A proposed mechanism for increasing transient therapeutic protein production in plant tissue is the use of replication initiator proteins. Transgenic N. benthamiana plant tissue containing a viral replication initiator protein (REP) from Bean Yellow Dwarf Virus has been generated and characterized. 15 lines were isolated based on an antibiotic resistance gene linked to REP: (5) ARPF, (3) ARPR, (4) ADPF, and (3) ADPR. Polymerase chain reaction testing showed that 10 of these lines successfully incorporated the REP gene along with the resistance gene. Transgenic root and cell cultures were generated from these plants, representing 3 of the 4 viral orientations in root culture and 1 in cell culture. Two other cell culture lines representing two different viral orientations are currently in liquid flasks but have failed to grow within a period of 15 weeks.
Along with the 2 sequential transformations for generating root cultures, a technique was developed to simultaneously add REP and the Ri genes to plant tissue, reducing the time required to form transgenic roots. A soybean root line was successfully generated using this cotransformation technique. Both Agrobacterium rhizogenes K599 and 15834 were used in these studies. Although no REP PCR (+) root lines were generated using the 15834 strain, several root lines were isolated that were not yet PCR tested (GMA+ARPF, ADPF; GMB+ARPF; GMD+ADPF, ADPR). The ability to produce “hairy roots” is in contrast to literature sources that have not had much success with strains other than K599. Three of the four cotyledons infected with strain 15834 produced roots in this study.
A genetic segregation study was used to determine the genotype of some of the N. benthamiana transformants. Antibiotic selection levels were screened and applied to transgenic seeds. Using statistical analysis, there is no indication that kanamycin levels up to 400 mg/L affect the germination percentage of N. benthamiana seeds. 300 mg/L was chosen for the selection pressure since this was sufficient to bleach and kill seeds that had not inherited the resistance gene within 18 days. Two lines segregated in a Mendelian fashion, indicating a single gene (or closely linked multiple-gene) insert. Plants were moved to soil to obtain healthier seeds for future segregation studies to generate homozygous plants.
The steps required in reverse transcriptase polymerase chain reaction were developed to test the tissue for expression of REP. Details of the RNA extraction, reverse transcription, and polymerase reaction steps have been optimized to provide clear reliable results. RNA extraction in the presence of the Trizol reagent prevents degradation of the plant tissue during the first critical moments of cell disruption. 2 pmol of primer during the reverse transcription reaction is sufficient to produce a cDNA construct, but it does not form significant non-specific binding products – avoiding a smear on the gel. At least 25 cycles are required for the PCR step, and 1 ?L of PCR product is sufficient to form distinct bands. A housekeeping gene has been successfully amplified with this system, showing the technique works. Successful amplification of REP mRNA has been demonstrated in E. coli using a plasmid with a constitutive promoter, although testing is needed to rule out the possibility of DNA contamination. No evidence of REP expression has been found in the plant lines tested up to this point.