Patent Application
20060037100
The present invention relates to transgenic pepper plants resistant to anthracnose fungus and a method for producing the said transgenic pepper plants. More specifically, the present invention relates to novel distinctive pepper transgenic lines carrying PepEST or PepDef genes as well as methods for their production.
Capsicum annuum L. (pepper) is an important vegetable crop characterized by high earning crop around the world. However, all commercial varieties are susceptible to anthracnose fungus, which may result in 10-15% losses in annual yield. So, current goal of pepper biotechnology is to increase pepper's resistance to anthracnose fungus. To date, improvement of pepper has been restricted to conventional breeding since the transformation of pepper was not routinely applicable to introduce valuable genes into the genome.
A transformation system of plants requires tissue cultures competent for efficient plant regeneration as well as an effective method of gene delivery. In pepper, tissue culture techniques have been used to produce somatic embryos and haploid plants. And its ‘in vitro’ regeneration has been reported by numerous laboratories. Despite of the tissue cultural advantage of this species, pepper has been known as a recalcitrant plant to be genetically transformed. There have been no reports on genetically stable transgenic peppers although a few attempts have been made to transform pepper plants in recent years. Here, we established an efficient transformation method for pepper using Agrobacterium tumefaciens and also demonstrated stable inheritance of the transgenes in transgenic peppers.
Plant transformation involves the transfer of desired genes into the plant genome and then the regeneration of a whole plant from transformed cells. To transfer genes into plants, Agrobacterium is widely used in many plant species. Agrobacterium infection and gene transfer normally occur at the site of a wound in the plant. In the case of pepper, Agrobacterium infection causes severe accumulation of phenolic compounds at the infection sites, and further growth of the tissues is thus arrested. So, the protocol for tissue culture method was designed to circumvent growth retardation after Agrobacterium infection. Transformants were then selected by their ability to divide and grow in tissue culture medium with antibiotics. Here, we report a reproducible method for the stable transformation of pepper plants using Agrobacterium.
With the advent of genetic engineering, introducing disease resistant genes such as PepEST (U.S. Pat. No. 6,018,038) and PepDef (U.S. Pat. No. 6,300,489) have led to the development of transgenic peppers. PepEST gene encoding a member of esterase was isolated from the ripe pepper fruits that showed incompatible interaction with anthracnose fungus. The PepEST protein plays dual roles in the plant-pathogen interaction, namely direct inhibition of fungal infection by arresting appressorium formation and by activating the disease-resistant signaling pathway. On the other hand, many plant defensins can inhibit the growth of a broad range of fungi at micromolar concentrations but are nontoxic to both mammalian and plant cells. Plant defensins are structurally related to insect defensins such as drosomycin, which is an antifungal peptide found in Drosophila melanogaster. The PepDef protein that belongs to a defensin family is small cysteine-rich peptides with antimicrobial activity. Thus, PepEST and PepDef genes, respectively, were introduced into pepper plants to control anthracnose fungal disease. The present invention relates to new distinctive pepper transgenic lines carrying PepEST or PepDef genes and the method for their production as well.
According to the invention, new transgenic pepper lines, designated as PepEST transgenic pepper (PepEST-TP) and PepDef transgenic pepper (PepDef-TP) respectively, are provided. This invention thus relates to plants of transgenic pepper lines, to seeds of transgenic pepper lines and to methods for producing the transgenic pepper plants mediated by Agrobacterium. This invention also relates to establishment for producing other transgenic pepper lines derived from transgenic PepEST-TP and PepDef-TP. This invention further relates to hybrid the plants produced by crossing the transgenic PepEST-TP and/or PepDef-TP with other pepper lines.
Followings are detailed description of embodiments of the present invention.
Plant Material
Pepper seeds were surface sterilized for 5 min in 0.2% sodium hypochlorite followed by several rinses with sterile distilled water and then germinated on Murashige and Skoog (MS) medium in the dark. After 7 days of incubation, seedlings were exposed to light for 6 hr. Then, cotyledons and hypocotyls were excised for inoculation of Agrobacterium.
Construction of the Transformation Vector
A full length cDNAs of PepDef gene (SEQ ID NO: 1) and PepEST gene (SEQ ID NO: 2) were isolated from pepper. The PepEST cDNA was amaplified by PCR using a forward primer sequence with a BamHI restriction site (5′-ggatccaaaatggctagccaaagttttgttcc-3′: SEQ ID NO: 3) and a reverse primer sequence (5′-aatttgtagtagcacatatgaa-3′: SEQ ID NO: 4). This fragment was subcloned into BamHI- and Smal-digested pBI121. Then, the expression cassette was restricted with HindIII and EcoRI and ligated into cloning sites of pCAMBIA 1300 and named as pCAM-EST. To clone PepDef cDNA, primers used were (5′-gggtctagaaaaatggctggcttttccaaagtg-3′: SEQ ID NO: 5) for the forward with XbaI and (5′-ctcggatcctaattaagcacagggcttcgt-3′: SEQ ID NO: 6) for the reverse with BamHI. Then, the PCR product was cloned into pCAMBIA1300 and named as pCAM-Def. Finally, the plasmid DNA was mobilized into A. tumefaciens GV3101, respectively.
For plant transformation, Agrobacterium strain GV3101 carrying the binary vectors were used. Agrobacteria were cultured to log phase in YEP medium at 28° C. The bacteria were resuspended and agitated for 4 hr in MS liquid medium containing 20 μM acetosyringon to induce the virulence.
Pepper Transformation
Pepper explants excised from the cotyledon were incubated on CIM medium (MS medium supplemented with 0.5 mg/l IAA and 0.2 mg/Zeatin) prior to inoculation with Agrobacterium. Following 48 hr incubation, the explants were submerged in the Agroacterium suspension for 5 min, blot-dried and co-cultured for 48 hr at 28° C. in the dark on CIM medium. Infected explants were transferred for selection to CIM medium with 500 mg/l cefotaxime and 20 mg/l hygromycin B for 2 weeks. Thereafter on every 2 weeks, the explants were subcultured onto SIM medium (MS medium supplemented with 0.2 mg/l IAA and 1 mg/Zeatin) containing both antibiotics to induce shoot regeneration. The shoots regenerated from calli were rooted on the MS medium containing 10 mg/l hygromycin B. The established plantlets were acclimated in a greenhouse for further analysis.
Inheritance Analysis of Transgenic Progenies
The transgenic pepper lines were maintained in greenhouse and self-fertilized to generate the seeds. The transgenic progenies were screened by hygromycin resistance that was provided by the hpt gene. Seeds of T0 transgenic pepper were surface sterilized and placed on a half strength MS medium containing 20 mg/l hygromycin B for 7 days for germination. Finally, healthy green plants were counted and transferred to soil.
Screening of Transgenic Pepper by PCR
Polymerase chain reaction (PCR) was performed with the genomic DNA from putative transgenic plants and their progenies to examine the presence of transgenes. Sets of specific primers were used to amplify GUS, PepEST, and PepDef, respectively. The primer set consists of (i) forward primer designed based on the sequence of CaMV35S promoter, corresponding to nucleotide positions 847-874 and (ii) reverse primer described above for each gene. The PCR conditions were 5 min at 94° C., then 35 cycles of 94° C. for 30 sec and 30 sec for annealing at 60° C. with 1 min extension period at 72° C. The amplified fragments were separated on 1% agarose gels.
Southern Analysis
Genomic DNA from selected transgenic pepper plants was used for Southern hybridization. Ten μg of genomic DNA was digested with 50 units of Hind III or EcoRI for overnight. DNA gel blotting was performed and then prehybridization was carried out at 65° C. for 2 hr, followed by hybridization at 65° C. overnight with the [α-32P] dCTP-labeled CDNA probe in the prehybridization solution. Radiolabeled probe was prepared by using a random primer-labeling kit. Then, the blots were washed once in 2×SSC, 0.1% SDS for 10 min at 65° C., and once in 0.1×SSC, 0.1% SDS. The blots were exposed to X-ray film.
Northern Analysis
Total RNAs were extracted from independent transgenic peppers by the RNeasy Plant Kit (QIAGEN) according to the manufacturer's instructions and stored at −80° C. RNA gel blotting was performed and prehybridization was carried out at 65° C. for 2 hr, followed by hybridization at 65° C. overnight with the [α-32P] dCTP-labeled cDNA probe in the prehybridization solution. The blots were washed once in 2×SSC, 0.1% SDS for 10 min at 65° C., and once in 0.1×SSC, 0.1% SDS. The blots were exposed to X-ray film. Radiolabeled probe was prepared by using a random primer-labeling kit.
GUS Enzymatic Assay
GUS histochemical staining of transgenic plants was performed as described by Jefferson et al (1987) in a solution of 50 mM NaPO4 (pH 7.0), 10 mM EDTA, 0.5 mM K3[Fe(CN)6], 0.5 mM K4[Fe(CN)6], 0.1% sarcosyl, 0.1% β-mercaptoethanol, 0.1% Triton X-100, 1 mg/ml X-gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid) at 37° C. overnight. GUS fluorogenic assays of tissue samples from various organs were performed as described by Jefferson et al (1987). Extracts were assayed for GUS activity and protein concentrations were determined by Bradford assay (Bio-Rad). Fluorescence at time intervals was measured with excitation at 320-390 nm and emission at 415-650 nm by using a TD-700 fluorometer (Turner Designs, USA) and the slope was determined. The specific activity of the GUS enzyme was calculated as pmol 4-methyl umbelliferone (MU) min/mg total protein. GUS activity was estimated from the average of three replicate assays.
SDS-PAGE and Western Blotting
Protein samples were extracted from leaves or fruits directly in 2× loading buffer and separated by SDS-PAGE. Protein concentrations were determined by Bradford assay (Bio-Rad). Proteins were transferred to PVDF membranes (Bio-Rad) and blocked in 5% skim milk powder in TBS (10 mM Tris (pH 8.0), 150 mM NaCl). A polyclonal anti-PepEST rabbit IgG was used at a 1:4000 dilution in 5% blocking solution. The anti- PepDef was used at a 1:3000 dilution. Proteins were detected using a 1:8000 dilution of mouse anti-rabbit IgG conjugated to peroxidase (Sigma) using ECL chemiluminescence blotting substrate (Amersham). The gel was stained with Coomassie Brilliant Blue.
ELISA
Proteins were isolated from leaf materials of transgenic plants and protein concentrations were determined in the crude extracts according to Bradford (1976). The soluble protein fractions were subjected to ELISA to determine the amount of PepEST or PepDef protein.
Resistance Evaluation of the Transgenic Plants
Spores of anthracnose fungus were cultured on a potato dextrose agar (PDA) medium at 28° C. The spores were collected and diluted in sterilized water. Then, the spore suspension was filtered through two layers of gauze, and the filtrate was centrifuged at 1,500 rpm for 5 min. The sediment composed of conidia was resuspended in sterilized water with the concentration adjusted to 5×105/ml.
Inoculation with C. gloeosporioides was done by applying 10 μl of a spore suspension on mature unripe-green fruits. The fruits with drops of spore suspension were placed at high humidity for 2 days to stimulate infection by hyphal germination in dark at 28° C. Thereafter, the fruits were incubated further in a growth chamber. The infected fruits were collected separately from the drop-inoculated area. For the control, 10 μl of distilled water was applied on the unripe pepper fruits.
A binary vector pCAMBIA1300 was used as a backbone for plant expression vectors. An expression cassette containing a resistance gene driven by CaMV35S promoter and Nos terminator was cloned into the multi-cloning site of pCAMBIA1300. The resulting expression vectors were carried PepEST and PepDef and named as pCAM-EST and pCAM-Def, respectively. The T-DNA region of the vector carries hygromycin phosphotransferase (HPT) gene driven by CaMV35S promoter and the gene expression cassette.
To optimize the regeneration condition for pepper explants, the explants were tested on various combinations of auxin and cytokinin; the best combination for shoot regeneration was IAA and Zeatin in 0.1-0.5 and 1-2 mg/L, respectively. Numerous genotypes tested were well regenerated under these conditions. The age of the plants had some influence on both regeneration and transformation. The aseptic plants should be germinated in dark for 7 days and then illuminated in light for 6 hours just before use. The explants should be isolated before the emergence of true leaf.
Agrobacterium tumefaciens was treated in a various manner, such as pH, temperature, chemicals, during the infection onto pepper explants. We then scored callus development on the infected explants under high dose of hygromycin B (20 mg/l). Callus formation efficiently occurred after inoculation on the medium at pH 5.5 at 26° C. The duration of incubation had effects on the transformation frequency. A longer incubation time resulted in browning of the pepper explants. Therefore, 2 days of incubation were optimal for cocultivation of the explants with Agrobacterium.
Since the explants were isolated from young hypocotyls and cotyledons, they retained efficient morphogenetic potentials for shoot development. Particularly, de novo regenerations occurred in the upper part of the explants. We can easily observe condensed axillary shoots from the green part of explants. Therefore, it is important to select the transformed cells under strict selection conditions because mild strength of antibiotics in the selection medium does not properly inhibit the growth of ‘false positive shoots’. Even more, the false positive shoots would completely block the division of transgenic cells. Greening of the explants can be inhibited by limiting light and with high concentrations of hygromycin B. Healthy transgenic callus developed from the cutting edge of the explants. Then, the transgenic callus was forced to regenerate shoots on the shoot induction medium containing 20 mg/l hygromycin B.
An efficient pepper transformation method was established on the basis of the shoot regeneration system of pepper on selection medium. To test the reliability of the transformation system, GUS reporter gene was introduced into pepper plants. The pepper explants inoculated with Agrobacterium carrying pCAMBIA1301 or 1304 was able to produce stably transformed callus and plants. The integration and expression of GUS gene were confirmed by Southern and Northern hybridizations, respectively (
The PepEST gene, encoding an esterase, was cloned from the ripe pepper fruit that showed resistance to anthracnose fungus (Kim et al., 2001). To assess the function of the PepEST gene in disease resistance, its gene was introduced into pepper using Agrobacterium-mediated transformation. To express the 36.5 kD PepEST protein in the plant, the cDNA sequence was ligated into plasmid pCAMBIA1300 between the CaMV35S promoter and the Nos terminator. In the transgenic plants, Southern and Northern analyses were carried out to confirm the presence and expression of the transgene using PepEST sequences (
The pepper defensin, PepDef, accumulated highly during fruit ripening. The role of PepDef was suggested to protect the reproductive organs against biotic and abiotic stresses (Oh et al., 1999). To generate transgenic resistant peppers against C. gloeosporioides based on the proposed function of PepDef, a chimeric construct was designed. Transcription of the PepDef gene was placed under the control of CaMV35S promoter and Nos terminator.
The construct was transformed into pepper using Agrobacterium strain GV3101. The regenerated plants displayed normal phenotypes compared with wild type peppers. To screen transgenic plants, PCR was conducted by the combination of a sequence from the CaMV35S promoter as a forward primer and a sequence from the 3′-untranslated region of PepDef cDNA as a reverse primer. Transgenic pepper seeds were collected from the individual transgenic lines (Data not shown).
To assay the disease resistance in the transgenic plants, conidia of the virulent C. gloeosporioides were used to inoculate the unripe fruits of transgenic peppers. Transgenic fruits remained healthy but the unripe fruits from wild type plant developed typical anthracnose symptoms. As shown in
Oh B J, Ko M K, Kostenyuk I, Shin B C and Kim K S (1999) Coexpression of a defensin gene and a thionin-like gene via different signal transduction pathways in pepper and colletotrichum gloeosporioides interactions. Plant Molecular Biology 41: 313-319
