Influence of culture vessel characteristics and agitation rate on gaseous exchange, hydrodynamic stress, and growth of embryogenic cork oak (Quercus suber L.) cultures

Abstract

Somatic embryogenesis can be induced in the leaves of cork oak (Quercus suber L.) trees. The use of this propagation system in multivarietal forestry requires the mass production of cloned plants at low cost. Investigations were made into the influence of three types of Erlenmeyer flask and three orbiting speeds (60, 110, and 160 rpm) on oxygen transfer rate (K L a), the shear force index (SFI), biomass production, and the proliferation of embryogenic clumps (EMCs) in cultures during the proliferation phase. K L a varied between 0.11 and 1.47 h −1 without biomass production being limited by oxygen availability. The EMCs grew even in hypoxic conditions, although the suppression of gaseous exchange strongly reduced biomass production. Cultures with different levels of hydrodynamic stress and SFI values (1.4·10 −3 -8.8·10 −3 cm min −1 ) were obtained. Proliferation rates of EMCs increased with agitation rate and the SFI. The largest number of EMCs was obtained in baffled flasks agitated at 160 rpm (K L a of 1.47 h −1 , and SFI of 8.8·10 −3 cm min −1 ) with mild hydrodynamic stress enhancing growth. Biomass production increased with agitation and hydrodynamic stress, but only when the SFI value was below 5·10 −3 cm min −1 . The greatest biomass production was obtained in smooth 100 ml flasks agitated at 160 rpm. The differentiation of embryos was favoured by the lowest K L a (0.11 h −1 ) and SFI (1.40·10 3 cm min −1 ) values, achieved using these flasks when agitated at 60 rpm.

Suspension Culture and Somatic Embryogenesis of Korean Pine

“…A similar phenomenon also exists in stone pine: when the orbiting speed increased from 50 rpm to 150 rpm, both SCV and DW were significantly reduced [22]. In Quercus suber L., when the stirring rate increased from 60 rpm to 110 rpm, the yield of SEs gradually increased [14]. It shows that the proliferation of Korean pine ET gradually decreases with the increase of orbiting speed.…”

“…Therefore, the appropriate orbiting speed is important for the normal proliferation of ET in suspension culture [27]. In Korean pine suspension culture, the orbiting speed significantly affects the proliferation of ET, and the amount of proliferation gradually decreases as the orbiting speed increases [14,22]. A similar phenomenon also exists in stone pine: when the orbiting speed increased from 50 rpm to 150 rpm, both SCV and DW were significantly reduced [22].…”

“…Stirring is a key factor on liquid cultures. It can not only regulate the growth and development of the culture by affecting the gas environment, but also promotes the full mixing of nutrients and metabolites [14]. On the other hand, the shear force generated by stirring can destroy the aggregation of ET, and promote either the formation or lysis of ET; excessive stirring can cause damage to cell development [26].…”

“…The liquid culture medium can effectively reduce the aggregation of cells by shaking, and make the distribution of nutrients and metabolites uniform. The cells can quickly absorb nutrients increase cell proliferation rate [14]. In addition, liquid suspension culture is conducive to breaking down ET into smaller ET aggregates, effectively increasing ET proliferation and SE yield.…”

“…Low inoculum-density could lead to low proliferation, while high inoculum-density could inhibit cell growth [18]. Similarly, the volume and orbiting speed of the Erlenmeyer flask can regulate the growth of cells by affecting the amount of gas exchange and hydrodynamic stress [14]. In the semi-solid medium, the diffusion of nutrients is a slow process, and a concentration gradient will be formed around the cells, which will slow down growth, so liquid medium is suitable for medium components with lower concentration [18].…”