Katahdin SP951 Research Articles

Bioinformatics Toxicity Assessment of RB Protein from Transgenic Potato Resistant to Phytophthora infestans

The genetically engineered potato cultivar Katahdin SP951 is resistant to late blight disease, which is caused by Phytophthora infestans . The biosafety and food safety of this cultivar should be assessed prior to its commercialization. Toxicity is one of the parameters tested in the food safety evaluation of transgenics. Toxicity testing includes the bioinformatics analysis of the homology of the RB protein of Katahdin SP951 with known proteins. Therefore, this study aimed to perform the bioinformatics analysis of Katahdin SP951 RB protein against homologous toxin proteins. Bioinformatics analysis was conducted by first translating the RB gene into an amino acid sequence by using the Emboss Transeq program from the EMBL-EBI website. The Shuffle Protein Program was then applied to obtain the randomized amino acid sequences of the RB protein. The Basic Local Alignment Search Tool (BLAST) Protein search program was utilized to identify proteins with high similarity and homology. Moreover, BLAST Suite-2 was used to analyze the homology between two or more sequence alignments. Results showed high homology between the sequences of the RB protein and those of known resistance ( R ) proteins with an E value of less than 0.22. This result may be attributed to the presence of numerous R proteins in plants. Further analysis indicated that the sequence of the RB protein had extremely nonsignificant homology with sequences of proteins known to be toxic in the Entrez protein database of National Center for Biotechnology Information. Therefore, the RB protein is nontoxic.

Penentuan Alergenisitas Protein Gen RB pada Kentang Produk Rekayasa Genetika Berdasarkan Studi Bioinformatika

<p>Genetically modified products (GMP) of Katahdin potato event SP951 containing RB gene resistant to late blight diseases<br />caused by Phytophtora infestans has been developed in the USA. This Katahdin SP951 potato has been crossed with local<br />varieties Atlantic and Granola for its development in Indonesia. In the release process, the GMP potato should be tested for<br />environmental and food safety. One of the food safety assessment needs to be done by determining allergenicity of RB protein<br />whether it is potential as allergen. This research aims to translate the RB gene sequence into RB protein sequence and<br />investigate the potential RB protein as an allergen through bioinformatic studies. This study was performed based on the<br />alignment with available protein allergens from available database websites. The predicted RB protein obtained from 2,913<br />amino acids RB gene was a 971 amino acids length protein with ATG as a start codon and TAA as a stop codon. Bioinformatics<br />studies of RB protein were performed using www.allergenonline.com, consisted of three searches, i.e. full-length search by<br />FASTA, 80 amino acids search by FASTA, and 8 amino acid exact matches. For full-length alignment search, there are three<br />allergen proteins similar with RB protein sequence with the percentage identity of <35%, while for alignment with 80 amino<br />acids and 8 amino acids did not show similarity with any allergen protein in the database. It can be concluded that RB protein<br />did not have any potential as an allergen, as according to Codex Alimentarius guidelines for full-length alignment search, only<br />protein with identity greater than >50% indicating possible cross reactivity with protein allergen.</p>

Identifikasi cDNA Gen RB pada Tanaman Kentang Produk Rekayasa Genetika Katahdin SP951

<p>To overcome the negative impact of the fungicide application, RB gene, originated from Solanum bulbocastanum, a resistance<br />gene to the disease has been introduced to the genome of potato plant Katahdin, which was later named as Katahdin SP951.<br />The aims of the research were to confirm the RB gene in the genetically modified plant (GMP) Katahdin SP951 through<br />resequencing the RB gene in the Katahdin SP951 genome and to align the RB genes isolated from the transgenic Katahdin SP951<br />and the cloning vector pLCD0454, which was used the vector to transform Katahdin cultivar. The RB gene has high similarity to<br />other resistance genes in potato plants, and therefore nontransgenic Katahdin, and Solanum bulbocastanum were used in the<br />analysis as the negative and positive control, respectively. Seven pairs of specific primers, which could differentiate the RB gene<br />were used for gene sequencing studies. The sequencing experiments obtained 2913 bases which showed 100% similarity to the<br />RB sequence isolated from the pLCD04541 plasmid. It is concluded that the RB gene in the Katahdin SP951 did not experience<br />mutation during transformation process and integration into the Katahdin genome.</p>

Analisis Integrasi dan Segregasi Gen Ketahanan terhadap Hawar Daun pada Progeni F1 Hasil Persilangan Tanaman Kentang Transgenik dengan Non Transgenik

Potato late blight, caused by Phytophthora infestans is one of the most devastating plant diseases. Potato yield losses due to this disease ranged from 47-100%. Frequent intervals and high rates of fungicide spray, currently practiced by potato growers to control the disease are expensive. Host resistance is an alternative control measure that is more economically and environmentally sustainable. Development of late blight resistant plants was conducted by crossing RB transgenic Katahdin SP904 and<br />SP951 as male and two susceptible (Atlantic, Granola) varieties as female parents. F1 progenies were molecularly characterized for the integration of the RB transgene and evaluated for their segregations. Crossing data of Atlantic x transgenic Katahdin SP904 and SP951 produced 71 (57.72%)<br />berries with average number of seeds per berry 139.58 and 83 (41.29%) berries with average number of seeds/berry 85.23, respectively. Granola x transgenic Katahdin SP904 and SP951 crosses gave higher results in terms of berry set (79.55 and 84.44%, respectively) than Atlantic x transgenic Katahdin crosses. A total of 554 F1 progenies were analyzed for the<br />presence of the RB PCR marker. An expected 619-bp and 840-bp band were amplified in the progenies that contain the RB gene. The RB gene was integrated in 65 (45.45%), 77 (47.83%), 47 (45.63%), and 71 (48.30%) F1 progenies of Atlantic x transgenic Katahdin SP904, Atlantic x transgenic Katahdin SP951, Granola x transgenic Katahdin SP904, and<br />Granola x transgenic Katahdin SP951, respectively. Chisquare tests showed that all the four cross combinations followed a 1 : 1 segregation ratio.

GENETIC STABILITY ANALYSIS OF RB GENE IN GENETICALLY MODIFIED POTATO LINES TOLERANT TO Phytophthora infestans

Development of potato cultivars with high levels of broad spectrum resistance is a key long-term management strategy against late blight disease caused by Phytophthora infestans. Six progeny lines of hybridization between transgenic potato Katahdin SP951 with non-transgenic Granola and Atlantic were selected based on agronomical characteristics and resistance to late blight disease. The study aimed to analyze the number of insertions and stability of inserted RB gene in the transgenic potato lines. The research was carried out through plant DNA extraction, southern blot analysis and polymerase chain reaction (PCR). Southern blot analysis was used to detect the number of inserts integrated into potato genome, while PCR analysis was used to detect stability of RB gene from generation to generation. The results showed that the progenies obtained from hybridization between Atlantic and transgenic Katahdin SP951 (lines No. 20 and 27) and between Granola and transgenic Katahdin SP951 (line No. 69) contained one copy number of RB gene, according to the probing of nptII. The result is similar to that of inserted RB gene found in the parental transgenic Katahdin SP951. The presence of RB gene in four different generations (G0, G1, G2 and G3) showed stable integration of the gene into the plant genome. The single copy number of RB gene will repress the occurrence of silencing gene expression. The stability analysis of RB gene can determine that the gene is still present in plant genome after several generations.

GENETIC STABILITY ANALYSIS OF RB GENE IN GENETICALLY MODIFIED POTATO LINES TOLERANT TO Phytophthora infestans

Development of potato cultivars with high levels of broad spectrum resistance is a key long-term management strategy against late blight disease caused by Phytophthora infestans. Six progeny lines of hybridization between transgenic potato Katahdin SP951 with non-transgenic Granola and Atlantic were selected based on agronomical characteristics and resistance to late blight disease. The study aimed to analyze the number of insertions and stability of inserted RB gene in the transgenic potato lines. The research was carried out through plant DNA extraction, southern blot analysis and polymerase chain reaction (PCR). Southern blot analysis was used to detect the number of inserts integrated into potato genome, while PCR analysis was used to detect stability of RB gene from generation to generation. The results showed that the progenies obtained from hybridization between Atlantic and transgenic Katahdin SP951 (lines No. 20 and 27) and between Granola and transgenic Katahdin SP951 (line No. 69) contained one copy number of RB gene, according to the probing of nptII. The result is similar to that of inserted RB gene found in the parental transgenic Katahdin SP951. The presence of RB gene in four different generations (G0, G1, G2 and G3) showed stable integration of the gene into the plant genome. The single copy number of RB gene will repress the occurrence of silencing gene expression. The stability analysis of RB gene can determine that the gene is still present in plant genome after several generations.

EFFECTS OF LATE BLIGHT RESISTANT POTATO CONTAINING RB GENE ON THE SOIL MICROBES, PESTS AND PLANT DISEASES

Late blight caused by Phytophthora infestans is an important disease on potato. Several potato hybrids have been generated by crossing local varieties (Atlantic and Granola) with Katahdin SP951 which contains late blight resistance gene RB. Prior to release, these hybrids need to be evaluated for their environ-mental effects on non-target organisms and natural pests and diseases. The objectives of the study were to investigate the effect of LBR potato hybrids on beneficial soil microbes, pests and diseases. The trial was conducted in the confined field trial (CFT) in Lembang, West Java. The parental non-transgenic (NT) clones (Granola, Atlantic and Katahdin) and LBR hybrids (four clones of Atlantic x Katahdin SP951 hybrids; 10 clones of Granola x Katahdin SP951) were planted at a plant spacing of 30 cm x 70 cm. Fungicide applications were used as treat-ments (no spray, five and twenty times sprays). The experi-ment was arranged in a randomized completely block design with three replications. The parameters determined were popula-tions of N2 fixing and P solubilizing bacteria, soil C/N ratio as well as natural pests and diseases. The results showed that the transgenic LBR potato hybrids did not have negative effect on N fixing bacteria. The bacterial populations were around 1010-11 cells g-1 soil before planting, 1012 cells at 1.5 months after planting (MAP) and 108 cells after harvest. For P- solubilizing bacteria, their populations were 1010 cells before planting, 1012 cells at 1.5 MAP and 1011 cells g-1 soil after harvest. The soil C/N ratio of the transgenic plot was not statistically different compared to non-transgenic plot, i.e. 12-15 before planting, 10-11 at 1.5 MAP, and 10 after harvest in non-spray plot. Pests and diseases such as Alternaria solani, Liriomyza, potato tubber moth, aphid and mites on the transgenic and non-transgenic plots were statistically not different. The resistance score for A. solani was 7.2 (parental tansgenic) and 7.6 (parental non-transgenic); for Liriomyza it was 2.07 (parental transgenic) and 2.32 insect per plant (parental non-transgenic), the PTM was 0.63 (parental transgenic) and 0.73 insect per plant (parental non-transgenic), aphid and mites were 0.75 (parental transgenic) and 1.68 insects per plant (parental non-transgenic). The study indicated that LBR potato hybrids did not have any negative impacts on non-target organisms.

EFFECTS OF LATE BLIGHT RESISTANT POTATO CONTAINING RB GENE ON THE SOIL MICROBES, PESTS AND PLANT DISEASES

Late blight caused by Phytophthora infestans is an important disease on potato. Several potato hybrids have been generated by crossing local varieties (Atlantic and Granola) with Katahdin SP951 which contains late blight resistance gene RB. Prior to release, these hybrids need to be evaluated for their environ-mental effects on non-target organisms and natural pests and diseases. The objectives of the study were to investigate the effect of LBR potato hybrids on beneficial soil microbes, pests and diseases. The trial was conducted in the confined field trial (CFT) in Lembang, West Java. The parental non-transgenic (NT) clones (Granola, Atlantic and Katahdin) and LBR hybrids (four clones of Atlantic x Katahdin SP951 hybrids; 10 clones of Granola x Katahdin SP951) were planted at a plant spacing of 30 cm x 70 cm. Fungicide applications were used as treat-ments (no spray, five and twenty times sprays). The experi-ment was arranged in a randomized completely block design with three replications. The parameters determined were popula-tions of N2 fixing and P solubilizing bacteria, soil C/N ratio as well as natural pests and diseases. The results showed that the transgenic LBR potato hybrids did not have negative effect on N fixing bacteria. The bacterial populations were around 1010-11 cells g-1 soil before planting, 1012 cells at 1.5 months after planting (MAP) and 108 cells after harvest. For P- solubilizing bacteria, their populations were 1010 cells before planting, 1012 cells at 1.5 MAP and 1011 cells g-1 soil after harvest. The soil C/N ratio of the transgenic plot was not statistically different compared to non-transgenic plot, i.e. 12-15 before planting, 10-11 at 1.5 MAP, and 10 after harvest in non-spray plot. Pests and diseases such as Alternaria solani, Liriomyza, potato tubber moth, aphid and mites on the transgenic and non-transgenic plots were statistically not different. The resistance score for A. solani was 7.2 (parental tansgenic) and 7.6 (parental non-transgenic); for Liriomyza it was 2.07 (parental transgenic) and 2.32 insect per plant (parental non-transgenic), the PTM was 0.63 (parental transgenic) and 0.73 insect per plant (parental non-transgenic), aphid and mites were 0.75 (parental transgenic) and 1.68 insects per plant (parental non-transgenic). The study indicated that LBR potato hybrids did not have any negative impacts on non-target organisms.

RESISTANCE EVALUATION ON POPULATIONS OF CROSSES BETWEEN TRANSGENIC POTATO KATAHDIN RB AND NON-TRANSGENIC ATLANTIC AND GRANOLA TO LATE BLIGHT (Phytophthora infestans) IN CONFINED FIELD TRIAL

Late blight resistance gene (RB gene) isolated from Solanum bulbocastanum, is a broad resistance gene against all races of Phytophthora infestans. The gene was transformed into Katah-din event SP904 and SP951 using Agrobacterium tumefaciens and these transgenic plants have been crossed with susceptible potato cultivars Atlantic and Granola. Populations of the crosses have been molecularly characterized for the integration of the RB transgene. The study aimed to evaluate the resistance of the populations of crosses between transgenic Katahdin RB and susceptible non-transgenic parents (Atlantic and Granola) to late blight in a confined field trial at Pasir Sarongge, Cianjur, West Java. A total of 84 clones originated from four popula-tions were evaluated for resistance to late blight. These included 22 clones of Atlantic x transgenic Katahdin SP904, 16 clones of Atlantic x transgenic Katahdin SP951, 19 clones of Granola x transgenic Katahdin SP904, and 27 clones of Granola x transgenic Katahdin SP951. Observations of the late blight infection were conducted when late blight symptoms were detected, i.e. at 56, 60, 63, 70, and 77 days after planting (DAP). The result showed there were high variations in the resistance level of all the 84 clones tested. Clones of crosses between susceptible parents (Atlantic or Granola) and resistant parents (transgenic Katahdin SP904 or Katahdin SP951) showed a similar pattern based on the area under disease progress curve (AUDPC) value, i.e. 377.2 greater than the AUDPC of the resistant parents (180.1), but smaller than that of the susceptible parents (670.7). Observation at 77 DAP resulted four resistant potato clones having resistance score of 7.0-7.6, higher than the transgenic parents Katahdin SP904 (4.6) and Katahdin SP951 (6.8), i.e. clone B8 (Atlantic x transgenic Katahdin SP951) with resistance score of 7.6 and clones B26 (Atlantic x transgenic Katahdin SP951), C183 (Granola x transgenic Katahdin SP904), and D89 (Granola x transgenic Katahdin SP951) with resistance score of 7. These four transgenic potato resistant clones need to be further developed as promising potato clones to late blight.<br /><br />

Efikasi Gen RB pada Tanaman Kentang Transgenik Katahdin SP904 dan SP951 terhadap Empat Isolat Phytophthora infestans dari Jawa Barat

<p>Efficacy of RB gene in transgenic potato Katahdin SP904<br />and SP951 to West Java isolates of Phytophthora<br />infestans. A. Dinar Ambarwati, S.M. Sumaraw, Agus<br />Purwito, M. Herman, E. Suryaningsih, and Hajrial<br />Aswidinnoor. Potato late blight, caused by Phytophthora<br />infestans is one of the most devastating plant disease. Potato<br />yield losses due to this disease ranged from 47-100%. A<br />major late blight resistance gene, called RB, previously was<br />identified in the wild potato species Solanum<br />bulbocastanum. RB gene has been integrated into cultivated<br />potato Katahdin using Agrobacterium-mediated transformation,<br />and showed durable and broad spectrum resistance<br />either in laboratory assay or in confined field trial. Evaluation<br />of transgenic Katahdin SP904 and SP951 was conducted to<br />verify whether the RB gene with broad spectrum to all<br />known races of P. infestans in the United States and in<br />Toluca, Mexico was also effective against P. infestans<br />isolates in Indonesia. Efficacy of RB gene was evaluated for<br />foliar and tuber resistance to West Java isolates. Transgenic<br />Katahdin were more resistant in foliar than non transgenic<br />plants, at 14 days after inoculation. Diseases intensity of<br />transgenic Katahdin SP904 and SP951 were 19.8-43.8%,<br />whereas non transgenic Katahdin, Granola, and Atlantic<br />were 46.9-100%. In contrast to the foliar resistance<br />phenotype, RB-containing tubers in transgenic Katahdin did<br />not exhibit increased resistance to Lembang, Pangalengan<br />and Galunggung isolates. Tubers of transgenic Katahdin<br />SP904, SP951, and non transgenic Katahdin showed lesion<br />volume of 0.93, 0.91, and 0.91 cm3, respectively. RB gene in<br />transgenic Katahdin showed efficacy against late blight P.<br />infestans in foliar, but did not showed efficacy in tuber.<br />Transgenic Katahdin RB thus providing a potential source of<br />resistance for breeding programs.</p>