Investigation of physiological components involved in low water conservation capacity of in vitro walnut plants

To obtain benefits of in vitro plant micropropagation, successful acclimatization to ex vitro atmosphere is always required. However, a huge number of in vitro-produced plants cannot survive during ex vitro acclimatization. This study aims to analyze the components that contribute to poor water conservation capacity of in vitro plants after exposure to ambient environment during ex vitro acclimation. Micropropagated shoots grown in vitro (in vitro plants) or after acclimation to greenhouse conditions (greenhouse plants) were microscopically and biochemically analyzed for differences in morphological and biochemical components. Furthermore, dynamic responses of leaves to ex vitro condition were gravimetrically analyzed during 1.5 h desiccation. Compared with greenhouse plants, thinner leaves together with bigger stomata with larger pore area, higher stomatal and epidermal densities were observed in in vitro plants. Plants cultured in vitro kept very high transpiration rate despite of dramatic decrease in leaf RWC during desiccation. Contribution of stomata was more pronounced than the role of cuticule in leaf water loss. Higher concentrations of proline and glycine betaine were observed in the leaves of in vitro plants. Higher (less negative) osmotic potential (ψs) in the leaves of in vitro plants was concurrent with lower levels of potassium and calcium in their leaves. In conclusion, higher compatible solute level in the leaf of in vitro plant does not contribute to water conservation during ex vitro acclimation and low foliar ion levels in in vitro plants can be due to low transpiration rate in plants as a result of in vitro production.