Acclimatization of in vitro plantlets is one of the key steps in successful tissue culture propagation. Gaseous atmosphere during in vitro culture can influence the rate of ex vitro acclimation of the plantlets produced. In the current study, effects of elevated CO2 concentration on the leaf water loss dynamic responses of in vitro–produced walnut leaves during ex vitro desiccation were investigated. Elevated CO2 concentration in the headspace of culture vessels caused a considerable decrease in stomatal aperture. Although the traits related to stomatal size were not influenced by CO2 elevation, the number of small stomata was increased, and the number of large stomata was decreased at elevated CO2 concentration. Higher CO2 concentration resulted in a lower transpiration rate and a higher relative water content (RWC) during ex vitro desiccation. This improvement was due to decreased stomatal aperture during the first phase of water loss. Osmotic potential (ψs) was decreased under an elevated CO2 concentration, but no influence was observed on the concentration of compatible solutes. In conclusion, increasing the CO2 concentration of culture vessel headspace can be an efficient tool for improving acclimation of in vitro–grown walnuts without negative effects on plantlet growth.
Kourosh Vahdati, Zeinab Maleki Asayesh, Sasan Aliniaeifard, and Charles Leslie
Mohammad Sadat-Hosseini, Kourosh Vahdati, and Charles A. Leslie
Somatic embryos (SEs) can play important roles in genetic manipulation and breeding. They can be used as targets for induced mutagenesis, as material for cryopreservation and germplasm conservation, and for transformation or gene editing in support of plant improvement and proof of gene function. However, germination rates of walnut (Juglans regia) SEs are low, and the genetic stability of plantlets regenerated from them has not been explored. Here, we studied first the effects of gibberellic acid (GA3) and low temperature storage (LTS) on germination of walnut somatic embryos. Second, we assessed the genetic fidelity of plantlets regenerated from these SEs by comparing them to each other and to their cultivar of origin. Results showed that GA3 and LTS increased the walnut SE germination rate. The best rate was observed when SEs were subjected to LTS for 60 d followed by culture on a medium with either 1 or 3 mg·L−1 GA3 (56.6% and 46.6% germination respectively). Genetic stability was evaluated, using flow cytometry and 15 sets of ISSR primers. Flow cytometry indicated that all samples (i.e., regenerated and parental counterpart) showed the same peak. Amplified fragments of inter simple sequence repeats (ISSR) primers ranged in size from ≈200 to 1800 bp. All ISSR profiles of regenerants were monomorphic. Results did not show any genetic differences among plantlets regenerated from SEs or from their parental counterpart. Due to this apparent genetic stability, walnut SEs can be useful for genetic transformation and germplasm conservation.
Naser Lotfi, Kourosh Vahdati, Bahman Kholdebarin, and Elaheh Najafian Ashrafi
Walnut (J. regia L.) is one of the most sensitive plants to abiotic stresses for which finding salt-tolerant genetic resources is very important. Effects of salt stress on seed germination of seven walnut cultivars (Serr, Lara, Pedro, Chandler, Hartely, Vina, and Roun de Montignac) were studied. Salt stress treatments were applied using NaCl solutions ranging from 50 to 250 mm. The increase in salinity levels resulted in a substantial decrease in root relative water content. The percent of germination was also affected by salinity level and cultivar and their interactions. The mean germination time differed among treatment media and cultivars and a significant interaction was observed between these two factors. According to the cluster analysis, ‘Chandler’ was classified as the most tolerant cultivar. ‘Hartley’, ‘Pedro’, and ‘Round de Montignac’ (‘RDM’) were also classified in another group (semisensitive), whereas ‘Vina’, ‘Serr’, and ‘Lara’ were classified as sensitive cultivars. With the increase in salt stress, resistant cultivars accumulated more potassium (K) and calcium, especially in their shoots. However, in the semitolerant cultivars, accumulation of K in the root was more than in the shoot. Differences in magnesium accumulation in roots and shoots of all samples were significant at all stress levels and were dependent on genotypes. Sodium levels in roots were higher than in the shoots in most cultivars, especially in the semisensitive and tolerant ones. There was a low correlation (r 2 ≤ 0.15) between walnut seed traits (seed weight and kernel weight) and growth indices with respect to salinity tolerance.
Kourosh Vahdati, Charles Leslie, Zabihollah Zamani, and Gale McGranahan
In vitro rooting of three commercial cultivars of Persian walnut (Juglans regia L.), `Sunland', `Chandler', and `Vina', was examined using a two-phase rooting procedure: root induction in the dark on Murashige and Skoog (MS) medium with 15 μm IBA followed by root development in the light on a mixture of one-quarter strength Driver Kuniyuki Walnut (DKW) basal medium and vermiculite (1:1.25, v/v). Rooting percentages were: `Sunland' (94%), `Chandler (55%), and `Vina' (27%). A positive relationship was observed between the vigor of cultivars and rooting ability, but shoot length did not affect rooting success. Rooting was optimum when shoots were cultured on root induction media for 6 to 8 days. Increasing the sucrose level in the root induction medium to 40 g·L-1 improved rooting, and shoots induced to root at 22 °C rooted more readily than those induced at 30 °C. Either increasing or decreasing the nitrogen level in the multiplication medium had a negative effect on rooting. Rooted walnut shoots often cease growth during acclimatization, resulting in shoot rosetting. Spray application of Promalin® (25 mL·L-1) caused buds to break and induced elongation of shoots. Chemical name used: indole-3-butyric acid (IBA).
Rohollah Karimi, Ahmad Ershadi, Kourosh Vahdati, and Keith Woeste
The genetic structure and diversity of natural populations of Juglans regia L. in Iran were characterized using 11 microsatellite loci. A total of 105 individuals from seven populations were sampled. A high level of genetic diversity was observed within populations with the number of alleles per locus (A) ranging from three to 11 (average = 5.73), the proportion of polymorphic loci was 100%, and the expected heterozygosity ranged from 0.598 to 0.848 (average = 0.707). The proportion of genetic differentiation present among populations accounted for 12% of the total variation. Such considerable interpopulation differentiation detected in J. regia L. could have resulted from several factors, including restricted gene flow between populations. Significant departures from Hardy-Weinberg equilibrium were observed for WGA276, WGA32, and WGA321 loci. The deviations were primarily the result of the surplus of heterozygotes. The unweighted pair group method with arithmetic mean cluster analyses based on Nei's unbiased genetic distances separated the seven populations into two main groups.
Asadolah Aslani Aslamarz, Kourosh Vahdati, Majid Rahemi, and Darab Hassani
The objective of this work was to determine the chilling and heat requirements of Persian walnut cultivars and genotypes using excised twigs. The experiment was carried out from Nov. 2006 and 2007 to Mar. 2007 and 2008. One-year-old twigs were prepared from four cultivars and four domestic genotypes of Juglans regia L. After leaf fall, the twigs were taken and placed in plastic bags and kept at 4 ± 1 °C to stimulate 400 to 1500 chilling hours. After chilling, the excised twigs were transferred to the greenhouse with a natural photoperiod and a temperature from 18 to 27 °C. The evaluation of budbreak was made three times a week and the number of accumulated growing degree hours (°C) was determined until the buds reached the balloon or green tip stage. The chilling requirements were lowest (400 h) for catkins and highest (1000 h) for lateral buds. The Serr cultivar and ‘Z30’ genotype had the lowest chilling requirements (650 and 650 h). ‘Lara’, ‘Z63’, ‘Z53’, ‘Pedro’, and ‘Z67’ showed intermediate chilling requirements with values of 900, 900, 800, 750, and 750 h, respectively. Finally, ‘Hartley’ completed its dormancy after an accumulation of 1000 h, being the walnut cultivar with the highest chilling requirement in our study. As the final result, the cultivars and genotypes were classified into three groups based on their heat requirements: low requirement (‘Z30’ and ‘Serr’), medium requirement (‘Z53’, ‘Z67’, ‘Lara’, and ‘Pedro’), and high requirement (‘Hartley’ and ‘Z63’).
Mohammad Sadat Hosseini Grouh, Kourosh Vahdati, Mahmoud Lotfi, Darab Hassani, and Nejat Pirvali Biranvand
We report the first successful regeneration of haploid lines in persian walnut (Juglans regia) developed by in situ parthenogenesis followed by embryo rescue. Female flowers of cultivars Hartley and Pedro and two native Iranian selections (Z63 and Z67) were pollinated using pollen of selections Z53 and Z30 that had been irradiated with gamma rays at five doses (50, 150, 300, 600, and 900 Gy). Gamma-irradiated pollen induced fruit set and development of some parthenogenetic embryos. The immature embryos were excised 30 and 45 days after pollination, cultured in vitro, and then stratified for 30 days at 4 °C to overcome dormancy. Ploidy level of the resulting plantlets was determined by chromosome counting and flow cytometry. Haploid plants were obtained from ‘Hartley’, ‘Pedro’, Z63, and Z67 after pollination using pollen irradiated at 300 and 600 Gy. Plants obtained from pollen irradiated at 50 and 150 Gy were all diploid. Molecular marker analysis using four simple sequence repeat (SSR) markers also showed that all the diploid plants recovered were zygotic and no spontaneous double haploid plants were obtained in this work. Also, the haploid plantlets presented only one allele of their female parents. These profiles confirmed the parthenogenetic origin of the obtained haploid plants. The techniques used to induce haploid walnut plants by irradiated pollen were successful and could be used in breeding programs and accelerate genome analysis in this plant in which the genome size is approximately three times the size of the human genome.
Azadeh Behrooz, Kourosh Vahdati, Farhad Rejali, Mahmoud Lotfi, Saadat Sarikhani, and Charles Leslie
Drought stress is one of the main constraints limiting worldwide crop production. Arbuscular mycorrhizae (AM) and plant growth-promoting bacteria (PGPB) such as Azotobacter chroococcum and Azospirillium lipofrum have been shown to alleviate drought stress effects. Therefore, the interaction effect of AM fungi [Glomus mosseae, G. etunicatum, and a mix of these (G. mix), and PGPB bacteria (Azotobacter chroococcum + Azospirillium lipofrum)] was investigated in 1-year-old walnut seedlings (cv. Chandler) under normal and drought stress conditions. Drought stress reduced growth (plant height, root length, number of leaves, and fresh weight) and leaf nutrient content (N, P, and Zn) significantly of walnut plants. In contrast, proline, total soluble sugar, starch peroxidase enzyme activity, and total phenolic content of walnut leaves increased under this stress. Application of fungi or bacteria, and especially their simultaneous use, alleviated the negative effects of drought stress on walnut seedlings. AM fungi and PGPB increased significantly the content of some metabolites, including total phenolic content, proline level, peroxidase activity, total soluble sugar, and starch content as well as peroxidase enzyme activity. This led to an increase in walnut plant growth under the drought stress condition. Among AM fungi, G. etunicatum was more effective in reducing drought stress symptoms than either G. mosseae or the G. mix of fungi. In conclusion, use of G. etunicatum, along with PGPB, can reduce negative effects of drought stress on walnut seedlings.
Karim Keshavarz, Kourosh Vahdati, Mahmoud Samar, Behzad Azadegan, and Patrick H. Brown
An experiment was conducted in a persian walnut (Juglans regia) orchard in the north of Iran to evaluate the effects of zinc (Zn) as zinc sulfate and/or boron (B) as boric acid in foliar spray with different concentrations and combinations. Three B and three Zn concentrations (0, 174, and 348 mg·L−1 for B and 0, 1050, and 1750 mg·L−1 for Zn) were applied either independently or in combination. Leaf nutrient concentrations, pollen germination, fruit set, leaf chlorophyll index, nut and kernel characteristics, vegetative growth, nut weight, and nut yield were measured to assess the effects of treatments. The results showed that all B and Zn applications and combinations had a significant effect on all traits except nut and kernel diameter, shell percent, husk thickness, and pistillate flower abscission (PFA). Pollen germination, fruit set, vegetative growth, nut weight, kernel percent, nut and kernel length, and chlorophyll index were highest when B and Zn were applied simultaneously at 174 and 1050 mg·L−1 concentrations, respectively.
Asadolah Aslani Aslamarz, Kourosh Vahdati, Majid Rahemi, Darab Hassani, and Charles Leslie
To study the cold-hardiness of Persian walnut cultivars and selections, three methods were compared: 1) thermal analysis; 2) evaluation of tissue health after controlled freezing; and 3) field observations after a severe midwinter freeze. Stem segments and buds were collected from eight Persian walnut genotypes (four commercial cultivars and four promising Iranian selections). Thermal analysis was conducted using thermoelectric modules (TEM) to measure the high (HTE) and low (LTE) temperature exotherms produced when water and tissues freeze. TEM signals were recorded as the temperature of the samples was decreased at a rate of 2 °C/h. Tissue injury under controlled temperatures was evaluated using pre-chilled stem segments cooled at 2 °C/h to set temperatures ranging from –5 to –30 °C and then held at these temperatures for 16 h. Frozen samples were thawed and visually evaluated for severity of injury. Cold damage under field conditions was evaluated after an unusually severe winter freeze. Twigs from affected trees were removed in mid-February and in April and visually rated for extent of injury and ability to recover. The occurrence of LTEs was correlated with death of the tissues as assessed by tissue browning. Both the capacity to supercool and the cold-hardiness of cultivars and selections tested increased with accumulated seasonal chilling and decreased as they approached spring budbreak. Thermal analysis showed a tendency for buds and stems to exhibit multiple LTEs at peak dormancy. The cultivars and selections were classified into three groups based on their cold-hardiness: sensitive (‘Z30’ and ‘Serr’), semihardy (‘Z53’ and ‘Z67’), and hardy (‘Lara’, ‘Hartley’, ‘Z63’, and ‘Pedro’).