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- Author or Editor: Yann-Jiun Liu x
An efficient method for the regeneration of shoots directly from cell suspensions of three commercial cultivars of lettuce (Lactuca sativa L. cv. Great Lakes 659-700, Salad Bowl, and Prize Head) is described. Cell suspensions were prepared by osterizing cotyledon-derived callus for 60 seconds. The effects of callus quality, light intensity, carbohydrate type and concentration, auxins, and cytokinins on cell growth and differentiation in the suspension culture were examined. Among these factors, callus quality and carbohydrates were the most critical. The optimal medium for regeneration of shoots in suspension culture was SH (Schenk and Hilderbrandt) basal medium containing 1000 mg myo -inositol/liter, 1.5% glucose, 0.44 μm BA, and 0.54 μm NAA. The pH of the medium was adjusted to 5.8. Under such condition, hundreds of shoots could be produced from 50 to 55 mg (dry wt) of cell aggregates within 2 weeks. Chemical names used: α-] naphthaleneacetic acid (NAA); indole3-acetic acid (IAA); 6-benzylaminopurine (BA).
A scale-up process of lettuce (Lactuca sativa L.) suspension culture in a 2-liter bioreactor was investigated. Factors that influenced cell growth and differentiation, including foaming, the wall effect (inoculum adhering onto the vessel wall above the medium level), aeration, and dissolved oxygen (DO), were tested. The wall effect resulted in severe inoculum loss (10%) in 24 hours. Inoculum loss significantly decreased shoot regeneration. The wall effect was caused by two factors: 1) foaming caused by the interaction between air bubbles and inoculum, and 2) the bubbles produced by aeration. Foaming could be prevented by sieving the inoculum through a 400-pm screen filter and then rinsing the inoculum thoroughly with distilled water to remove single cells, cell debris, and the contents of broken cells. The wall effect caused by air bubbles could be prevented by putting a 150-μm screen column in the center of the bioreactor to isolate the aeration area from the inoculum. After the wall effect was removed, shoot regeneration in the bioreactor increased significantly to a level similar to that in 125-ml flasks at an aeration rate of 1 to 2 vvm (liters air/liters medium per rein). DO for this shoot regeneration level was ≈ 70% to 80%of saturation at the end of bioreactor culture.
A bioreactor was used to establish a scale-up system for somatic embryogenesis in `Scarlet' carrot (Daucus carota L.). At a cell density of 1–2 × 106 cells/ml, mature and germinating embryos could be observed within 4 to 5 weeks. As cell density exceeded 2 × 106 cells/ml, the culture turned darker yellow, and embryo development was inhibited. Cell densities below 106 cells/ml resulted in abnormal embryos. Bioreactor design had a critical impact on somatic embryogenesis due to various types and the strength of shear forces generated. In this study, an air-lift bioreactor was selected from three different types (spinner flask, screen column bioreactor, and air lift) because it resulted in the highest biomass production and somatic embryogenesis. Foaming was eliminated by preculture of embryogenic cells in flasks; cells were then sieved on a 60-μm polyester screen and thoroughly rinsed with distilled water before being transferred to the bioreactor. Such preculture for at least 10 days significantly increased the regeneration of somatic embryos. During somatic embryogenesis, dissolved O2 concentrations decreased to 33% of saturation, and then increased up to 80% when embryo development approached maturity and mature embryos germinated. Bioreactor-cultured embryos germinated with relatively short cotyledons and long roots, whereas flask-cultured embryos germinated with relatively long cotyledons and short roots.