Low CO2 Levels Are Detrimental for In Vitro Plantlets through Disturbance of Photosynthetic Functionality and Accumulation of Reactive Oxygen Species

Abstract

Photosynthesis of plantlets in tissue culture containers is not considered important, compared to photosynthesis of ex vitro plants, due to the exogenous source of carbohydrates present in tissue culture media. However, CO2 starvation can generate a burst of reactive oxygen species (ROS). We examined this phenomenon in tissue culture, since CO2 levels may become very low during the light period. The research was carried out with lily scales, regenerating adventitious bulblets, and with Arabidopsis seedlings. CO2 starvation was achieved by placing a small vial of concentrated KOH solution in the culture container. CO2 removal reduced the growth of regenerated lily bulblets by 33% or 23%, with or without sucrose in the medium, respectively. In Arabidopsis seedlings, CO2 removal decreased growth by 50% or 78% in the presence or absence of sucrose in the medium, respectively. Therefore, the addition of sucrose as a replacement for photosynthesis resulted in only partial recovery of growth. Staining with nitroblue tetrazolium (NBT) showed little to no ROS in ex vitro growing seedlings, while abundant ROS were detected in seedlings grown under in vitro CO2 starvation. Seedlings grown under normal tissue culture conditions (no CO2 withdrawal) showed low levels of ROS. In lily tissue culture, CO2 starvation decreased the maximum quantum efficiency of photosystem II (Fv/Fm) from 0.69 to 0.60, and in Arabidopsis from 0.76 to 0.62. Fv/Fm of ex vitro lily and Arabidopsis seedlings was 0.77 and 0.79, respectively. This is indicative of a disturbance in photosynthesis functionality and the occurrence of in vitro stress under reduced CO2 concentrations. We conclude that poor growth, in the absence of CO2, was partly due to strongly reduced photosynthesis, while the detrimental effects were most likely due to a burst of ROS.

Unlocking the Potential of In Vitro Photoautotrophy for Eryngium foetidum: Biomass, Morphophysiology, and Acclimatization

“…In the photomixotrophic system with a lower gas exchange rate (or without natural ventilation), the microenvironment of the flask is the factor with the greatest influence on the growth and development, since carbon is made available through the supply of sucrose in the medium [33][34][35]. Undeniably, sealing the bottles (rigid lids or without membranes) provides high humidity inside them, greater accumulation of the phytohormone ethylene, reduced gas exchange, and low CO 2 concentration [36][37][38][39]. These factors lead to the development of plants with morphophysiological disorders, such as leaf formation displaying mesophyll with large intercellular spaces, poorly developed vascular system, lack of epicuticular wax formation, and non-functional stomata [16,18,19,40].…”

In Vitro Propagation Journey of Ornamental Gladiolus (Gladiolus Species): A Systematic Review Analysis Based on More Than 50 Years Research

“…It is challenging for in vitro plantlets to survive ex vivo due to their aberrant morphology, anatomy, and physiology [152,[166][167][168]. Tissue-cultured plants are unlikely to be actively photosynthesizing, despite the fact that they might appear normal in vitro [169]. Due to the persistence of pathogenic microorganisms, conventional vegetative propagation in bulbous crops like gladiolus can result in phytopathological and physiological deterioration; epigenetic changes are the cause of physiological deterioration [170,171].…”

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

“…Improper development of the photosynthetic system and malfunctioning of stomata during in vitro culture may lead to a series of morphological and physiological disorders of plant tissue (Urban et al, 2017). Although tissue-cultured plants may look normal in vitro, they are unlikely to be actively photosynthesizing (Askari et al, 2022). This is due to the provision of sucrose as the source of carbon and different environments than ex vitro, which disturb the natural development of the photosynthetic apparatus and stomatal responses afterward (Gago et al, 2014).…”

“…It is wrongly claimed that due to the presence of sugar in the medium, in vitro, plants do not need photosynthesis. It has been shown that the removal of light and CO 2 as the two main inputs of photosynthesis is harmful for the growth of tissue cultured plants (Askari et al, 2022). Adaptation of plantlets after in vitro growth needs practices such as reducing RH, increasing light level, and CO 2 concentration, or reducing the osmotic potential of the culture medium (Aliniaeifard et al, 2020).…”