# US 8,772,033 B2 · Methods of multiplying plant embryogenic tissue in a bioreactor

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- Number: US 8,772,033 B2
- Document ID: US8772033B2
- Status: Granted
- Kind: B2
- Application number: 13/039,638
- Filed: 2011-03-03
- Published: 2011-10-06
- Granted: 2014-07-08
- Earliest claimed: 2010-03-31
- Assignees: Weyerhaeuser NR Company
- Applicants: Anthony P. Swanda
- Inventors: Anthony P. Swanda
- Web page: https://patentgrep.com/patent/US8772033B2

## CPC

- C12N 5/04: Plant cells or tissues {(culture media C12N5/0025)}
- A01H 4/002: {Culture media for tissue culture}
- A01H 4/005: {Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques}
- C12N 5/0025: {Culture media for plant cell or plant tissue culture}
- C12N 2500/34: Sugars
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A01H: NEW PLANTS OR {NON-TRANSGENIC} PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES

## Abstract

The present invention provides methods of multiplying plant embryogenic tissue in a bioreactor.

## Claims

### Claim 1

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).

### Claim 2 (depends on 1)

The method of claim 1, wherein the parameter is dry weight concentration.

### Claim 3 (depends on 1)

The method of claim 1, wherein the parameter is selected from the group comprising sugar concentration, Brix, and electrical capacitance.

### Claim 4 (depends on 1)

The method of claim 1, further comprising tuning the feedback controller.

### Claim 5 (depends on 1)

The method of claim 1, wherein the plant embryogenic tissue is embryonal suspensor mass.

### Claim 6 (depends on 1)

The method of claim 1, wherein the additional multiplication medium is delivered to the bioreactor about once per day.

### Claim 7 (depends on 1)

The method of claim 1, wherein the additional multiplication medium is delivered to the bioreactor about hourly.

### Claim 8 (depends on 1)

The method of claim 1, wherein the additional multiplication medium is continuously delivered to the bioreactor.

### Claim 9 (depends on 1)

The method of claim 1, wherein the plant embryogenic tissue is Loblolly pine.

### Claim 10 (depends on 2)

The method of claim 2, wherein the effective growth rate (keff) is calculated using the equation:
k
eff
=
1
t
f
⁢
1
⁢
n
⁡
(
V
f
⁢
X
f
V
o
⁢
X
o
)
wherein V0 and Vf are initial and final culture volume, respectively; X0 and Xf are initial and final dry weight biomass concentration, respectively; and tf is time in days between start and end of a batch.

### Claim 11 (depends on 3)

The method of claim 3, wherein the parameter is Brix, and wherein the flow rate factor (kf) is determined using the following equation:
k
f
n
=
k
f
n
-
1
+
K
e
⁡
[
(
B
n
-
1
-
B
n
)
+
Δ
⁢
⁢
t
τ
l
⁢
⁢
(
B
sp
-
B
n
)
]
wherein n is a current sample time; n−1 is a previous sample time; Kc is controller gain; B is a Brix value of a culture supernatant sample; Bsp is a target Brix value; Δt is a time between Brix samples; and τI is a controller reset day.

### Claim 12 (depends on 3)

The method of claim 3, wherein the parameter is electrical capacitance, and where the flow rate factor (kf) is determined using the equation:
k
f
n
=
k
f
n
-
1
+
K
c
⁡
[
(
C
n
-
1
-
C
n
)
+
Δ
⁢
⁢
t
τ
l
⁢
(
C
sp
-
C
n
)
]
wherein n is a current sample time; n−1 is a previous sample time; Kc is a controller gain; C is a capacitance value of a culture supernatant sample; Csp is a target capacitance value; Δt is a time between capacitance sample; and τI is a controller reset day.