Heating protocols media

Reactions between A -(l-chloroalkyl)pyridinium chlorides 33 and amino acids in organic solvents have a low synthetic value because of the low solubility of the amine partner. A special protocol has been designed and tested in order to circumvent this drawback. Soon after the preparation of the salt, an aqueous solution of the amino acid was introduced in the reaction medium and the two-phase system obtained was heated under reflux for several hours. However, this was not too successful because sulfur dioxide, evolved during the preparation of the salt, was converted into sulfite that acted as an 5-nucleophile. As a result, A -(l-sulfonatoalkyl)pyridinium betaines such as 53 were obtained (Section IV,B,3) (97BSB383). To avoid the formation of such betaines, the salts 33 were isolated and reacted with an aqueous solution of L-cysteine (80) to afford thiazolidine-4-carboxylic acids hydrochlorides 81 (60-80% yields). 

The Verax system comprises a bioreactor (fluidization tube), a control system (for pH, oxygen, medium flow rates), gas and heat exchanger, and medium supply and harvest vessels. The system is run continuously for long periods (typically over 100 days). In the authors laboratory it produced 15 x 10 cells/ hire and 540 mg mAb/litre/day (compared to 166 in the fixed bed described above, 25.5 in a stirred reactor, and 18.5 mg in an airlift fermenter). Protocols for its operation come with the equipment and versions have been published (41). In summary it is probably the most productive system available giving the cells a very high specific production rate but does require some skill to operate to its maximum potential... 

In terms of DH, a microspore culture protocol has been developed for E. sativa based on the B. napus protocol (Leskovsek et al., 2008). Buds (4-5 mm in length) in the late uninucleate to early binucleate stage of microspore development were selected for the experiments. NLN basal medium (Lichter, 1982) with 13% sucrose was used as the wash medium and the culture medium. Activated charcoal was added to the NLN culture medium. A heat shock of 32°C for 24 h was beneficial. Embryos were observed 14-21 days after initial culture however, conversion to plants was low most likely due to secondary embryogen-esis. The majority of the resulting plants were diploid (Leskovsek et al., 2008). 

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