Methods of multiplying plant embryogenic tissue in a bioreactor

A method of multiplying plant embryogenic tissue in a bioreactor comprising the steps of:

• (a) inoculating plant embryogenic tissue and multiplication medium into a bioreactor;
• (b) measuring a parameter that is a direct measure of, or correlated to, a concentration of the plant embryogenic tissue in the bioreactor;
• (c) determining a target value of the parameter;
• (d) taking the measurement obtained in step (b) and the target value determined in step (c) to calculate a flow rate of additional multiplication medium,
• wherein when the parameter is dry weight concentration, the flow rate of additional multiplication medium is calculated using the equation:
• f
• =
• -
• 100
• Δ
• ⁢
• ⁢
• B
• target
• ⁢
• x
• o
• ⁢
• k
• eff
• Y
• x
• /
• s
• ⁢
• exp
• ⁡
• (
• k
• eff
• ⁢
• t
• )
• wherein ΔBtarget is the difference between a Brix value of a target culture supernatant (Bsp) and a Brix value of fresh medium to be added (Bm);
• Yx/s is an amount of biomass produced divided by an amount of sugar consumed;
• biomass (x) is culture volume (V) multiplied by dry weight biomass concentration (X);
• x0 is the initial biomass;
• keff is effective growth rate;
• and t is time;
• wherein when the parameter is sugar concentration, Brix or electrical capacitance, the flow rate of additional multiplication medium is calculated using the equation:
• f=Vo·kfexp└kft┘
• wherein Vo is an initial volume of a bioreactor, kfis a flow rate factor, and t is time;
• wherein a proportional integral feedback controller is used to calculate the flow rate factor, and wherein the proportional integral feedback controller is a non-linear, discrete proportional integral controller;
• (e) delivering additional multiplication medium to the bioreactor based on the flow rate calculated in step (d);
• and
• (f) periodically repeating steps (b) to (e).