the single Agrobacterium-mediated transformation - the most common for rape transformation with a range from 4.0 to 6.6%, which was produced from hypocotyl explants reported previously (Halfhill et al. 2001; Das et al. 2006). Our transgenosis efficiency of 2-16% is also higher than the 0-5.03% reported by Pavingerov and Ond ej (1995) in the seed transformation. This is probably related to the microwound caused by ultrasonic, which secretes more phenolic compounds to activate vir genes that are responsible for the transfer of DNA from bacterium to

F ig.1 Stages of transgenic B. n a p u s production by seed transformation and the histochemical analysis of GUS gene expression in the transformed line. A, sonication treatment of rape seeds in sonifier with an ice bath; B, the inoculation treatment of seed explants wit h Ag roba cte riu m tum efa cien s in a homoeothermic water bath at 28C; C, germination of the seeds by sonication treatment on a wet filter paper at 27C; D, transformed seedling grew in the pot with commercial soil; E, F and G, blue stain (indicated by arrows) exhibited GUS activity in leaf, stem, and root tissues of the line 15 respectively.

Fig. 2 Detection of ChIFN- gene in T0 rapeseed plants by nested PCR assays. The figure of 573 bp is the expected size of PCR product. M, marker; C1, plasmid; T1-T3, DNA of transgenic plants based on sonication treatment; C2 , DNA from no-transformation control plant.

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plant in the infection process (Pavingerov and Ond ejn 1995; Ananthakrishnan et al. 2007; Beranov et al. 2008). DNA damage repair of fragmentation induced by ultrasonic wave in cells also facilitate extern genes to integrate into the plant chromosome. Additionally, sonication ruptured the teguments and embryonic tissues of the seed, thus allowing for gene introduction easily. In summary, use of ultrasound increases the transformation efficiency in this SAAT transformation. Transgenic plants could be produced by seed transformation method (Feldmann 1991; Pavingerov and Ond ej 1995). Pavingerov and Ond ejn (1995) obtained transformants of Arabidopsis thaliana by this transformation and further found that the genetic characteristic (kanamycin resistance marker) can transmit into T2 and T3 progeny. Although the offspring of T0 was not analysed by further molecular and genetic analysis for revealing the stable transgene expression in this study, some data on the seed transformation of B. napus were obtained. This could be very important, especially in the case of that rape seed (embryo) was sensitive to A. tumefaciens infection and the treatment with ultrasound facilitated the transfer of exogenous gene into the seed (embryo) cells, but more studies are needed to establish reliable protocols for this method. Using A. tumefaciens system, Ponstein et al. (2002) have obtained transgenic rape from the petioles and hypocotyls, which needed about several months (Das et al. 2006). However, transgenic lines were obtained from the kanamycin resistant plants survied by seed transformation method less than 2 months in this study. Seed transformation via SAAT method in the experiment can get directly transgenic lines from seeds and avoid complex tissue culture under sterile conditions. This method is relatively fast, simple, and economical, as well as easy to apply in plant genetic transformation. This is the first report of rape seed transformation using seed explants. It shows the potential use in large scale for introduction of exogenous genes in the biorector of B. napus and possibly in other plant species.

(2007DFA31260), and the Science Foundation for the Excellent Youth Scholars of Guizhou Province of China to Zhao Degang (20030312).

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Acknowledgements
This work was supported by research grants from the National Key Technology Research and Development Program of China (2007BAD59B06), the International Science and Technology Cooperation Program of China

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