Growth, photosynthetic function, and stomatal characteristics of Persian walnut explants in vitro under different light spectra.

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.

Natural variation in photosynthesis and water use efficiency of locally adapted Persian walnut populations under drought stress and recovery.

Persian walnut is a drought-sensitive species with considerable genetic variation in the photosynthesis and water use efficiency of its populations, which is largely unexplored. Here, we aimed to elucidate changes in the efficiency of photosynthesis and water content using a diverse panel of 60 walnut families which were submitted to a progressive drought for 24 days, followed by two weeks of re-watering. Severe water-withholding reduced leaf relative water content (RWC) by 20%, net photosynthetic rate (Pn) by 50%, stomatal conductance (gs) by 60%, intercellular CO2 concentration (Ci) by 30%, and transpiration rate (Tr) by 50%, but improved water use efficiency (WUE) by 25%. Severe water-withholding also inhibited photosystem II functionality as indicated by reduced quantum yield of intersystem electron transport (φEo) and transfer of electrons per reaction center (ET0/RC), also enhanced accumulation of QA (VJ) resulted in the reduction of the photosynthetic performance (PIABS) and maximal quantum yield of PSII (FV/FM); while elevated quantum yield of energy dissipation (φDo), energy fluxes for absorption (ABS/RC) and dissipated energy flux (DI0/RC) in walnut families. Cluster analysis classified families into three main groups (tolerant, moderately tolerant, and sensitive), with the tolerant group from dry climates exhibiting lesser alterations in assessed parameters than the other groups. Multivariate analysis of phenotypic data demonstrated that RWC and biophysical parameters related to the chlorophyll fluorescence such as FV/FM, φEo, φDo, PIABS, ABS/RC, ET0/RC, and DI0/RC represent fast, robust and non-destructive biomarkers for walnut performance under drought stress. Finally, phenotype-environment association analysis showed significant correlation of some photosynthetic traits with geoclimatic factors, suggesting a key role of climate and geography in the adaptation of walnut to its habitat conditions.

Clonal Propagation of Walnuts (Juglans spp.): A Review on Evolution from Traditional Techniques to Application of Biotechnology.

Walnuts (Juglans sp.) are allogamous species. Seed-derived plants are not always superior to the selected parent. Clonal propagation of selected stock plants is an essential requirement for the clonal fidelity of the descendants and to maintain their genetic structure. Selection of the desired plant is realized only after reaching maturity, and characterizing and evaluating the performance of adult trees require a long time. Clonal propagation methods ensure proper transmission of characters to descendants and can be used effectively in breeding programs. The commercialization of a cultivar or rootstock depends on the success of vegetative propagation. Walnuts, like other tree species, are recalcitrant to conventional vegetative propagation methods and even non-conventional in vitro culture (micropropagation). Elucidation of factors determining the success of cloning of desired plants would contribute to understanding current limitations for most genotypes of Juglans. We outline the role of grafting and cuttings and stool layering, as well as in vitro culture on walnut multiplication. These techniques are, in practice, entirely different; nevertheless, they are affected by common factors. The incompatibility of stock-scion and the reduced ability of stem cuttings to root are the main bottlenecks for grafting and cutting, respectively. Genotype, age, and physiological status, reinvigoration or rejuvenation-treatment of donor plant, period of harvesting and processing of explants critically affect the results of methods followed. The in vitro culture technology is the most suitable for walnut cloning. This also has constraints that affect commercial propagation of most desired genotypes. We describe comprehensive results and synthesis in this review on the asexual reproduction of walnuts, providing a better comprehension of the limiting factors and the ways to overcome them, with direct implications on commercial propagation and the releasing of outstanding genotypes.

Genome-wide association analysis and pathway enrichment provide insights into the genetic basis of photosynthetic responses to drought stress in Persian walnut.

Uncovering the genetic basis of photosynthetic trait variation under drought stress is essential for breeding climate-resilient walnut cultivars. To this end, we examined photosynthetic capacity in a diverse panel of 150 walnut families (1500 seedlings) from various agro-climatic zones in their habitats and grown in a common garden experiment. Photosynthetic traits were measured under well-watered (WW), water-stressed (WS) and recovery (WR) conditions. We performed genome-wide association studies (GWAS) using three genomic datasets: genotyping by sequencing data (∼43 K SNPs) on both mother trees (MGBS) and progeny (PGBS) and the Axiom™ Juglans regia 700 K SNP array data (∼295 K SNPs) on mother trees (MArray). We identified 578 unique genomic regions linked with at least one trait in a specific treatment, 874 predicted genes that fell within 20 kb of a significant or suggestive SNP in at least two of the three GWAS datasets (MArray, MGBS, and PGBS), and 67 genes that fell within 20 kb of a significant SNP in all three GWAS datasets. Functional annotation identified several candidate pathways and genes that play crucial roles in photosynthesis, amino acid and carbohydrate metabolism, and signal transduction. Further network analysis identified 15 hub genes under WW, WS and WR conditions including GAPB, PSAN, CRR1, NTRC, DGD1, CYP38, and PETC which are involved in the photosynthetic responses. These findings shed light on possible strategies for improving walnut productivity under drought stress.

Characterization of plant growth-promoting rhizobacteria (PGPR) in Persian walnut associated with drought stress tolerance.

There is a lack of information on the rhizosphere of nut-bearing trees where microbial populations can benefit roots and tree growth. The current research aimed at discovering plant growth-promoting rhizobacteria (PGPR) in the rhizosphere of soil samples from around the root zone of six walnut trees, each of which was considered as a genotype, i.e. 'TT1', 'TT2', 'SS2', 'ZM1', 'Chandler' and 'Haward'. The trees grew in different arid and semiarid regions of Iran and Turkey. The strains were isolated and identified based on different morphological and biochemical markers. Drought-stress tolerance was assessed in the case of each isolate through their transfer to culture medium, containing polyethylene glycol (PEG6000) at 0 and 373.80 g L-1. Resilient strains were analyzed for measuring their ability to produce siderophore, hydrogen cyanide (HCN), Indole-3-acetic acid (IAA) and Gibberellic acid (GA3). In sum, 211 isolates were identified, of which a large number belonged to the Bacillus genus and, specifically, 78% of the strains were able to grow under drought stress conditions. The genus Arthrobacter was only detected in the rhizosphere of 'ZM1', 'Haward' and 'TT1' genotypes. In 4% of the strains, IAA production exceeded 53 mg L-1, while a high level of phosphorus solubility was verified in 6% of the strains. No strain was found to have the capability of producing HCN. The strains were screened for drought-tolerance, which resulted in the discovery of two promising strains, i.e. ZM39 and Cha43. Based on molecular identification through amplification and sequencing of the 16S rDNA gene, these two strains seemed to belong to Bacillus velezensis and Bacillus amyloliquefaciens, respectively. The discovery of new PGPR strains could probably assist walnut trees in improving their mechanisms of adaptation to drought stress.

Inhibition of browning via aqueous gel solution of Aloe vera: a new method for preserving fresh fruits as a case study on fresh kernels of Persian walnut.

Aloe Vera (AV) gel is commonly used as a natural, inexpensive, edible coating that can improve the quality and shelf life of fruits. The objective of this study was to evaluate how two methods of applying AV, i.e. as an edible coating (dry environment) and as a gel solution (aqueous environment: a new method), prevent browning and maintain quality characteristics of fresh kernels of Persian walnut for 60 days during cold storage. Distilled water was used as a control group for both environments. In general, AV caused a reduction in the peroxide value (POV) of kernels, while preserving Total Phenolic Compound and Total Antioxidant Activity (TAA). The AV treatment slowed down the process of color change and maintained sensory properties during storage, compared to the control groups of both methods. The AV gel solution performed better than the AV edible coating in terms of POV, color (L* and h°) and microbial growth. In contrast, the AV edible coating was more effective in preserving TPC and TAA. Also, TAA was found to have a significant, positive correlation with L* and, simultaneously, a negative correlation with POV. As far as we know, this is the first instance that the AV gel was used as a formulated solution and as an edible coating on fresh fruits. This innovative method can be used in commercial practice, while being ecofriendly and non-chemical as a treatment for the maintenance of postharvest quality in fruits.

Predictive modeling of Persian walnut (Juglans regia L.) in vitro proliferation media using machine learning approaches: a comparative study of ANN, KNN and GEP models.

BACKGROUND: Optimizing plant tissue culture media is a complicated process, which is easily influenced by genotype, mineral nutrients, plant growth regulators (PGRs), vitamins and other factors, leading to undesirable and inefficient medium composition. Facing incidence of different physiological disorders such as callusing, shoot tip necrosis (STN) and vitrification (Vit) in walnut proliferation, it is necessary to develop prediction models for identifying the impact of different factors involving in this process. In the present study, three machine learning (ML) approaches including multi-layer perceptron neural network (MLPNN), k-nearest neighbors (KNN) and gene expression programming (GEP) were implemented and compared to multiple linear regression (MLR) to develop models for prediction of in vitro proliferation of Persian walnut (Juglans regia L.). The accuracy of developed models was evaluated using coefficient of determination (R2), root mean square error (RMSE) and mean absolute error (MAE). With the aim of optimizing the selected prediction models, multi-objective evolutionary optimization algorithm using particle swarm optimization (PSO) technique was applied. RESULTS: Our results indicated that all three ML techniques had higher accuracy of prediction than MLR, for example, calculated R2 of MLPNN, KNN and GEP vs. MLR was 0.695, 0.672 and 0.802 vs. 0.412 in Chandler and 0.358, 0.377 and 0.428 vs. 0.178 in Rayen, respectively. The GEP models were further selected to be optimized using PSO. The comparison of modeling procedures provides a new insight into in vitro culture medium composition prediction models. Based on the results, hybrid GEP-PSO technique displays good performance for modeling walnut tissue culture media, while MLPNN and KNN have also shown strong estimation capability. CONCLUSION: Here, besides MLPNN and GEP, KNN also is introduced, for the first time, as a simple technique with high accuracy to be used for developing prediction models in optimizing plant tissue culture media composition studies. Therefore, selection of the modeling technique to study depends on the researcher's desire regarding the simplicity of the procedure, obtaining clear results as entire formula and/or less time to analyze.

Molecular and morphological evaluation of transgenic Persian walnut plants harboring Fld gene under osmotic stress condition.

BACKGROUND: Soil drought stress is a limiting factor of productivity in walnut (Juglans regia L). Ferredoxin (Fd) level decreases under adverse environmental stress. Functional replacement of decreased Fd by Fld (Flavodoxin) had been shown to have protective effect under abiotic stress condition. This study aimed to evaluate four transgenic lines (L3, L4, L13 and L17) along with non-transgenic line under three osmotic stresses levels (0, 10 and 12% PEG). METHODS AND RESULTS: This experiment carried out based on a completely randomized design with four replications. To confirm that the Fld gene is successfully integrated into the walnut genome, PCR and dot blot analysis were carried out. The transgenic lines of walnut expressing Fld displayed increased tolerance to osmotic stress at 10 and 12% PEG condition. Lines expressing Fld exhibited increasing tolerance to drought stress and maintained health of plants under osmotic conditions. Results of real time PCR showed that expression level of Fld gene in L4 was higher than the others. Among transgenic lines, L4 was more tolerant than other lines under osmotic stress. CONCLUSIONS: These findings indicate that expression of Fld gene can increase tolerance to osmotic stress in Persian walnut and is useful tool for walnut production in arid and semi-arid regions.

Advances in Rootstock Breeding of Nut Trees: Objectives and Strategies.

The production and consumption of nuts are increasing in the world due to strong economic returns and the nutritional value of their products. With the increasing role and importance given to nuts (i.e., walnuts, hazelnut, pistachio, pecan, almond) in a balanced and healthy diet and their benefits to human health, breeding of the nuts species has also been stepped up. Most recent fruit breeding programs have focused on scion genetic improvement. However, the use of locally adapted grafted rootstocks also enhanced the productivity and quality of tree fruit crops. Grafting is an ancient horticultural practice used in nut crops to manipulate scion phenotype and productivity and overcome biotic and abiotic stresses. There are complex rootstock breeding objectives and physiological and molecular aspects of rootstock-scion interactions in nut crops. In this review, we provide an overview of these, considering the mechanisms involved in nutrient and water uptake, regulation of phytohormones, and rootstock influences on the scion molecular processes, including long-distance gene silencing and trans-grafting. Understanding the mechanisms resulting from rootstock × scion × environmental interactions will contribute to developing new rootstocks with resilience in the face of climate change, but also of the multitude of diseases and pests.

Anti-seizure effects of walnut peptides in mouse models of induced seizure: The involvement of GABA and nitric oxide pathways.

Epilepsy is one of the foremost medical disorders. Oxidative stress is a well-known mechanism in epileptogenesis, and many studies suggest that oxidative stress affects the onset and evolution of epilepsy. Here we evaluated the walnut peptide extracts' anti-seizure property in three different mouse seizure models including pentylenetetrazole-induced clonic seizure, chemical kindling, and maximal electroshock. Walnut peptides (20 mg/Kg) were administered by intraperitoneal (IP) injection of mice 60 min before seizure induction in the three models. To delineate the mechanisms of walnut peptides anti-seizure activity, we evaluated the impact of diazepam, flumazenil, and a NOS inhibitor on this activity. Intraperitoneal administration of walnut peptides significantly increased the seizure threshold. Our results also demonstrated that walnut peptides exert their anti-seizure properties through the modulation of benzodiazepine receptors. Thus, walnut peptides may be considered as a new anti-convulsion agent, which can reduce seizure occurrence and slow down seizure progression.

Production of Haploid and Doubled Haploid Lines in Nut Crops: Persian Walnut, Almond, and Hazelnut.

This chapter deals with induction of haploidy via parthenogenesis in Persian walnut and via microspore embryogenesis in almond and hazelnut. Haploid induction through in situ parthenogenesis using pollination with irradiated pollen to stimulate the embryogenic development of the egg cell, followed by in vitro culture of the immature haploid embryos. Microspore embryogenesis allows the induction of immature pollen grains (microspores), to move away from the normal gametophytic developmental route in the direction of the sporophytic one, yielding homozygous organisms (embryos in this case). Unlike other fruit crops (such as Citrus), regeneration of entire plants has not yet been obtained in our studied nut crops; however, it gives the methodology should be used to continue the roadmap.

Combining independent de novo assemblies to optimize leaf transcriptome of Persian walnut.

Transcriptome resources can facilitate to increase yield and quality of walnuts. Finding the best transcriptome assembly has not been the subject of walnuts research as yet. This research generated 240,179,782 reads from 11 walnut leaves according to cDNA libraries. The reads provided a complete de novo transcriptome assembly. Fifteen different transcriptome assemblies were constructed from five different well-known assemblers used in scientific literature with different k-mer lengths (Bridger, BinPacker, SOAPdenovo-Trans, Trinity and SPAdes) as well as two merging approaches (EvidentialGene and Transfuse). Based on the four quality metrics of assembly, the results indicated an efficiency in the process of merging the assemblies after being generated by de novo assemblers. Finally, EvidentialGene was recognized as the best assembler for the de novo assembly of the leaf transcriptome in walnut. Among a total number of 183,191 transcripts which were generated by EvidentialGene, there were 109,413 transcripts capable of protein potential (59.72%) and 104,926 were recognized as ORFs (57.27%). In addition, 79,185 transcripts were predicted to exist with at least one hit to the Pfam database. A number of 3,931 transcription factors were identified by BLAST searching against PlnTFDB. Furthermore, 6,591 of the predicted peptide sequences contained signaling peptides, while 92,704 contained transmembrane domains. Comparison of the assembled transcripts with transcripts of the walnut and published genome assembly for the 'Chandler' cultivar using the BLAST algorithm led to identify a total number of 27,304 and 19,178 homologue transcripts, respectively. De novo transcriptomes in walnut leaves can be developed for the future studies in functional genomics and genetic studies of walnuts.

Flowering in Persian walnut: patterns of gene expression during flower development.

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.

Combining phenotype, genotype, and environment to uncover genetic components underlying water use efficiency in Persian walnut.

Walnut production is challenged by climate change and abiotic stresses. Elucidating the genomic basis of adaptation to climate is essential to breeding drought-tolerant cultivars for enhanced productivity in arid and semi-arid regions. Here, we aimed to identify loci potentially involved in water use efficiency (WUE) and adaptation to drought in Persian walnut using a diverse panel of 95 walnut families (950 seedlings) from Iran, which show contrasting levels of water availability in their native habitats. We analyzed associations between phenotypic, genotypic, and environmental variables from data sets of 609 000 high-quality single nucleotide polymorphisms (SNPs), three categories of phenotypic traits [WUE-related traits under drought, their drought stress index, and principal components (PCs)], and 21 climate variables and their combination (first three PCs). Our genotype-phenotype analysis identified 22 significant and 266 suggestive associations, some of which were for multiple traits, suggesting their correlation and a possible common genetic control. Also, genotype-environment association analysis found 115 significant and 265 suggestive SNP loci that displayed potential signals of local adaptation. Several sets of stress-responsive genes were found in the genomic regions significantly associated with the aforementioned traits. Most of the candidate genes identified are involved in abscisic acid signaling, stomatal regulation, transduction of environmental signals, antioxidant defense system, osmotic adjustment, and leaf growth and development. Upon validation, the marker-trait associations identified for drought tolerance-related traits would allow the selection and development of new walnut rootstocks or scion cultivars with superior WUE.

Applying the AOGCM-AR5 models to the assessments of land suitability for walnut cultivation in response to climate change: A case study of Iran.

Due to higher temperatures and lower water availability, climate change is likely to have a major impact on walnut production in the near future. Climate change will alter the land suitability for walnut cultivation around the world, especially in arid and semi-arid regions like Iran. Here, land suitability for the cultivation of walnut (Juglans regia L.) in Iran was determined using the GIS for present and future conditions (2020-2049) with an approach to climate change. Accordingly, data from 375 synoptic stations throughout Iran were gathered for climatic factors including average, minimum and maximum temperatures, relative humidity and chilling requirement. Also, ASTER sensors (Advanced Spaceborne Thermal Emission and Reflection Radiometer) and their data provided this research with cells that make a precision of 150 m (5 s), and the data were used for gauging geological parameters such as altitude and land slope. The electrical conductivity (EC) of soil and water were informed by the data bank of the Iranian Water Resources Management. The results of temperature simulations for the future (2020-2049) were analyzed by 21 AOGCM-AR5 models under the RCP4.5 emission scenario. In the first phase of evaluations, the maps of land suitability were constructed for present conditions by considering a network of the above-mentioned parameters. By combining these layers of information, the final map of land suitability was illustrated for walnut cultivation. In the second phase, the NEX-GDDP was used in order to determine land suitability for the future (2020-2049). The results showed that Iran currently has 582844 km2 of land suitable for walnut cultivation. However, the future will see less suitable lands: the current area will be reduced by 6.19%, from 582844 km2 to 546710 km2. In general, the northern, northwestern and western margins of Iran are currently suitable for walnut cultivation. By approximation, these lands will also be major areas for prospective cultivations of walnut in the future (2020-2049), even though their current stretch will be reduced.

Genome-wide patterns of population structure and association mapping of nut-related traits in Persian walnut populations from Iran using the Axiom J. regia 700K SNP array.

Persian plateau (including Iran) is considered as one of the primary centers of origin of walnut. Sampling walnut trees originating from this arena and exploiting the capabilities of next-generation sequencing (NGS) can provide new insights into the degree of genetic variation across the walnut genome. The present study aimed to explore the population structure and genomic variation of an Iranian collection of Persian walnut (Juglans regia L.) and identify loci underlying the variation in nut and kernel related traits using the new Axiom J. regia 700K SNP genotyping array. We genotyped a diversity panel including 95 walnut genotypes from eight Iranian provinces with a variety of climate zones. A majority of the SNPs (323,273, 53.03%) fell into the "Poly High Resolution" class of polymorphisms, which includes the highest quality variants. Genetic structure assessment, using several approaches, divided the Iranian walnut panel into four principal clusters, reflecting their geographic partitioning. We observed high genetic variation across all of the populations (HO = 0.34 and HE = 0.38). The overall level of genetic differentiation among populations was moderate (FST = 0.07). However, the Semnan population showed high divergence from the other Iranian populations (on average FST = 0.12), most likely due to its geographical isolation. Based on parentage analysis, the level of relatedness was very low among the Iranian walnuts examined, reflecting the geographical distance between the Iranian provinces considered in our study. Finally, we performed a genome-wide association study (GWAS), identifying 55 SNPs significantly associated with nut and kernel-related traits. In conclusion, by applying the novel Axiom J. regia 700K SNP array we uncovered new unexplored genetic diversity and identified significant marker-trait associations for nut-related traits in Persian walnut that will be useful for future breeding programs in Iran and other countries.

Antioxidant and Anticancer Activities of Walnut (Juglans regia L.) Protein Hydrolysates Using Different Proteases.

Walnut (Juglans regia L.) contains approximately 20-25 % protein with abundant essential amino acids. The enzymatic hydrolysate of Persian walnut (Chandler) seed proteins was prepared by incubation with three different proteases, including pancreatic chymotrypsin and trypsin, and a microbial enzyme proteinase K. The hydrolysates were found to possess excellent antioxidant capacities. The peptide fractions scavenged the 2, 2'-anizo-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) free radicals and inhibited the activity of reactive oxygen species. Walnut protein hydrolysates were also tested, for the first time, against the viability of human breast (MDA-MB231) and colon (HT-29) cancer cell lines. MTT, [3-(4, 5dimethylthiazolyl)-2,5-diphenyl-tetrazolium bromide], assay was used to assess in vitro cancer cell viability upon treatment with the peptide fractions. The peptide fractions showed cell growth inhibition of 63 ± 1.73 % for breast cancer and 51 ± 1.45 % for colon cancer cells. Thus, a direct correlation between antioxidant and anticancer activities of walnut peptide fractions exists and supports their potential therapeutic benefit.