Potato tuberworm, Phthorimaea operculella (Zeller), is the most serious insect pest of potatoes worldwide. The introduction of the Bacillus thuringiensis (Bt) toxin gene through genetic engineering offers host plant resistance for the management of potato tuberworm. We report on the field and storage studies to evaluate Bt-cry5 potato lines for resistance to potato tuberworm in Egypt under natural infestations and their agronomic performance in both Egypt and Michigan. From 1997 to 2001, field experiments were conducted at the International Potato Center (CIP) Research Station, Kafr El-Zyat, Egypt, and/or Agricultural Genetic Engineering Institute (AGERI), Giza, Egypt, to evaluate resistance to tuberworm. A total of 27 Bt-transgenic potato lines from six different Bt constructs were evaluated over a 5-yr period. After harvest and evaluation of the agronomic trials, storage evaluation of potato tuberworm damage was done at the CIP Research Station. The 1997 field trial was the first field test of genetically engineered crops in Egypt. Field tests to assess potato tuberworm resistance in Egypt were able to differentiate between the Bt-transgenic lines and the nontransgenic lines/cultivars in 1999, 2000, and 2001. The Bt-cry5-Spunta lines (Spunta-G2, Spunta-G3, and Spunta-6a3) were the most resistant lines in field with 99-100% of tubers free of damage. In the 2001 storage study, these lines were also over 90% free of tuberworm damage after 3 mo. NYL235-4.13, which combines glandular trichomes with the Bt-cry5/gus fusion construct, also had a high percentage of clean tubers in the field studies. In agronomic field trials in Michigan from 1997 to 2001, the Bt-transgenic lines in most instances performed similar to the nontransgenic line in the agronomic trials; however, in Egypt (1998-1999), the yields were less than one-half of those in Michigan. Expression of the Bt-cry5 gene in the potato tuber and foliage will provide the seed producer and grower a tool in which to reduce potato tuberworm damage to the tuber crop in the field and storage.

A dose-effect relationship has been established for high-energy neutrons (maximum energy 600 MeV) within a dose range of 0.2 to 80 cGy and for low-energy neutrons produced by a 252 Cf source (mean energy 2.35 MeV) for doses between 0.2 and 5 cGy. The number of micronuclei produced by neutron radiations was found to increase linearly with dose. The Relative Biological Effectiveness (RBE) values calculated using 6 °Co radiation as a reference were, in the high-dose ·region, 4.7 ± 0.4 and 11.8 ± 1.3 for the high-and low-energy neutrons, respectively. At doses below 1 cGy constant values of 25.4 ± 4.4 and 63.7 ± 12 were reached for the respective neutron energies. (Submitted to Int. J. Radiat. Biol.) *) Present address: Rad. Environ. Lab., Brookhaven National Laboratory, Upton, NY, USA.-iii-RESUME Nous avons etabli des courbes d'effet de dose pour des neutrons de haute energie (energie max.: 600 MeV) pour des doses comprises entre 0,2 et 80 cGy, et pour des neutrons de basse energie, provenant d'une source de 252 Cf (energie moyenne: 2,35 MeV), pour des doses entre 0,2 et 5 cGy. Le nombre de micronoyaux augmente lineairement avec la dose. Aux hautes energies, les valeurs de l'efficacite bio-logique relative (EBR) calculee en utilisant les rayons y du 60 co comme radiations de reference etaient de 4,7 ± 0,4 et 11,8 ± 1,3 respectivement pour les neutrons de haute et de basse energie. A des doses inferieures a 1 cGy, nous avons trouve, pour les deux energies, des valeurs constantes de l'EBR: 25,4 ± 4,4 et 63,7 ± 12. ABRISS Eine Dosis-Effekt-Beziehung wurde aufgestellt fur hochenergetische Neutronen (max. Energie 600 MeV) bei Bestrahlungsdosen von 0,2 bis 80 cGy und.fur Neutronen niederer Energien (mittlere Energie 2,35 MeV), die aus einer 252 Cf Quelle produziert wurden, mit Bestrahlungsdosen zwischen 0,2 und 5 cGy. Es zeigte sich, dass die Zahl der durch die Neutronenbestrahlung produzierten Mikrokerne linear mit der Dosis ansteigt. Die Werte fur Relative Biologische Wirksamkeit (RBW) wurden unter Bezugnahme auf Resultate mit 60 co Gammastrahlen berechnet und betrugen bei hohen Bestrahlungsdosen 4,7 ± 0,4 fur hochenergetische Neutronen und 11,8 ± 1,3 fur Neu-tronen niederer Energien. Bei Bestrahlungsdosen unter 1 cGy ergaben sich fur beiden Neutronenenergien konstante Werte von 25,4 ± 4,4 bzw. 63,7 ± 12.

Background The potato tuber starch trait is affected by the composition of amylose and amylopectin. The synthesis of amylose in amyloplasts is catalyzed by granule-bound starch synthase (GBSS). GBSS gene expression was inhibited via CRISPR-Cas9-mediated genome editing in leaves of the Desiree Potato cultivar. Constructs containing the Cas9 gene and sgRNAs targeting the GBSS gene were introduced by Agrobacterium-mediated transformation delivery into protoplasts. Methods and results Outcomes included lines with mutations in all or only some of the homoeoalleles of GBSS genes and lines in which homoeoalleles carried different mutations and multiple alleles that were up to 21 % of regenerated shoots. Mutations were found in one bp from the used guide sequence, verifying the high homology between a guide sequence and a target region near the protospacer adjacent motif (PAM) site. DNA delivery into protoplast resulted in mutants with no detectable Cas9 gene. Microscopic evaluation of iodine-stained starch granules was shown to be a sensitive system for qualitative and quantitative examination of amylose formation in starch granules of transgenic potato tubers. Conclusion This study presented the successful application of CRISPRCas9 to fully knockout a GBSS gene function in a potato tetraploid plant in one round of transfection, without a stable introduction of DNA into the genome. In this study, the region of the GBSS gene was targeted, for driving the guide sequences. all the different experiments yielded high mutation frequencies, in the same order of magnitude, demonstrating the robustness of the CRISPR-Cas9 technique for potato research and breeding. Here, also the use of this new technology was demonstrated to develop a trait of commercial interest, an amylopectin potato starch, with uses in both food and technical applications.

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Plants are severely affected by many biotic stresses, which cause a reduction in crop quality and quantity. One of the strategies to manage biotic stresses is the generation of transgenic plant lines that can be used as biosensors. These biosensor plants can trigger an early warning upon any pathogen infection. Two promoters with β-glucuronidase reporter gene fusions were constructed. The first contained the flagellin sensing 2 gene promoter, whereas the second contained synthetic promoter containing four repeats of cis-acting elements from the pathogen-related protein 1 gene and two transcription enhancers from the 35S promoter. Transformed leaves were treated with a phytohormone salicylic acid to mimic the occurrence of biotic stress. Validation of reporter gene expression induced from both constructs in transformed potato leaves displayed an increase upon salicylic acid treatment. The results reflect that both constructs could serve in the production of potato biotic stress biosensors.