In vitro induction and selection of mutants obtained through gamma irradiation with improved processing traits in potato (Solanum tuberosum L.).

PURPOSE: This manuscript aimed for the generation of γ-irradiation derived mutants of potato genotype PAU/RR-1501 possessing desirable processing traits. MATERIALS AND METHODS: Nodal cuttings from virus-free explants were established on basal MS medium and irradiated with different doses (0, 5, 10 and 20 Gy) of γ-irradiation. The 5 and 10 Gy treated plantlets were multiplied and used for micro-tuber induction. Harvested micro-tubers were planted in pots for the selection and evaluation of mutants in M1V2 generation. RESULTS: Four weeks post-treatment, plantlets (5 Gy) showed enhanced growth as compared to the control while 20 Gy treatment exhibited completely ceased shoot growth. The highest number and weight of mini-tubers per plant was recorded for 10 Gy followed by 5 Gy treatment as compared to control. The γ-irradiation treatments caused changes in the skin color and shape of M1V2 tubers. CONCLUSION: Under the 5 Gy treatment 49.9% of clones produced exhibited cream and 8.53% brown skin color. Nine putative mutants were identified in genotype PAU/RR-1501 exhibiting promising processing traits.

Exploring the Potential Role of Ribosomal Proteins to Enhance Potato Resilience in the Face of Changing Climatic Conditions.

Potatoes have emerged as a key non-grain crop for food security worldwide. However, the looming threat of climate change poses significant risks to this vital food source, particularly through the projected reduction in crop yields under warmer temperatures. To mitigate potential crises, the development of potato varieties through genome editing holds great promise. In this study, we performed a comprehensive transcriptomic analysis to investigate microtuber development and identified several differentially expressed genes, with a particular focus on ribosomal proteins-RPL11, RPL29, RPL40 and RPL17. Our results reveal, by protein-protein interaction (PPI) network analyses, performed with the highest confidence in the STRING database platform (v11.5), the critical involvement of these ribosomal proteins in microtuber development, and highlighted their interaction with PEBP family members as potential microtuber activators. The elucidation of the molecular biological mechanisms governing ribosomal proteins will help improve the resilience of potato crops in the face of today's changing climatic conditions.

Solanum tuberosum Microtuber Development under Darkness Unveiled through RNAseq Transcriptomic Analysis.

Potato microtuber (MT) development through in vitro techniques are ideal propagules for producing high quality potato plants. MT formation is influenced by several factors, i.e., photoperiod, sucrose, hormones, and osmotic stress. We have previously developed a protocol of MT induction in medium with sucrose (8% w/v), gelrite (6g/L), and 2iP as cytokinin under darkness. To understand the molecular mechanisms involved, we performed a transcriptome-wide analysis. Here we show that 1715 up- and 1624 down-regulated genes were involved in this biological process. Through the protein-protein interaction (PPI) network analyses performed in the STRING database (v11.5), we found 299 genes tightly associated in 14 clusters. Two major clusters of up-regulated proteins fundamental for life growth and development were found: 29 ribosomal proteins (RPs) interacting with 6 PEBP family members and 117 cell cycle (CC) proteins. The PPI network of up-regulated transcription factors (TFs) revealed that at least six TFs-MYB43, TSF, bZIP27, bZIP43, HAT4 and WOX9-may be involved during MTs development. The PPI network of down-regulated genes revealed a cluster of 83 proteins involved in light and photosynthesis, 110 in response to hormone, 74 in hormone mediate signaling pathway and 22 related to aging.

Eradication of Potato Virus S, Potato Virus A, and Potato Virus M From Infected in vitro-Grown Potato Shoots Using in vitro Therapies.

Certain viruses dramatically affect yield and quality of potatoes and have proved difficult to eradicate with current approaches. Here, we describe a reliable and efficient virus eradication method that is high throughput and more efficacious at producing virus-free potato plants than current reported methods. Thermotherapy, chemotherapy, and cryotherapy treatments were tested alone and in combination for ability to eradicate single and mixed Potato virus S (PVS), Potato virus A (PVA), and Potato virus M (PVM) infections from three potato cultivars. Chemotherapy treatments were undertaken on in vitro shoot segments for four weeks in culture medium supplemented with 100 mg L-1 ribavirin. Thermotherapy on in vitro shoot segments was applied for two weeks at 40°C (day) and 28°C (night) with a 16 h photoperiod. Plant vitrification solution 2 (PVS2) and cryotherapy treatments included a shoot tip preculture followed by exposure to PVS2 either without or with liquid nitrogen (LN, cryotherapy) treatment. The virus status of control and recovered plants following therapies was assessed in post-regeneration culture after 3 months and then retested in plants after they had been growing in a greenhouse for a further 3 months. Microtuber production was investigated using in vitro virus-free and virus-infected segments. We found that thermotherapy and cryotherapy (60 min PVS2 + LN) used alone were not effective in virus eradication, while chemotherapy was better but with variable efficacy (20-100%). The most effective result (70-100% virus eradication) was obtained by combining chemotherapy with cryotherapy, or by consecutive chemotherapy, combined chemotherapy and thermotherapy, then cryotherapy treatments irrespective of cultivar. Regrowth following the two best virus eradication treatments was similar ranging from 8.6 to 29% across the three cultivars. The importance of virus removal on yield was reflected in "Dunluce" free of PVS having higher numbers of microtubers and in "V500' free of PVS and PVA having a greater proportion of microtubers > 5 mm. Our improved procedure has potential for producing virus-free planting material for the potato industry. It could also underpin the global exchange of virus-free germplasm for conservation and breeding programs.

In vitro evaluation of some high yield potato (Solanum tuberosum L.) cultivars under imposition of salinity at the cellular and organ levels.

Salinity and drought stress, which combines a lack of water and sodium toxicity, are more of the problems faced by plants and agricultural crops in newly reclaimed lands. Therefore, the direction of our research is to produce salinity-tolerant plants to increase the productivity of crops under conditions of salt stress. Potato callus was studied using different concentrations of NaCl (0.0, 50, 75, 100, 125, 150 and 200 mM). Shoot induction was obtained from callus treated with MS medium containing 4.0 and 5.0 mg l-1 TDZ + 0.5 mg l-1 GA3 with NaCl up to 125 mM and 150 mM for Rosetta and Victoria, respectively. When plantlets were cultured on MS medium containing 3.0 mg l-1 kinetin and 1.0 mg l-1paclobutrazol (PBZ) with 80 or 90 g l-1 sucrose after two months gave a good microtuber per explant of Rosetta and Victoria cultivar which gave number of microtuber/plantlet (1.85) and (2.40) when plantlets treated with 125 mM and 150 mM NaCl of Rosetta and Victoria cultivar, respectively. In general, the results were shown in each treatment of NaCl and that amounts of proline at 125 and 150 mMNaCl were significantly more than 0.0, 50, 75 and 100 mM NaCl. This result is related to the role of proline in the osmotic adjustment of a higher concentration of salinity. The results showed that the amounts of sodium increased with increasing the salt concentration, but the amount of potassium decreased and also increased the Na+/K+ ratio with increasing the salt concentration. This research is important for in vitro potato plant regeneration, which requires optimization before genetic transformation can be achieved.

Highly Efficient Agrobacterium-Mediated Transformation of Potato (Solanum tuberosum) and Production of Transgenic Microtubers.

The following method enables the rapid production of transgenic potato plants and microtubers for gene validation and expression, or promoter studies. The method is highly efficient, with reproducible transformation efficiencies of at least 50% to 60% with potato cv. Desiree, and can produce transgenic microtubers within 6 months of initiation of the experiment. Microtubers are produced in the absence of hormones, giving an in vitro gene testing system broadly analogous to the natural state. © 2018 by John Wiley & Sons, Inc.

Efectos de nitratos y sacarosa en la propagación in vitro de tres variedades de papa nativa / Effect of nitrates and sucrose on the in vitro propagation of three cultivars of venezuelan potatos

RESUMEN En Venezuela es prioridad rescatar las papas nativas por representar un gran alimento y un valioso recurso genético. Pero, desafortunadamente estas papas fueron desplazadas por variedades comerciales introducidas y las pocas semillas existentes están degeneradas y reutilizadas en continuos ciclos de siembra, No obstante, estandarizar las condiciones del cultivo aséptico es garantía de "semillas" rehabilitadas con mejor calidad fitosanitaria. Se planteó, entonces investigar el efecto de distintas concentraciones de nitratos y sacarosa en la propagación in vitro de variedades venezolanas ̀̀Cucubaʹ ̀̀Arbolona Negraʹ y ̀̀Rosadaʹ. Segmentos uninodales fueron cultivados e incubados en fotoperíodo de16 horas de luz (76 μmol m-2 s-1) y temperatura 19º C ± 1. Fue implementado el diseño estadístico factorial, estableciendo tres tratamientos partiendo del medio básico Murashige & Skoog (MS), sólido. Hubo un efecto significativo entre los tratamiento, al menos una de las medias es diferente a las otras, para las tres variedades la mayor inducción de crecimiento ocurrió, al modificar MS aumentando sus concentraciones de nitrato de amonio a 1,98 g L-1, nitrato de potasio 2,28 g L-1y bajando sacarosa a 20 g L-1, los vástagos desarrollaron 5,82 cm. de longitud promedio y las raíces 3 cm. Mientras, el MS (tratamiento control), presentó vástagos de 2,94cm. longitud promedio y sin enraizar. Entre las variedades ̀̀Cucuba´ obtuvo el mayor crecimiento. Posteriormente, las vitroplántulas resultantes, tratadas en MS líquido, con sacarosa al 8 % fueron inducidas a producir microtubérculos a los 90 días. Obteniendo así resultados prometedores para la propagación in vitro de las papas nativas.

ABSTRACT The recovery of native potato varieties is a current priority in Venezuela, given their value as a genetic resource of high nutritional quality. Unfortunately, native potato varieties were progressively replaced by exotic commercial, varieties. To date, the limited existing germplasm of native potato varieties is impoverished and of low quality, due to its continual reutilization in crop cycles. Nevertheless, efforts can be made to recover and standardize the production of quality propagules under adequate sanitary conditions. The aim of this study was to assay the effect of varying concentrations of nitrates and saccharose in tissue culture media of three Venezuelan varieties "Cucuba", "Arbolona Negra" and "Rosada". Unimodal segments were planted and incubated using a photoperiod of 16 h light (76 μmol photon m-2 s-1) at 19 ± 1 ºC. The experiment was designed following a standard factor analysis, consisting of three treatments, parting from the basic Murashige & Skoog (MS) medium and data were submitted to an multifactor ANOVA. Our findings indicate significant statistical differences amongst all of the treatments assayed, confirming that all of the varieties reached maximum physiological response under increasing concentrations of nitrates. Such was the case with 1.98 g L-1 ammonium nitrate and 2.28 g L-1 potassium nitrate using a concentration of saccharose 20g L-1. Mean shoot and root lengths under optimal concentrations were 5.82 cm and 3.0 cm, respectively. In contrast, MS basic culture media represented the treatment of least growth induction; yielding un rooted shoots of a mean length of 2.94 cm. Of these three native varieties, "Cucuba" proved to have the highest growth rates. All of the Vitroplantlets were then transferred to liquid MS media, with a saccharose concentration of 8 %, originating microtubers after 90 days. We conclude that these findings may be of use for massive in vitro production of native potato varieties.

Analysis of genetic stability of in vitro propagated potato microtubers using DNA markers.

The genetic stability of in vitro propagated potato microtubers was assessed using random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR), simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers. Microtubers were developed through in vitro from potato microplants using standardized protocols. The microtubers were conserved for 1 year under three different culture media and consequently microplants were regenerated for the DNA analyses. During the study, a total of 38 (10 RAPD, 11 ISSR, 12 SSR and 5 AFLP) primers produced a total of 407 (58 RAPD, 56 ISSR, 96 SSR and 197 AFLP) clear, distinct and reproducible amplicons. Cluster analysis revealed 100 % genetic similarity among the mother plant and its derivatives within the clusters by SSR, ISSR and RAPD analyses, whereas AFLP analysis revealed from 85 to 100 % genetic similarity. Dendrogram analysis based on the Jaccard's coefficient classified the genotypes into five clusters (I-V), each cluster consisting of mother plant and its derivatives. Principal component analysis (PCA) also plotted mother plant and its genotypes of each cluster together. Based on our results, it is concluded that AFLP is the best method followed by SSR, ISSR and RAPD to detect genetic stability of in vitro conserved potato microtubers. The in vitro conservation medium (T2) is a safe method for conservation of potato microtubers to produce true-to-type plans.

Evaluación en campo de plantas de ñame (Dioscorea alata L) obtenidas de los microtubérculos formados en sistema de inmersión temporal / Field evaluation of yam plants (Dioscorea alata L) obtained from microtubers produced in a temporary immersion system

Los microtubérculos en algunas especies de plantas constituyen una importante alternativa como material vegetal de plantación. Se definió como objetivo de trabajo evaluar en campo la respuesta morfoagronómica de las plantas obtenidas de los microtubérculos de ñame formados en Sistema de Inmersión Temporal (SIT). Como variantes experimentales se plantaron tres categorías de microtubérculos, clasificados según su masa fresca (I. de 0,5 a 0,9 g; II. de 1,0 a 2,9 g; III. igual o mayor de 3,0 g), plantas in vitro previamente aclimatadas y corona de tubérculo. Se evaluó el efecto de la masa fresca de los microtubérculos sobre su brote, supervivencia y posterior desarrollo de las plantas derivadas de ellos en campo. Con los microtubérculos de ñame, con una masa fresca igual o superior a 3,0 g, se alcanzó el más alto porcentaje de brotación (91,30%) y supervivencia de las plantas (96,50%), así como las mejores respuestas en los caracteres cuantitativos que se evaluaron en campo. Estos resultados confirmaron la importancia de la masa fresca de los microtubérculos para ser empleados como material vegetal de plantación directo en campo.

Microtubers in some plant species represent an important alternative crop-planting material. The presentwork involved field work for evaluating the morphoagronomic response of plants obtained from yam microtubersproduced in a temporary immersion system (TIS). Three categories of microtuber were planted asexperimental variants; they were classified by fresh mass (1 - 0.5 to 0.9 g, 2 - from 1.0 to 2.9 g and 3 - equal to or greater than 3.0 g), previously in vitro-acclimated plants and tuber crowns. The effect of microtuber freshweight on their sprouting, survival and later development of the plants derived from them in the field were evaluated. The highest sprouting (91.30%) and plant survival percentages (96.50%) and the best response in quantitative traits evaluated in the field were obtained with yam microtubers having a fresh mass equal to or greater than 3.0 g. These results confirmed the importance of microtubers’ fresh weight for using them as plant material in direct planting in the field.