Kimmo Koivu

Potato plants transformed with a potato virus Y P1 gene sequence are resistant to PVYO

The cloned P1 sequence of PVYO was transferred in sense orientation into the potato cultivar Pito usingAgrobacteriummediated transformation. Sixteen of the putatively transformed plants (NPTII positive) were assayed for PVYO resistance. No PVYO was detected in four plants, representing two lines, 21 days after two sap-inoculations and 35 days after graft-inoculation, and the plants remained symptomless, whereas other tested plants showed mosaic symptoms and had high PVY titers similar to those of the control plants. No line was resistant to PVYN and potato viruses A and X. Southern analysis confirmed the presence of the transgene(s) in the two PVYO-resistant and one susceptible line examined, but no signal was detected in nontransformed Pito. These results suggest a high level of protection against PVYO in potato transformed with P1 sequence of PVYO.CompendioLa cadena clonada del PI de PVYO fue transferida, en orientatión positiva, al cultivar Pito de papa, utilizando la transformatión mediante Agrobacterium. Dieciséis de las plantas transformadas putativamente (NPTII positivo) fueron evaluadas para resistencia a PVYO. No se detectó PVYO en cuatro plantas, representando dos líneas, 21 días después de dos inoculaciones con savia y 35 días después de inoculatión por injerto, y las plantas permanecieron sin síntomas, mientras que otras de las plantas probados mostraron síntomas de mosaico y tuvieron titulaciones altas de PVYsimilares a aquellas de las plantas testigo. Ninguna línea fue resistente al PVYN o a los virus A y X de la papa. El análisis del RNA en placas de nitrocelulosa confirmó la presencia del (o de los) transgene (s) en las dos líneas resistentes y en una susceptible al PVYO examinadas, pero no se detectó señal alguna en las plantas de Pito no transformadas. Estos resultados sugieren un alto nivel de protección contra el PVYO en la papa transformada con el encadenamiento del PI de PVYO.

Transgenic crop plants expressing synthetic cry9Aa gene are protected against insect damage

A synthetic gene sequence of cry9Aa was made to achieve high expression levels in a plant cell. Tobacco, potato, cauliflower and turnip rape plants were transformed with this synthetic gene driven by the double 35S promoter using Agrobacterium tumefaciens LBA4404. The presence and expression of the synthetic cry9Aa gene was evaluated in Southern, Northern and Western analysis and with insect bioassays. The expression of the gene in tobacco plants reached a level of 5 pg of mRNA per 1 µg of total RNA and 0.3% of soluble protein or 1.4 µg of Cry9Aa protein per 1 g of leaf material. The expression level in the other species was three to ten times lower. Tobacco plants were also transformed with a truncated native cry9Aa gene construct and with a translational fusion construct of the truncated native cry9Aa and the uidA (GUS) gene sequence. The constructs were transformed in tobacco plants under the control of the same promoter as the synthetic cry9Aa. The expression level of the native cry9Aa gene constructs ranged from 0.03 to 1 pg of cry9Aa mRNA per 1 µg of total RNA. The protein was undetectable in Western analysis. In comparison to the native constructs the expression level of the synthetic cry9Aa gene was five to ten times higher at the mRNA level and at least 50 times higher at the translational level. Bioassays against Plutella xylostella performed with transgenic cauliflower showed high insecticidal activity of the plants expressing the synthetic cry9Aa gene.

High level of resistance to potato virus Y by expressing P1 sequence in antisense orientation in transgenic potato

Potato cultivar Pito was transformed with the P1 sequence of potato virus Y (PVYO) in antisense orientation. Five transgenic lines (ASR) showed a high level of resistance to PVYO on mechanical and side-graft inoculation, expressed as no symptoms, and no detectable amounts of PVY in the inoculated and the upper uninoculated leaves, as determined by ELISA 21 and 35 days after inoculation. The possibility of accumulation of low levels of PVYO not detected by ELISA was tested by grafting a scion of untransformed Pito (WT) on each of the PVYO-inoculated ASR plants. With this method, no PVY was detected in the WT plants grafted on the PVYO-inoculated ASR plants. Two of the six plants of the ASR line AI 0623-2 showed PVY accumulation when grafted on the PVYO-inoculated WT plants. Resistance was specific to PVYO, as the ASR lines were fully susceptible to PVYN, PVA and PVX. The ASR lines and PVY-susceptible lines contained 1–5, and 1–3 P1 gene inserts, respectively, as determined by Southern analysis. All lines expressed similarly low levels of the antisense P1 transcript. These results show that effective, virus strain-specific resistance to PVYOcan be achieved by expressing the P1 sequence in antisense orientation in potato. The low transgene transcript levels suggest that resistance in the ASR plants may be based on gene silencing.

Cloning of new rubisco promoters from Brassica rapa and determination of their activity in stably transformed Brassica napus and Nicotiana tabacum plants

The aim of our study was to identify the highest expressing rubisco small subunit (RbcS) promoters (pRbcS) from the cotyledons of germinating seedlings of Brassica rapa var. oleifera to drive high-level and preferably stage-specific transgenic protein expression in Brassicaceae plants. We cloned four new pRbcS promoters using several approaches, including the construction of a cDNA library and use of genome walking technique. Real-time PCR analysis of RbcS mRNA expression clearly showed that two of these promoters exhibited the highest activity on the germination stage of plant development. We used gusA expression as a reporter of promoter activity in Brassica napus and Nicotiana tabacum plants that were transformed with the constructs using an Agrobacterium-mediated transformation strategy. The mRNA level of RbcS and of gusA was quantified in transformed plants. The data obtained demonstrate that the promoter most active in seedlings under native conditions was also most active in transgenic constructs at the same stage of plant development. The fine structure of the promoters is discussed herein.

Modified resistance of Solanum brevidens to potato Y potyvirus and tobacco mosaic tobamovirus following genetic transformation and explant regeneration

Abstract Infected plants of Solanum brevidens accumulate extremely low titres of potato Y potyvirus (PVY) and moderate titres of tobacco mosaic tobamovirus (TMV); but, in plants infected with both viruses, accumulation of PVY is enhanced, suggesting that TMV assists the cell-to-cell spread or replication of PVY. Cell-to-cell movement of TMV is mediated by the virus-coded 30-kDa movement protein. In this study, S. brevidens was transformed with the 30-kDa gene of TMV-204. Systemic spread of the temperature-sensitive mutant TMV-Ls1 occurred in four transgenic lines carrying the 30-kDa gene sense construct at the non-permissive temperature (33°C) indicating expression of functional wild type 30-kDa protein capable of complementing defective movement functions of TMV-Ls1. PVY accumulation was enhanced in three of these lines, in four transgenic ‘sense’ lines in which no complementation of TMV-Ls1 occurred, in nine ‘antisense’ lines, two lines lacking the 30-kDa gene insert, and nine non-transgenic callus-derived lines. No detectable infection or systemic spread of TMV-204 occurred in most of the callus-derived lines. Thus, enhanced accumulation of PVY and increased resistance to TMV appeared to be associated with tissue culture-induced changes rather than the 30-kDa gene insert.

Agrobacterium tumefaciens-Mediated Transformation of Solarium brevidens and S. tuberosum cv. Pito

Abstract An Agrobacterium tumefaciens -mediated transformation procedure was developed for the non tuber-bearing, diploid wild potato species Solanum brevidens, and the tetraploid S. tuberosum cv. Pito. Cointegrative transformation vectors pGV2260 and pGV3850, and binary vector pGUS-INT were employed in transformation of S. brevidens and Pito, respectively. Leaf and stem explants of S. brevidens and microtuber discs of Pito were precultured 24 h, and cocultivated 48 h on solidified callus induction medium with a 24-h liquid culture of A. tumefaciens C58C1 diluted 1:10 with liquid MS medium. Explants were rinsed with cefotaxime solution (500 mg/1) to remove Agrobacterium, grown without the selective agent kanamycin on solified callus induction media for 14 days, and then exposed to kanamycin selection for the first seven days on callus induction medium and subsequently on shoot regeneration medium. Shoots regenerated faster from stem explants than from leaf explants. Up to 49% and 57% of the transformed le...