Automated plant culture proliferation system

An automated plant tissue proliferation system in which mature plants within a culture vessel are cut into a plurality of plugs using a die type cutter block. The cutter block is moved using a pick and place actuator to a work station where any extraneous nutrient media or plant material adhering to the sides of the cutter are removed using an indexed membrane sheet. A pair of opposed articulated wiping arms cause the membrane sheet to be wrapped around the cutter and pulled down vertically along its sides to remove the excess material. Thereafter, the cutter is indexed to deposit some fraction of the cut cells into a container having culture medium. Fluid pressure is used to eject material from the appropriate cells onto the media material. A number of additional containers are indexed into position with the cutter until all of the plugs of material are removed. the actuator and ejection system are so operated to distribute the plugs of plant material in a preselected array in a number of containers. The system provides rapid plant proliferation minimizing labor requirements and the liklihood of tissue culture contamination.

BACKGROUND OF THE INVENTION 
This invention relates to an automated process and device for propagation 
of plants using tissue culture (in vitro) techniques. 
In conventional procedures for plant cloning or propagations by tissue 
culture, a portion of a plant is placed in a nutrient medium in an aseptic 
controlled environment and caused to grow. The medium typically has 
hormones to encourage plant growth of a desired type. Once the plant 
reaches a given stage of growth, it is divided and separated and placed in 
additional container having growth medium, thus proliferating the plant. 
Aseptic conditions are required to discourage bacterial and fungal 
contamination which can cause significant losses. The most widely used 
current process for aspectic plant tissue culture propagation requires an 
operator to manually remove plant tissue from culture medium and divide it 
into a number of parts which are manually aseptically replanted into 
containers containing new media. This procedure is slow, highly labor 
intensive and exposes the plant to the potential for contamination. The 
high labor costs associated with such processes have limited the 
commercial viability of this approach. 
In order for in vitro proliferation systems to be commercially viable on a 
larger scale, an automated system is needed in which plant material is cut 
into uniformly sized pieces. Following the cutting process, the cut 
material should be distributed in a precisely controlled and uniform 
manner in growth vessels. Distribution of the cut plant material within 
the growth vessels must be uniform in order to better predict maturation 
time, and to more efficiently utilize the culture media required. Various 
systems for automated plant tissue culture proliferation systems are 
presently known. These systems typically employ a blender which finely 
divides the plant tissue into small components which are thereafter 
deposited on a suitable growth medium. Although these processes work well 
for some applications, they are unsuited for certain plant species and, 
further, are believed to provide lower yield due to the indiscriminate 
manner in which the plants are divided (causing much damage). Moreover, 
problems of needing excess liquid to facilitate blending and difficulties 
with cleaning and sterilication of numerous components are present with 
such processes. 
In view of the foregoing, there is a need to provide an automated 
proliferation system which divides plant tissue and distributes it in a 
uniform manner under aseptic conditions while minimizing the above noted 
problems with prior art systems. 
SUMMARY OF THE INVENTION 
In accordance with the present invention, an automated plant culture 
proliferation system is provided utilizing a pick and place actuator 
having a movable gripper unit which removably engages a die cutting block 
which forms a number of cells arranged in a two-dimensional array. When 
the die cutter block is forced into a tissue culture container containing 
plant tissue of appropriate maturity, cut plant tissue and medium is 
forced into the cells of the cutter, thus forming a number of plugs of 
material. The cutter is then lifted out of the container and indexed to a 
wipe and feed mechanism which removes excess plant tissue and culture 
medium adhering to the side surfaces of the cutter in an aseptic manner. 
Thereafter, the pick and place actuator is indexed to another work station 
where the material of a fraction of the total number of cells of the 
cutter block are ejected into a new container containing culture medium. 
Additional containers receive material from the remaining cells of the 
cutter block until they are emptied. The actuator and ejection system are 
controlled so that each of the new containers receive plugs of plant 
tissue distributed in a uniform manner. Since the system uses a cutter 
block for the purposes of cutting and holding the material for 
distribution, only a single component needs to be cleaned and sterilized. 
Additional benefits and advantages of the present invention will become 
apparent to those skilled in the art to which this invention relates from 
the subsequent description of the preferred embodiments and the appended 
claims, taken in conjunction with the accompanying drawings.

DETAILED DESCRIPTION OF THE INVENTION 
A device for implementing the plant tissue culture proliferation system 
according to this invention, is shown in FIGS. 1 and 2, and is generally 
designated by reference number 10. Device 10 is supported by table 11 and 
principally comprises, a pick and place actuator 12 which has gripper head 
20 that is movable into position at the pickup and cutting station 14, and 
a wipe and feed station 16, and a dispensing station 18. Pick and place 
actuator 12 rotates gripper head 20 about a vertical axis between the 
various work stations and also enables its vertical position to be 
controlled through vertical slide unit 26. Suitable control means, 
preferably of a programable type would be employed to control movement of 
actuator 12. 
Now with reference to FIGS. 3 through 9, the configuration of gripper head 
20 and the die cutter block 24 which it engages will be described. Gripper 
head 20 is adapted to provide a quick change capability for engaging 
cutter block 24. A number of pins designated by reference number 28 (four 
shown in the figures) are vertically movable through bores 30 within 
gripper head 20 and have elastomeric tips 32, preferably made of neoprene, 
at their ends. Each of pins 28 are connected to plate 34 and slide 36 of 
an air cylinder such that the pins can be actuated from the extended 
position shown in FIG. 3 to a retracted position where tips 32 are in 
engagement with the lower surface of gripper head 20. Cutter block 24 has 
blind bores 38 for accepting pins 28 which have a tapered section 39 
defining an entrance bore diameter equal to the diameter of tips 32, as 
best shown in FIG. 7. Operation of the quick change mechanism is shown 
beginning at FIG. 3 where pins 28 are oriented in registry with bores 38, 
and thereafter the unit is displaced downwardly causing tips 32 to be 
received by the bores. Thereafter, slide 36 is actuated raising tips 38 
and causing them to engage the lower surface of gripper 20. As shown in 
FIG. 7, compression of tips 32 causes them to expand radially, thus 
increasing their frictional engagement with the inside surfaces of bores 
38, and thereby connecting cutter block 24 to gripper 20. FIG. 6 
illustrates vertical slide unit 26 being actuated to lift cutter block 24. 
When the radial compression of tip 32 is relieved, a slight degree of 
frictional engagement between the tips and bores 38 is provided to retain 
cutter block 24 in place until an operator forceably removes it. 
Gripper head 20 includes a number of fluid passage bores 40 opening at its 
upper surface and connected to lines 42. The opposed ends of bores 40 open 
at the lower surface of the gripper head. Die cutter block 24 is machined 
to form a two-dimensional array of individual square shaped cells 52, as 
shown with reference to FIG. 9. In the example shown, a six-by-six array 
is provided, thus forming thirty-six individual cells 52. As will be 
apparent from the following description, other dimensions and shapes of 
arrays could be used. A thin wall 54 separates cells 52 and forms a 
sharpened knife edge lower surface 56. Fluid passage bores 58 communicate 
gripper bores 40 with each of cells 52. When gripper head 20 and cutter 
block 24 are connected as shown in FIG. 8, bores 40 and 58 are in registry 
enabling fluid pressure pulses (preferably air) applied through lines 42 
to communicate with cells 52. 
The details of pickup and cutting station 14 are best explained by 
returning to FIGS. 1 and 2. Station 14 includes an indexible carrier 44 
having a first machined pocket 46 for positioning and holding plant 
culture container 50, and a second machined pocket 48 for positioning and 
retaining cutter block 24. Container 50 is of conventional configuration 
(generally a square parallelopiped) and has an open top enclosed by a lid 
(not shown). During operation, indexible carrier 44 is moved to a position 
with gripper head 20 in registry with cutter block 24. Actuation of 
vertical slide unit 26 causes gripper 20 to engage cutter block 24, as 
previously explained. Vertical slide unit 26 is thereafter actuated to 
lift cutter block 24, and indexible carrier 44 is moved to position in 
registry with a container 50 containing plant medium with plant tissue of 
appropriate maturity. Vertical slide unit 26 is again actuated to force 
die cutter block 24 into container 50, as best shown with reference to 
FIGS. 10 through 12. 
During the steps shown in FIGS. 10 and 11 in which cutter block 24 is 
stroked into container 50, the material within the container is cut into 
individual plugs within cells 52 in a die cutting or "cookie cutter" 
fashion. Friction and vicous forces maintain the individual plugs of cell 
tissue and medium within the cutter block cells 52. Container 50 at 
station 14 contains plant tissue arranged at nine principal plant sites 
arranged in a square three-by-three array (other arrangements are 
possible). As shown in FIG. 9, the plant site hearts or centers 60 are 
located such that they are each divided into four equal quadrants by 
cutter block 24. For reasons which will become apparent later in this 
description, it is necessary for cutter block 24 to be dimensioned to 
provide clearance around its side surfaces from the inside surfaces of 
container 50. Therefore, a perimeter ring of excess plant tissue and 
medium remains in the container after withdrawal of cutter 24. The 
existence of the excess material causes some to transfer to the cutter 
side surfaces. 
After slide unit 26 is stroked vertically to pull cutter block 24 out of 
container 50, pick and place actuator 12 is rotated to move cutter block 
24 into registry with wipe and feed station 16, best explained with 
reference to FIGS. 13 through 18. The functions of station 16 are to 
ensure separation of the culture plugs, to remove the material adhering to 
the side surfaces of cutter 24, and to carry away the excess tissue. 
Wipe and feed station 16 is mounted to machine table 11 and includes an 
elevated platform or pad 66, preferably having a layer of elastic material 
such as urethane on its upper surface. On the four side surfaces of pad 
66, articulated wiping arms 68 are provided which are pivotably mounted to 
carrier 76 and are normally biased into the position shown in FIGS. 13 
through 15 by springs 70. A plastic membrane such as a Mylar sheet 78 is 
wrapped on drum 72 and passes over the top surface of pad 66 and is guided 
by rollers 74. In operation, cutter block 24 is pushed against pad 66 but 
is isolated from direct contact with the pad by Mylar sheet 78. Pick and 
place actuator 12 exerts a downward force slightly compressing pad 66 to 
insure that the plant medium and tissue is forced fully into cells 52 and 
separated. Cylinder 80 is thereafter actuated to pull carrier 76 downward. 
This action causes the outer surfaces of wiping arm 68 to engage risers 82 
having cam surfaces, causing them to be actuated to rotate toward the side 
surfaces of cutter block 24. A wipe pad 84 carried by each arm 68 contacts 
sheet 78 and presses it against cutter 24. Continued downward motion of 
carrier 76 causes pads 84 to be drawn down the sides of cutter block 24, 
wiping them clean of excess material. Wipe pads 84 frictionally engage 
Mylar sheet 78, thus forming a pouch of trapped waste material as best 
shown in FIG. 18 as the wiping arms are drawn along the sides of the 
cutter block. Arms 68 are relieved to provide clearance for the trapped 
pouch. Thereafter, cutter block 24 is withdrawn from station 16 with its 
side surfaces virtually free of potentially contaminating tissue culture 
and medium. 
In order to insure sterility of cutter block 24 during the wiping 
operation, the surface of Mylar sheet 78 which engages cutter block 24 is 
continually renewed. With reference to FIG. 13, the indexing mechanism is 
shown and includes jaw 86 which engages Mylar sheet 78 and pulls it in a 
downward direction by actuation cylinder 88. The contaminated Mylar sheet 
is deposited within container 90 for disposal. Indexing of sheet 78 causes 
the excess material to be carried away since it has a gel like consistency 
making it stick to the sheet. By continually presenting a clean portion of 
Mylar sheet 78, the likelihood of contamination of the tissue culture 
within cutter block 24 is minimized. 
The principles of isolating cutter block 24 from contamination of station 
16 could also be employed at pickup and cutting station 14. An indexed 
plastic sheet interposed between cutter block 24 and carrier 44 would 
prevent the transference of contaminants between successive cutter blocks. 
FIGS. 19 through 22 illustrate the unique positioning of cutter block 24 
with respect to containers 50 set in each of the pockets 102 of the rotary 
turntable. As shown in FIG. 19, cutter block 24 is disposed in the lower 
left-hand corner of container 50 and nine individual cells 52 shown in 
shaded lines are caused to be ejected onto the tissue culture medium as 
shown in the figure. Each of the nine ejected pockets 48 is located in the 
upper right-hand quadrant of each of the plant heart centers 60 which 
originally cut at station 14. A blast of air through selected lines 42 is 
conducted into the appropriate gripper bores 30 and cells 52 to eject 
material from the shaded cells shown in FIG. 19. Cutter block 24 is 
thereafter withdrawn from one container 50 and turntable 98 is indexed to 
present a new container. For the next container, the position of cutter 
block 24 is displaced such that a different quadrant, in this case the 
lower right-hand quadrant of each heart center 60, is properly positioned 
with respect to the container and an appropriate air signal causes the 
tissue to be ejected from these cells 52. In similar fashion, two 
additional containers shown by FIGS. 21 and 22 are positioned relative to 
cutter block 24 so that, as the remaining quadrants are ejected, they are 
again deposited in a predetermined orientation with respect to containers 
50. The result is that each container has an array of tissue plugs 
identically oriented at the positions shown in FIG. 9, i.e., centered with 
respect to the containers. This uniform placement of the ejected cell 
material plugs is desired since, after these plant cultures have matured 
sufficiently for division, cutter block 24 can be symmetrically positioned 
with respect to the container to again be located so that plant centers 60 
can be cleaved into four generally equal quadrants. 
Once each of containers 50 by carry rotary turntable 98 are planted, cutter 
block 24 is vertically withdrawn from the last container and is 
rotationally indexed 90 degrees to again be in registry with pickup and 
cutting station 14. While cutter block 24 is in its upward displaced 
position, an operator manually removes the used cutter block and loads a 
disinfected cutter block along with another container 50 with mature 
plants at that work station. In addition, the containers at rotary 
turntable 98 are removed and replaced with disinfected containers 
including only culture medium. 
Apparatus 10 is preferably enclosed within a sealed compartment (not shown) 
to minimize the likelihood of contamination. The compartment would have 
access doors to permit intervention by an operator or robot when 
necessary. 
Various steps of one process implementing the proliferation system in 
accordance with this invention are illustrated in block diagram form in 
FIG. 23. Block 110 is an inspection step at which a technician examines 
containers 50 taken from a growth chamber prior to loading them into 
apparatus 10 at pickup and cutting station 14. The cutting and 
distributing steps 112 and 114 are accomplished using apparatus 10 at work 
stations 14 and 18, as previously discussed. Following the cutting 
operation 112, containers 50 are sent to washing station 116 where 
remaining debris is removed. Additional containers 50 and lids can be 
added at station 116 as needed. Assuming that the containers and their 
lids pass inspection step 118, the containers are moved to dispensing 
station 120 where nutrient media is added. Thereafter, containers 50 are 
capped at station 122 and sent to autoclave 124 for disinfection. The 
disinfected containers and lids are then uncapped at station 126 and the 
containers are loaded at stations 14 and 18 and the lids are again added 
at capping station 128 after they receive tissue culture. 
The processing of cutter block 24 begins with loading a disinfected block 
at station 14 prior to the cutting operation. After cutter 24 is recycled 
back to station 14, it is removed for cleaning. Preferably an element 
similar to gripper 20 would be used to engage cutter 24 and to blow a 
cleaning medium such as steam through each of cells 52. In addition, scrub 
brushes could be used to remove matter from the internal and external 
surfaces of the cutter block. 
While the above description constitutes the preferred embodiments of the 
present invention, it will be appreciated that the invention is 
susceptible to modification, variation and change without departing from 
the proper scope and fair meaning of the accompanying claims.