Agrobacterium rhizogenes-mediated transformation and plant regeneration of four Gentiana species

Shoots of micropropagated Gentiana acaulis, G. cruciata, G. lutea, and G. purpurea were inoculated with suspensions of Agrobacterium rhizogenes cells, strains ATCC 15834 or A4M70GUS. Adventitious roots appeared at the sites of inoculation in all 4 species. Root tips were excised and cultured on growth regulator-free media for 2-6 years. They exhibited very high branching and plagiotropism. Spontaneous bud initiation occurred in roots of G. cruciata. Roots of G. lutea, G. acaulis and G. purpurea were cultured on media with high kinetin concentration, which induced the formation of friable callus tissues. Only in G. purpurea were these calluses organogenic. Regenerated shoots of G. cruciata and G. purpurea gave rise to plants, that displayed the typical phenotypes of A. rhizogenes-transformed plants: short internodes and rolled leaves. In the roots of G. acaulis and G. cruciata, transformed with A. rhizogenes A4M70GUS, a positive reaction with X-gluc indicated the activity of beta-glucuronidase. The DNA extracted from hairy roots and from the roots of transgenic plants hybridized with the appropriate genomic probes in Southern blotting. This is taken as evidence of the stable genetic transformation in the 4 Gentiana species

Production of anthraquinones by immobilized Frangula alnus Mill. plant cells in a four-phase air-lift bioreactor

The production of anthraquinones by Frangula alnus Mill. plant cells was used as a model system to evaluate the performance of a liquid-liquid extractive product-recovery process. The shake flask experiments have shown higher production of anthraquinones in cell suspension and flask cultures of calcium-alginate-immobilized cells when silicone oil was incorporated into the medium, compared to a control without silicone oil. An external-loop air-lift bioreactor, developed and designed for the production and simultaneous extraction of extracellular plant cell products, was regarded as a four-phase system, with dispersed gas, non-aqueous solvent and calcium-alginate-immobilized plant cells in Murashige and Skoog medium. Continuous extraction of anthraquinones by silicone oil and n-hexadecane inside the bioreactor resulted in 10–30 times higher cell productivity, compared to that of immobilized cells in a flask. Based on the mixing pattern, immobilized biocatalyst extraparticle and intraparticle diffusional constraints and the kinetics of growth, substrate consumption and product formation, a mathematical model was developed to describe the time course of a batch plant cell culture

Hydrodynamics and Mass Transfer in a Four‐Phase External Loop Air Lift Bioreactor

An external loop air lift reactor (ELALR) was developed and designed for continuous production and extraction of extracellular secondary metabolites by immobilized Frangula alnus Mill. plant cells. This system was considered as a four‐phase system, with dispersed gas, nonaqueous liquid solvent, and calcium alginate particles in water or Murashige and Skoog medium. Analysis of the four‐phase ELALR revealed the influence of gas, solvent, calcium alginate particles and MS medium composition on gas and solvent holdup, liquid circulation velocity, mixing in aqueous liquid phase, and mass transfer coefficients of oxygen and plant cell product. It was shown that the gas holdup appears to be the principal variable determining the properties of the four‐phase system. An unified correlation for the gas holdup, based on drift flux flow in a three‐phase system, has been extended to the four‐phase system. Bubble size and bubble size distribution observed in the bubbly flow regime were the same in water as in the MS medium system (Sauter mean diameter 2.5 mm). Consequently, gas holdup and all related parameters, such as liquid circulation velocity, oxygen mass transfer coefficient, and axial liquid dispersion in MS medium, were similar to those in water for the four‐phase system