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The main aim of the present investigation is modeling and optimization of a new culture medium for in vitro rooting of G×N15 rootstock using an artificial neural network-genetic algorithm (ANN-GA). Six experiments for assessing different media culture, various concentrations of Indole – 3- butyric acid, different concentrations of Thiamine and Fe-EDDHA were designed. The effects of five ionic macronutrients (NH 4 + , NO 3 − , Ca 2+ , K + and Cl − ) on five growth parameters [root number (RN), root length (RL), root percentage (R%), fresh (FW) and dry weight (DW)] were evaluated using the ANN-GA method. The R 2 correlation values of 0.88, 0.88, 0.98, 0.94 and 0.87 between observed and predicted values were acquired for all five growth parameters, respectively. The ANN-GA results indicated that among the input variables, K + (7.6) and NH4 + (4.4), K + (7.7) and Ca 2+ (2.8), K + (36.7) and NH 4 + (4.3), K + (14.7) and NH 4 + (4.4) and K + (7.6) and NH 4 + (4.3) had the highest values of variable sensitivity ratio (VSR) in the data set, for RN, RL, R%, FW and DW, respectively. ANN-GA optimized LS medium for G×N15 rooting contained optimized amounts of 1 mg L −1 IBA, 100, 150, or 200 mg L −1 Fe-EDDHA and 1.6 mg L −1 Thiamine. The efficiency of the optimized culture media was compared to other standard media for Prunus rooting and the results indicated that the optimized medium is more efficient than the others.

Background Flower development and sufficient fruit set are important parameters with respect to walnut yield. Knowledge about flowering genes of fruit trees can help to conduct better molecular breeding programs. Therefore, this study was carried out to investigate the expression pattern of some flowering genes ( FT , SOC1 , CAL , LFY and TFL1 ) in Persian walnut (cv. Chandler) during the growing season and winter dormancy. Results The results showed that walnut flower induction and initiation in Shahmirzad, Iran occurred in early June and late September, respectively. After meeting chilling and heat requirement, flower differentiation and anthesis occurred in late-March and mid-April to early-May, respectively. Study of flowering gene expression showed that the expression of the FT gene increased in three stages including before breaking of bud dormancy, from late March to late April (coincided with flower differentiation and anthesis) and from late May to mid-June (coincided with flower induction). Like FT , the expression of SOC1 gene increased during flower induction and initiation (mid-May to early-August) as well as flower anthesis (mid-April to early-May). LFY and CAL genes as floral meristem identity genes are activated by FT and SOC1 genes. In contrast with flowering stimulus genes, TFL1 showed overexpression during winter dormancy which prevented flowering. Conclusion The expression of FT gene activated downstream floral meristem identity genes including SOC1 , CAL and LFY which consequently led to release bud dormancy as well as flower anthesis and induction. Also, TFL1 as a flowering inhibitor gene in walnut showed overexpression during the bud dormancy. Chilling accumulation reduced TFL1 gene expression and increased the expression of flowering genes which ultimately led to overcome dormancy.

Background Pistachio ( Pistacia vera L.) growth, yield and quality are affected by abiotic stress especially drought. Understanding the strategies that improve dehydration tolerance is essential for developing resistant pistachio rootstocks. In the experiment, nine-month-old saplings of seven clonal interspecies hybrids of Pistacia atlantica × P. integerrima (C1, C2, C16-1, C8-3, C4-2, C9-4 and UCB1) were assessed for growth and physiological responses to water withholding and recovery. Result Water deficit negatively impacted growth parameters, including shoot dry weight, root dry weight and leaf area, in all hybrids; however, the C1 demonstrated relatively minor reductions compared to the other hybrids. Glycine betaine content in leaves increased by 49.4% in C9-4 and 47% in C1, while only 7% and 11% increases were found in the most sensitive clones, C8-3 and C4-2. Notably, C9-4, identified as the most tolerant clone, displayed the highest proline levels, with increases of 29.5% in leaves and 41.5% in roots, in contrast to C8-3, which showed minimal increases of 6% and 11% in leaves and roots, respectively. Clones with higher compatible solutes maintained higher relative water content (RWC), lower osmotic potential and smaller reductions in leaf water potential. RWC declined by just 6% in C9-4, whereas it dropped by 88% in C8-3. Osmotic potentials in C9-4 were − 1.61 MPa in leaves and − 0.271 MPa in roots, while in C8-3, they were − 0.93 MPa and − 0.11 MPa in leaves and roots, respectively. Following recovery, evaluations of growth, physiological traits and visual observations indicated that C8-3 had poor recovery ability. Heatmap and PCA analyses categorized the clones into three groups: “tolerant” (C9-4, C1 and C2), “moderately tolerant” (UCB1) and “sensitive” (C8-3, C4-2 and C16-1). Conclusion The results of this study underscore the significance of osmotic adjustment as a more critical trait compared to growth and stomatal parameters in effectively differentiating tolerant clones from sensitive ones.

Persian walnut ( Juglans regia ) has a considerable economic importance worldwide. However, the vigor and vitality of walnut trees were heavily affected by bark canker during the last few years. Irregular longitudinal cankers in the outer bark, stem tissue necrosis, and bleeding with black-colored exudates walnut trees were observed in Kermanshah, Hamedan, Markazi, Alborz, Isfahan, Qom, Semnan, and Razavi Khorasan provinces in western, central and eastern Iran during 2018 and 2019. A total of 150 symptomatic samples were collected from affected walnut trees in order to identify bacteria associated with walnut decline. Two-hundred sixty strains with a metallic green sheen were isolated on EMB-agar medium. The pathogenicity of all strains was proved by inoculating a suspension of the bacterial strains under the bark of immature walnut fruits cv. ‘Hartley’. Ninety-five strains caused necrosis and a dark-colored region in the mesocarp around the inoculation site 14 days post-inoculation. Moreover, 12 representative strains induced necrotic and black-colored tissues in the bark of young green twigs of two-year old walnut seedling cv. ‘Chandler’. The strains were classified into four categories based on conventional phenotypic characters confirmed with the 16S rRNA gene sequences. A phylogenetic tree based on the concatenated sequences of two housekeeping gene fragments, gyrB and infB , indicated that strains including I1, Q6, and S6 were grouped in a cluster with Gibbsiella quercinecans FBR97 T as well as strains I2, I5, and KE6 were clustered with Rahnella victoriana FRB 225 T . Moreover, strains MR1, MR3, and MR5 were grouped with the Enterobacter hormaechei subsp. hoffmannii DSM 14563 T . The phylogenetic analyses based on the partial sequencing of housekeeping genes including fusA , pyrG , and leuS revealed that strains KH1, KH3, and KH7 belong to Citrobacter braakii species. To the best of our knowledge, this is the first report of C. braakii and E. hormaechei as plant pathogens and R. victoriana associated with walnut decline.

Seed and seedling traits are affected by the conditions of the maternal environment, such as light, temperature, and nutrient availability. In this study, we have investigated whether different maternally applied nitrate and phosphate concentrations affect the seed and seedling performance of two closely related tomato species: Solanum lycopersicum cv. Money maker and Solanum pimpinellifolium accession CGN14498. We observed large differences for seed and seedling traits between the two species. Additionally, we have shown that for nitrate most of the seed and seedling traits were significantly affected by genotype–environment interactions (G×E). The effect of the maternal environment was clearly visible in the primary metabolites of the dry seeds. For example, we could show that the amount of γ-aminobutyric acid (GABA) in Money maker seeds was affected by the differences in the maternal environments and was positively correlated with seed germination under high temperature. Overall, compared with phosphate, nitrate had a larger effect on seed and seedling performance in tomato. In general, the different responses to the maternal environments of the two tomato species showed a major role for G×E in shaping seed and seedling traits.

Water scarcity, intensified by climate change, poses a major challenge to pistachio production in arid and semi-arid regions of the world. Water stress in these areas drastically reduces yield and endangers the long-term sustainability of orchards. Deficit irrigation (DI) is widely recognized as a water-saving strategy to optimize crop production under limited water resources. This study investigated and compared the key components of drought adaptation strategies in four pistachio varieties—‘Badami’, ‘Akbari’, 'Ahmad-Aghaei’ and 'Kale-Ghochi’—under DI in controlled conditions. A factorial experiment was conducted using a completely randomized design (CRD) to assess the effects of three DI levels: full irrigation (control), mild (DI30%) and severe (DI60%), on two-month-old pistachio seedlings over 10 weeks. We comprehensively assessed morphological and physiological characteristics, including root and shoot growth and stomatal traits. Photosynthetic pigment concentrations and relative water content were also measured. Based on the results, distinct varietal adaptation mechanisms emerged: ‘Badami’ exhibited a well-developed root system and enhanced osmotic adjustment, facilitating drought avoidance; ‘Akbari’ demonstrated robust aerial resistance through efficient stomatal regulation and sustained pigment retention, indicative of dehydration tolerance; 'Ahmad-Aghaei’ showed a balanced root–shoot coordination, especially under mild DI, suggesting moderate adaptability; whereas 'Kale-Ghochi’ displayed the least drought resistance, characterized by restricted root growth and impaired water status. Multivariate analyses integrating these traits effectively discriminated the varieties based on their drought response profiles. These results indicate the potential of drought-tolerant varieties, particularly ‘Badami’ to achieve up to 30% water savings under DI, a practical approach to sustaining pistachio production in regions facing water scarcity.

Climate change may cause a significant decline in winter chill over the coming decades and is becoming an important issue for walnut ( Juglans regia L.) breeding programs to address. The current study was conducted to identify superior walnut genotypes with low-chilling requirements in western Iran (Ilam province). In the first step, approximately 200 walnut trees of seed-origin were pre-selected based on a questionnaire. Based on this primary evaluation for phenotypic and horticultural traits, 55 of the 200 genotypes were selected for further study. Morphological and phenological traits of these 55 were evaluated for two consecutive years and 11 genotypes were selected as superior. These superior genotypes were moderate to late-leafing with high yield, lateral fruitfulness, thin shells, and light to extra light kernel color. Nut weight, kernel weight and kernel percentage of the 11 superior genotypes ranged between 12.6–15.9 g, 6.9–8.2 g, and 49.7–62.0%, respectively. These superior genotypes had high levels of protein (13.3–18.2%), oil (55.3–65.9%) and PUFAs content. Assessment of chilling requirement of these superior genotypes showed that eight (IrIlZg1, IrIlZg13, IrIlZg7, IrIlZg12, IrIlAa92, IrIlAa47, IrIlAa48 and IrIlAa27) had significantly lower chilling requirements than the cultivar ‘Chandler’. Although having low-chill requirements, these genotypes were moderate to late-leafing. This may be due to their high heat requirement, since chilling requirement together with heat accumulation determines the leafing and flowering date. In conclusion, the selected low chilling requirement genotypes having moderate to late leafing could be used in walnut breeding programs to release commercial cultivars which are suitable for areas with spring frost concerns and expected future loss of winter chill due to climate change.

Light plays a crucial role in photosynthesis, which is an essential process for plantlets produced during in vitro tissue culture practices and ex vitro acclimatization. LED lights are an appropriate technology for in vitro lighting but their effect on propagation and photosynthesis under in vitro condition is not well understood. This study aimed to investigate the impact of different light spectra on growth, photosynthetic functionality, and stomatal characteristics of micropropagated shoots of Persian walnut (cv. Chandler). Tissue-cultured walnut nodal shoots were grown under different light qualities including white, blue, red, far-red, green, combination of red and blue (70:30), combination of red and far-red (70:30), and fluorescent light as the control. Results showed that the best growth and vegetative characteristics of in vitro explants of Persian walnut were achieved under combination of red and blue light. The biggest size of stomata was detected under white and blue lights. Red light stimulated stomatal closure, while stomatal opening was induced under blue and white lights. Although the red and far-red light spectra resulted in the formation of elongated explants with more lateral shoots and anthocyanin content, they significantly reduced the photosynthetic functionality. Highest soluble carbohydrate content and maximum quantum yield of photosystem II were detected in explants grown under blue and white light spectra. In conclusion, growing walnut explants under combination of red and blue lights leads to better growth, photosynthesis functionality, and the emergence of functional stomata in in vitro explants of Persian walnuts.

Persian walnut ( Juglans regia L.) frequently encounters simultaneous drought and heat stress in its natural habitat, impacting its physiology and biochemistry. Therefore, understanding the physiological and molecular responses to these combined stresses is essential for improving stress resilience in this species. The present study investigates the effects of drought stress (DS), heat stress (HS), combined drought-heat stress (DH) and control condition (CC) on the antioxidant defenses and gene expression in own-rooted walnut plants (cv. Chandler) under controlled greenhouse conditions. Combined stress significantly increased malondialdehyde and hydrogen peroxide by 2.55- and 2.05-fold, respectively, while reducing membrane stability index by 0.43-fold compared to CC. Total chlorophyll declined by 0.18- and 0.09-fold under DS and HS, respectively, with the greatest reduction (0.37-fold) under DH. Levels of phenylalanine ammonia-lyase, polyphenol oxidase, superoxide dismutase, catalase, and guaiacol peroxidase rose 2.5- to sixfold under DH compared to CC. Similarly, phenolic compounds and antioxidant capacity increased between 1.49- and 3.55-fold relative to CC. Gene expression analyses of NCED6 , PPO , and GST revealed 2- to sixfold upregulation under both individual and combined stress conditions compared to control. These results highlight the crucial roles of these genes in activating antioxidant defense pathways and detoxification processes that mitigate oxidative damage in walnut under stress. Following stress relief, walnut seedlings displayed notable recovery in photosynthetic function and antioxidant activity. These findings indicate that combined drought and heat stress exerts a stronger impact than either stress alone, underscoring key adaptive mechanisms that may enhance walnut resilience to climate variability.

Background Root system architecture is a key determinant of plant establishment, particularly in species such as Pistachio ( Pistacia vera L.), which inherently display limited lateral root formation and low survival rates after transplanting. This study evaluated the impact of radicle-tip excision on root architecture and overall seedling vigor in ‘Ohadi’ pistachio cultivar. A factorial experiment was conducted using five distinct radicle length categories at the time of excision: L1 (0.5–1.0 cm), L2 (1.01–2.0 cm), L3 (2.01–3.0 cm), L4 (3.01–4.0 cm) and L5 (4.01–5.0 cm), and three cutting sizes of 1 mm (CS1), 3 mm (CS3) and 5 mm (CS5) while untreated seedlings were used as the control group. Following radicle-tip excision, the seedlings were grown in perlite-filled pots and irrigated weekly with half-strength Hoagland’s nutrient solution. Growth, vitality, and root architectural traits were assessed at end of the experiment. Result Remarkably, the L3CS3 treatment involving excision of 3 mm from the radicle tip when its length reached L3 (2.01–3.0 cm) resulted in significant enhancements in key morphological traits, including shoot and root fresh/dry weights, leaf area and plant height. In addition, root system characteristics such as the number of lateral roots (NLR), network depth (NWDP), network volume (NWVL), and convex area (NWCA) showed substantial improvement. Ordinal regression analysis revealed a strong relationship between lateral root traits and overall seedling vigor, confirming the physiological relevance of the root manipulation. Complementary correlation analysis further supported the notion that improved root traits positively influence shoot growth, indicating an integrative effect. Conclusion These findings underscore the potential of radicle-tip excision as a cost-effective and scalable approach to improve root functionality and transplant performance in pistachio, with broader applicability to other species that exhibit limited lateral root development in nursery production systems. Graphical abstract