ABSTRACT

A variety of Brassica napus L., H165, was used as recipient of foreign DNA. The foreign DNA used for transformation was a plasmid pBLGC, extracted from E.coli DH5a and containing a chitinase gene and¦Â-1,3-glucanase gene, driven by two CaMV 35S promoters, a terminator NOS and a marker, the kanamycin-resistant gene, NPTll. The laser microbeam puncture method was used for introducing foreign DNA into cotyledonary petiole cells of rape. At fist, the petioles were treated with high osmosis, and then put them into Rose culture chamber, containing plasmid DNA (10-100¦Ìg/ml). The laser microbeam puncture was carried out with wavelength 355 mm, output energy 10 mJ, pulse wide 10 ns and diameter of beam spot 1.38¦Ìm. After the puncture, the plasmid DNA could get into cells. The petioles were then cultured in a MS medium, containing kanamycin and 6-BA 4mg/L. After 20 days of culture, green and white bud grew up. The green buds grew into intact plantlets after one month, and transplanted into soil. The sclerotia challenge was carried out with 220 kanamycin-resistant plants. Among them, 75 plants showed resistance to sclerotia. The total genomic DNA of 20 plantlets was screened by PCR analysis, and 5 of them gave a positive result. Southern analysis was further performed and showed that 2 plantlets were positive in Southern hybrization and resistant to sclerotia. When a total number of 150 plants of T₁ generation were challenged with sclerotia, 65 plants showed resistance. The T₂ generation plants were challenged with sclerotia and most of them showed resistance.

KEYWORDS: Brassica napus, Chitinase,¦Â-1,3-glucanase, Plasmid DNA, Transformation, Southern hybrization

Sclerotia (Sclerotinia sclerotiorium, Lib de Bary) is one of the most serious pathogens of rape. It spreads in all of rapeseed production countries. In South China, the infected areas generally loss about 30% of the rapeseed prodution and even high up to about 80% in the heavily prevalent years, and the quality of oil and germination rate of these seeds are low. So it is very urgent to breed new sclerotia-resistant varieties. Grison et al. succeeded in transferring the chitinase gene into Brassica napus and obtaining the fungi tolerant transgenic plants (Grison et al. 1996). In this paper we report our recent results on transferring bivalent of ¦Â-1,3-glucanase gene from tobacco and chitinase gene into B. napus of variety H165. In transgenic plants and their offspring the resistance to sclerotia was markedly improved .

Materials and Methods

1. Materials

(1) Plants, genes and sclerotia: The H165 with low erucic acid and low glucosinolates contents used as receptor was selected by Institute of Genetics, CAS, in cooperation with Institute of Biotechnology of Yunnan Province. The sclerotia fungi used for challenge was provided by Professor Biwen Zhou. The bean chitinase gene was kindly provided by Dr. J. Gaynor, and the¦Â-1, 3-glucanase gene was cloned by our lab (Lan et al. 1998).

(2) Bacterium vector and primers: The plasmid pBLGC in E. coli DH5a contains a bivalent of ¦Â-1, 3-glucanase gene and chintinase gene, driven by two CaMV 35S promoters, a terminator NOS and a marker, the kanamycin-resistant gene, NpTll (Km^r).

(3) PCR primers: The genes in transformed B. napus were examined by two pairs of PCR primers: the first pairprimers derived from both ends of chitinase gene 3´: 5´-GGGATGACAGTGTCACTGAG-3´and partly complementary sequence of 35S promoter 5´:5´>CTGACGTAAGGGATGACGCC-3´ the second pairprimers derived from both ends of¦Â-1,3-glucanase gene, 3´:’55´-GCGGATCCGACCATGGCTGCTATCACTCC-3´ and 5´: 5´’-CGGTCGACCTCACATCTCACTTACGAGA-3´.

2. Methods

(1) The establishment of plantlet regeneration system of B.napus: The surface sterilized seeds of H165 variety were inoculated onto the plates which contains MS agar medium and incobated in culture room at 24-26¡æ for several days. The cotyledonary petioles were excised and transplanted on the MS medium supplemented with 6BA 4 mg/L (MS1). After 20 days of culture the shoots regenerated from the potioles.

(2) Transformation of B.napus: We followed the methods of Wang et al. (1993). In brief, E. coli DH5a was cultured overnight in LB medium (Km 50¦Ìg/ml) with shaking at 250 rpm and 37¡æ. The plasmid was extracted and purified by the method of Maniatis et al. (1982) and dissolved in TE buffer with the concentration of 500¦Ìg/ml. The laser microbeam puncture method was used for introducing foreign DNA into cotyledonary petiole cells of rape. At first, the petioles were treated with high osmosis (10mmol/L Tris; pH 7.2; 0.6 mol/L sorbitol) and then put them into Rose culture chamber, containing plasmid DNA (10-100¦Ìg/ml). The laser microbeam puncture was carried out with wavelength 355 mm, output energy 10 mJ, pulse wide 10 ns and diameter of beam spot 1.38¦Ìm. After the puncture, the plasmid DNA could get into cells. The petioles were then cultured in MS1 medium with Km 30 mg/L.

(3) Genomic DNA molecular analysis: The genomic DNAs from green plantlets were extracted according to Junghams (1990). Referring to the methods reported in Molecular Cloning we used the PCR to amplify and examine the integration of chitinase gene and¦Â-1,3-glucanase gene in the green plantlets genomic DNA. One¦Ìl of plant total DNA were used as PCR templets and the positive and negative control templets are the pBLGC plasmid and the genomic DNA of untransformed H165. The PCR products were detected by 1% agarose electrophoresis. In order to find out if the chitinase gene and ¦Â-1,3-glucanase gene had been integrated into the genomic DNA of the recipient plant. The transformants giving positive result of PCR screening were further detected by complete Southern blotting .

(4) Challenge with sclerotia: When the plants resistant to Km grew to 40-50 cm in height, they were challenged with the sterile sclerotia which had been cultured on the potato dextrose agar medium at 25¡æ for 3 to 5 days.

(5)The harvested seeds were screended for Km resistance: The seeds from T₀ plants were sowed. A tender leaf was selected from each T₁ plant when they grew to have 5 leaves. On one side of the selected leaf, 5¦Ìl of kanamycin (5%) solution was applied, and at the other side a small hole was made to mark the treatment.

Results

1. Introduction pBLGC into B.napus

Young cotyledonary petioles were the best material for gaining numbers of regenerated shoots in a short time. Our results showed that the 4-5 days old seedlings are optimal for cutting cotyledonary petioles. The cutting surfaces of young petioles must be treated with high osmosis solution. The treatment duration was a very important influencing factor for regeneration frequency. The experiment showed that after 20-40 minutes of treatment with high osmosis, the shoot regene- ration frequency was normal (100/100, No. of shoots regenerated / No. of petioles inoculated), while after 60 minutes of treatment, the regeneration frequency was 35% and usually no shoot regenerated after more than 60 minutes of treatment.

The concentration of plasmid DNA was also optimized. The experiment showed that 10-100¦Ìg/ml plasmid DNA for transformation by laser microbeam puncture method appeared to be adequate. After the puncture, the plasmid DNA could get into the cells. The petioles were then cultured in a MS medium containing Km and 4 mg/L of 6-BA (selective medium).

The concentration of Km in the selective medium was increased gradually. In the first selective medium the Km concentration was 10-15 mg/L, under which the generation frequency of green bud was high. After 15-20 days of culture, the white and green buds regenerated. The green buds were transferred to the second selected medium with 20-25 mg/L of Km for eliminating the¡°escape¡± buds. After 15-20 days of culture, 10% of green buds turned white and light purple, while the other green buds were transferred to the third selective medium with 30 mg/L of Km. After 2 weeks, about 5% green buds turned white and light purple, and they underwent necrosis progressively by the third or forth week of culture. The remaining green shoots were transplanted to the rooting medium containing 10 mg/L of Km for inducing roots. Finally, the rooting plants were transplanted into the soil and grew up into morphologically normal plants. Results presented that about 55% of transformed rape cotyledonary petioles could develop the green shoots resistant to Km and about 80-90% of these shoots could root and develop normally.

When the T₁ generation plants developed 5 true leaves, 5¦Ìl of 5% Km solution was dripped on one side of the selected young leaves. After 3-4 days the treated leaves on untransformed plants showed white spot, but the treated leaves on the transgenic plants still showed green colour. In T₁ generation, 150 Km-resistant plants were selected from 320 T₁ plants planted in the field. The selected method of Km resistant plants in T₂ generation was the same with that in T₁ generation, and 310 Km-resistant plants were selected from 420 T₂ plants planted in the field.

2. Sclerotia challenge of Km resistant plants

The sclerotia challenge was carried out with T₀, T₁ and T₂ generations of Km resistant plants. The sclerotia challenge was carried out with 220 T₀ Km-resistant plants. Among them, 75 plants showed resistance to sclerotia. When a total number of 150 plants of T₁ generation were challenged with sclerotia, 65 plants showed resistance. Among 310 T₂ generation plants challenged with sclerotia, most of them showed resistance.

3. Test of integration of chitinase gene and ¦Â-1, 3-glucanase gene into the rape genomic DNA

The genomic DNA from 20 Km-resistant plants of T₀ generation was screened by PCR analysis and 5 of them gave a positive result. Southern analysis was further performed and showed that 2 plants were positive results and resistant to sclerotia. The results indicated that the chitinase gene and glucanase gene had been integrated into the rape genome.

Acknowledgement

We thank Dr.J.Gaynor for kindly providing the bean chitinase gene.

Reference

1. Grison R. et al. 1996, Nature Biotechnology 14:643-646.

2. Junghams, H. 1990, BioTechniques 8(2): 176

3. Lan, H. et al. 1998, Chinese J Biotech 14:63-69 (in Chinese).

4. Maniatis, T. et al. 1982, Molecular Cloning. A Laboratory Manual. Cold Spring Harbor, New York.

5. Wang, L. et al. 1995, Acta Genetica Sinica 22:394-399 (in Chinese).