Salt Tolerance Potential in Orchid Oncidium hybridum Plants Revealed by Halophenotyping and Genotyping

Orchid plants are thought to be glycophytes. var. Gower Ramsey (OhGR) plants were tested for the halotolerance by cultivation in 0-5000mM NaCl solutions. Phenotypes such as leaf morphology and electrical conductivity of the salt treated plants for a week, and gene expression spectrums of different parts of those plants were analyzed. Electron transfer carrier genes OhFNR and OhFD as an example of house-keeping genes, a transcription factor OhMYB1R1 and a phospholipase D gene OhPLDζ1 as tolerance responsive genes, were investigated with qPCR. It was found that OhGR plants absorbed salt without secretion, the leaves turned yellow in the salt treatment with a basal to upper manner and the electrical conductivity of the leave tissues increased accordingly with the leaf morphological change. The genes assessed showed stress timing and tissue specific differential spectrums in which OhFD-OhF-NR were co-inhibited by salt stress, OhMYB1R1 and OhPLDs were stress inducible and enhanced. At the meanwhile, the upper leaves, the pseudobulbs and the roots kept morphologically normal and alive in most tested OhGR plants in various salt conditions tested, even a high amount of salt had been accumulated in the plants as revealed by electroconductivity measures. Our results show that OhGR plants have high salt tolerance potential based on their ordered cell and molecular mechanisms.

Leaf morphology was analyzed with microscopy and electric conductivity were measured by electric conductivity testers (Asmik, Hangzhou Asmik Sensor Technology Co. Ltd.). Different leaf tissues of different morphological characteristics were sampled for electric conductivity measure and RNA isolation. Total RNA was isolated according to manufacturer's protocol for RNA extraction kids, from control (0mM NaCl) leaf tissues and different salt-treated leaf parts, i.e. yellow vs. green, or upper vs. basal, at different time lengths during 2 weeks.
For quantitative real time PCR (qPCR), the ferredoxin OhFD [4] and its coupled electron carrier gene OhFNR (KX461908.1) [5], an MYB transcription factor OhMYB1R1 cloned by Li et al. [4], and the cDNA sequence of phospholipase D gene OhPLDζ1 was obtained from OhGR transcriptome data, and the primers of the cDNAs were designed based on the cDNA sequences. The qPCR was performed according to Li et al. [4].

Morphological phenotyping
OhGR Plants showed normal development in the investigated population in the present work (Figure 1.1), and at younger stage, 2-3-year age, it showed a typical 4-leaf patten in which 2 leaves positioned near the apical part, 2 leaves near the basal part ( Figure  1A). The 4-leaf stage kept stable for months before it turned to multi-leaf and flowering stages ( Figure 1B) in our cultivation condition and was proper for our phenotyping analysis. The morphology of the salt stressed OhGR plant leaves showed yellowing starting from the basal leaves, and particularly from the basal part of the basal leaves (Figure 1.3). The severe stressed OhGR plant showed basal leaf yellowing and death (Figure 1.3 & 1.5). The upper leaves exhibited limited yellowing areas in a dispersed distribution patten in severe stress conditions (Figure 1.5).
In the other variety, Kutoo plants treated with 1000 mmol/L [NaCl] solutions, showed similar phenotypic characters.

Physiological phenotyping
Electroconductivity of OhGR plants were examined to verify any absorption of NaCl in the plant tissues. As the NaCl concentration increased the plant tissues showed increased electroconductivity ( Figure 2).

Discussion and Conclusion
All orchids are listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora, CITES [6] and preservation, reproduction and development of orchids are of immediate importance. Development of salt-tolerant orchids or even halophytic varieties is a fascinating prospective in future seawater horticulture, providing great possibilities for sustainable development on the saline soil and seashore.
The present work reports salt tolerance in OhGR plants that are never considered or recorded as a kind of halotolerant plant. To verify if the salt tolerance is due to prevention of the salt via absorption, electroconductivities are measured and show that NaCl is in did penetrated into the inner tissues of the salt treated plants. The salt pre-treatment work shows the salt tolerance cannot be induced or enhanced by low [NaCl] solutions, i.e. the salt is accumulated in the plant and leaves degenerate when salt concentration passes a certain limit.
The expression of the electron transfer carrier gene OhFD with its coupled carrier gene OhFNR [7] showed a stress induced coinhibition pattern, indicating a progressive power-supply housekeeping disorder. Both OhMYB1R1 and OhPLDζ1 [8] are stress response genes at early stages of response [4]. The expression of OhMYB1R1 was found inducible in OhGR plants at lower salt concentration stress condition [4], and the present work showed that it was more enhanced by severe salt stresses. The OhPLDζ1 at the present work showed a co-expression pattern of OhMYB1R1 that points to similar responsive position in OhGR response to salt stresses.
The upper leaves, the pseudobulbs and the roots kept morphologically normal and alive in most tested OhGR plants in various salt conditions tested, even a high amount of salt had been accumulated in the organs as revealed by electroconductivity measures. Our results show that OhGR plants have high salt tolerance potential based on their ordered cell and molecular mechanisms.
The investigated degeneration caused by salt treatment was in the 4-leaf stage of the OhGR plant development, and further analysis should be made to check the salt stress effects on whole plant life including e.g. seedling and florescence stages. Even though, the high tolerance of OhGR plants to such high salt concentration, i.e. several times of seawater [NaCl], already is the potential to explore in orchid breeding and cultivation.