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.
Sanliang Gu, Sunghee Guak, Leslie H. Fuchigami, and Charles H. Shin
Seedling plugs of `Better Boy' tomato plants (Lycopersicon esculentum Mill.) were potted in processed fiber:perlite (60:40% by volume) media amended or nonamended with either crystalline or powdered hydrophilic polymer (2.4 kg·m–3), and treated with one of the several concentrations (0, 2.5, 5, 7.5, and 10%) of antitranspirant GLK-8924, at the four true-leaf stage. Plants were either well-irrigated or subjected to short-term water stress, water withholding for 3 days, after antitranspirant GLK-8924 application. Leaf stomatal conductance, transpiration rate, whole plant transpirational water loss, and growth were depressed by short-term water stress and antitranspirant GLK-8924. In contrast, hydrophilic polymer amendment increased plant growth, resulting in higher transpirational water loss. The depression of stomatal conductance and transpiration rate by short-term water stress was reversed completely in 2 days after rewatering while the reduction of plant growth rate diminished immediately. The effects of antitranspirant GLK-8924 were nearly proportional to its concentration and lasted 8 days on stomatal conductance and transpiration rate, 4 days on plant growth rate, and throughout the experimental period on plant height and transpirational water loss. Plant growth was reduced by antitranspirant GLK-8924 possibly by closing leaf stomata. In contrast, hydrophilic polymer amendment resulted in larger plants by factors other than influences attributed to stomatal status. Hydrophilic polymer amendment did not interact with antitranspirant GLK-8924 on all variables measured. The application of antitranspirant GLK-8924 was demonstrated to be useful for regulating plant water status, plant growth and protecting plants from short-term water stress.
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).
Daniel Potter, Fangyou Gao, Giovanna Aiello, Charles Leslie, and Gale McGranahan
The utility of intersimple sequence repeat (ISSR) markers for identification of English or Persian walnut (Juglans regia L.) cultivars was explored. Four cultivars were screened with 47 ISSR primers; eight of these primers, which generated reproducible and informative data, were selected for further study. Two individuals from each of 48 cultivars, including many currently important in the California walnut industry as well as accessions from Europe and Asia, were then examined with the eight ISSR primers. Polymerase chain reaction (PCR) products were separated on agarose gels and stained with ethidium bromide. Fifty-four bands were scored as present or absent in each cultivar; of these, 31 (57%) were polymorphic among the 48 cultivars. Combined data from the eight ISSR primers provided a unique fingerprint for each of the cultivars tested. Fifteen of the cultivars could be distinguished from all others with just one primer, 31 with a minimum of two primers, and two required three primers. Pairwise genetic distances between the cultivars were calculated and a dendrogram was generated using the neighbor-joining algorithm. Some of the groupings in the dendrogram corresponded to groups which, based on known pedigrees, are genealogically closely related. Others included accessions from diverse genetic and/or geographic origins. These results can be attributed to a combination of the limitations of the ISSR method for inferring genetic relationships, on the one hand, and the complex history of walnut cultivar development involving extensive exchange and breeding of germplasm from different geographic regions, on the other.
Sanliang Gu, Leslie H. Fuchigami, Sung H. Guak, and Charles Shin
Seedling plugs of `Better Boy' tomato plants (Lycopersicon esculentum Mill.) were potted in 60% processed fiber: 40% perlite (by volume) media amended or nonamended with either crystalline or powdered hydrophilic polymer (2.4 kg·m-3), and treated with one of several concentrations (0%, 2.5%, 5%, 7.5%, and 10%) of antitranspirant GLK-8924, at the four true-leaf stage. Plants were either well-irrigated or subjected to short-term water stress, withholding water for 3 days, after antitranspirant GLK-8924 application. Leaf stomatal conductance, transpiration rate, whole-plant transpirational water loss, and growth were depressed by short-term water stress and antitranspirant GLK-8924. In contrast, hydrophilic polymer amendment increased plant growth, resulting in higher transpirational water loss. The depression of stomatal conductance and transpiration rate by short-term water stress was reversed completely in 2 days after rewatering while the reduction of plant growth rate diminished immediately. The effects of antitranspirant GLK-8924 were nearly proportional to its concentration and lasted 8 days on stomatal conductance and transpiration rate, 4 days on plant growth rate, and throughout the experimental period on plant height and transpirational water loss. Plant growth was reduced by antitranspirant GLK-8924 possibly by closing leaf stomata. In contrast, hydrophilic polymer amendment resulted in larger plants by factors other than influences attributed to stomatal status. Hydrophilic polymer amendment did not interact with antitranspirant GLK-8924 on all variables measured. The application of antitranspirant GLK-8924 was demonstrated to be useful for regulating plant water status, plant growth, and protecting plants from short-term water stress.
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.
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’).
Reza Amiri, Kourosh Vahdati, Somayeh Mohsenipoor, Mohammad Reza Mozaffari, and Charles Leslie
Correlation and causal relationships among 21 horticultural traits were determined using 71 walnut genotypes selected from seven valleys in Kerman Province, Iran. Pearson's correlation coefficient was calculated. Kernel percentage and blight susceptibility were used as dependent variables in a stepwise regression model to determine predictor variables. Direct and indirect effects of each independent variable were calculated using path analysis. A highly significant correlation was observed between lateral bearing habit and yield. Lateral-bearing trees were also more susceptible to blight and winter cold than terminal bearers. Kernel and nut weights, shell thickness, and difficulty of extracting kernel halves were the most important traits accounting for kernel variation. Kernel weight and difficulty extracting kernel halves had the strongest positive direct effects and nut weight the most negative. Flowering habit, nut shape, and leafing date had positive direct effects on blight susceptibility, but the large residual effects suggest there are other important determinant traits for blight susceptibility, which were not considered in this study.
Sanliang Gu, Leslie H. Fuchigami, Lailiang Cheng, Sung H. Guak, and Charles C.H. Shin
Seedling plugs of `Early Girl' tomato plants (Lycopersicon esculentum Mill.) were potted in peatmoss and perlite (60:40% by volume) medium, fertilized with 8, 16, 24, or 32 g NutriCote Total controlled-release fertilizer (type 100, 13N–5.67P–10.79K plus micronutrients) per pot (2.81 l), and treated with 0%, 2.5%, 5%, or 7.5% antitranspirant GLK-8924 solution, at the four true-leaf stage. Plants were tipped at the second inflorescence and laterals were removed upon emergence. Leaf stomatal conductance, transpiration rate, and growth were depressed by GLK-8924. In contrast, higher fertilization rate increased plant growth but leaf stomatal conductance and transpiration rate were not affected until 3 weeks after GLK-8924 treatment. With 24 g NutriCote per pot, lamina N concentration in GLK-8924 treated plants was 12.5-fold of that in untreated plants, regardless of GLK-8924 concentration. Lamina P, K, Fe, and Cu were greater while S, Ca, Mg, Mn, B, and Zn were not affected by GLK-8924. The reduced growth by GLK-8924 may be due to the reduced stomatal conductance while the increased growth by high fertilization may be due to influences on plant nutritional status.