WO1996029856A1

WO1996029856A1 - Heat sealed container and method of use in plant culture

Info

Publication number: WO1996029856A1
Application number: PCT/US1996/004216
Authority: WO
Inventors: Robert Levin
Original assignee: Osmotek Ltd.
Priority date: 1995-03-28
Filing date: 1996-03-27
Publication date: 1996-10-03
Also published as: AU5375996A

Abstract

Apparatus (10) is provided for tissue culture including a vessel adapted for use in tissue culture including a container (12) and a lid (32) removably securable and sealable to the container, and gas exchange apparatus (80) for providing gas exchange between the vessel and an external environment, the gas exchange apparatus operative to substantially prevent passage of bacterial and fungal contamination.

Description

HEAT SEALED CONTAINER AND METHOD OF USE IN PLANT CULTURE

The present invention relates in general to

apparatus and a method for plant tissue culture and more

particularly to plant tissue culture vessel apparatus

and a method for culture of plant cells, tissue and

organs, and microhydroponics.

Various vessels for plant tissue culture which

maintain aseptic conditions within the vessel are known

in the art. Some of these vessels provide a mechanical

closure but are not amenable to sealing. For example U.S.

Patent 4,358,908 to Song describes a rigid container and

lid where the lid is secured to the container by a me- chanical closure. Venting to the interior of the vessel

is provided by a space between the lid and the container,

which space includes a barrier to bacterial and fungal

contamination of the interior of the vessel. Another

vessel is described by Song in U.S. Patent 4,224,756.

This vessel includes a container and lid where the lid is

secured to the vessel by a mechanical closure and aseptic

venting to the interior of the vessel is provided by a

filter in the lid.

Other vessels are sealable but do not provide a

mechanical closure. For example, M. Tanaka et al., "The

Use of Disposable Fluocarbon Polymer Film Culture Vessel

in Micropropagation", Acta Horticulturae, 230, 1988,

pages 73 - 80,describes a vessel formed from gas trans-

missive fluorocarbon polymer films supported by a rigid

frame. The vessel may be heat sealed and venting is

provided by gas transfer through the walls of the vessel.

Another sealed and vented vessel is described in U.S. Patent 5,008,231 to Kertz. The Kertz vessel includes

translucent plastic membrane sheeting formed into a sac

which may be heat sealed. Venting is provided by gas

transfer through the walls of the vessel.

Plant tissue culture is well known in the art.

Early attempts at automation are described and claimed in

applicant/inventor's U.S. Patents U.S. 4,855,236

Generally plants that are propagated by tissue

culture are subsequently transplanted to greenhouse trays

and are then hardened in a controlled environment ini¬

tially characterized by high humidity.

Apparatus particularly suitable for plant

propagation is described and claimed in assignee's U.S.

Patent 5,324,657. The present invention provides for an improved

vessel apparatus and method for tissue culture, and for

plant propagation which provide high throughput and

enhanced cost effectiveness.

There is thus provided in accordance with a

preferred embodiment of the present invention, apparatus

for tissue culture including a vessel adapted for use in

tissue culture including a container and a lid removably

securable and sealable to the container, and gas exchange

apparatus for providing gas exchange between the vessel

and an external environment, the gas exchange apparatus

operative to substantially prevent passage of bacterial

and fungal contamination.

In accordance with a preferred embodiment of

the present invention, the vessel is autoclavable.

Preferably, the lid is heat sealable to the

container. In accordance with a preferred embodiment of

the present invention, the container and the lid are

fabricated from a relatively rigid, relatively clear

plastic.

Preferably, the gas exchange apparatus is

disposed in the lid.

In accordance with a preferred embodiment of

the present invention, the gas exchange apparatus in¬

cludes a sterilizing membrane filter.

Preferably, the gas exchange apparatus further

includes gas exchange control apparatus operative to

control a rate of gas exchange through the gas exchange

apparatus.

In accordance with a preferred embodiment of

the present invention, the gas exchange apparatus further

includes a sterilizing filter, a film substantially

impermeable to passage of gases therethrough, the film

being fixed to the lid and covering the filter, and apparatus for selectively removing a portion of the film.

There is also provided in accordance with a

preferred embodiment of the present invention a method of

tissue culture including providing a container adapted

for tissue culture therein, providing a lid sealably and

removably securable to the container, thereby to define

therewith a vessel, providing gas exchange apparatus for

providing gas exchange between the vessel and an external

environment, the gas exchange apparatus operative to

substantially prevent passage of bacterial and fungal

contamination, sterilizing the vessel, aseptically plac¬

ing a medium suitable for a tissue culture in the vessel,

removably securing the lid to the container, aseptically

placing the tissue culture in the vessel, and sealing the

lid to the container.

There is also provided in accordance with a

preferred embodiment of the present invention a method of

tissue culture including providing a vessel adapted for tissue culture therein including a container and a lid

sealably and removably securable to the container, pro¬

viding gas exchange apparatus for providing gas exchange

between the vessel and an external environment, the gas

exchange apparatus operative to substantially prevent

passage of bacterial and fungal contamination, placing a

medium suitable for a tissue culture in the vessel,

removably securing the lid to the container, autoclaving

the vessel with the medium therein, aseptically placing

the tissue culture in the vessel, and sealing the lid to

the container.

Preferably, the sealing of the lid to the

container is performed by heat sealing.

In accordance with a preferred embodiment of

the present invention, the medium is stored in the vessel

before aseptically placing the tissue culture in the

vessel.

There is also provided in accordance with a preferred embodiment of the present invention apparatus

for plant propagation including a sealable vented vessel

for plant tissue culture which is adapted to contain a

liquid tissue culture medium, and a support substrate for

supporting growing plant material above and in contact

with the surface of the liquid tissue culture medium,

wherein the vessel is formed with at least one sealable

port therein at a height suitable for providing liquid

communication with the interior thereof under the support

substrate, thereby to permit sterile exchange of at least

a portion of the liquid tissue culture medium therein.

In accordance with a preferred embodiment of

the present invention, the at least one sealable port is

at a height suitable for providing liquid communication

with the interior thereof under the support substrate,

thereby to permit exchange of the liquid tissue culture

medium therein with a different nutrient solution suit¬

able for plant tissue culture hardening. Additionally in accordance with a preferred

embodiment of the present invention, a tray is provided

for supporting a plurality of vessels in communication

with the nutrient solution suitable for plant tissue

culture hardening following opening of the selectably

openable ports thereof.

There is also provided in accordance with a

preferred embodiment of the present invention, a method

for tissue culture including providing a sealable vented

vessel for plant tissue culture, the vessel being formed

with at least one selectably openable port, supplying a

liquid tissue culture medium to the vessel, placing a

support substrate for supporting growing plant material

above and in contact with the liquid tissue culture

medium, placing plant material on the support substrate

sealing the vessel closed and allowing the plant material

to grow, and sterilely exchanging at least a portion of

the liquid tissue culture medium within the vessel during growth of the plant material.

In accordance with a preferred embodiment of

the present invention, the method further includes,

following initial plant growth, opening the at least one

selectably openable port, replacing the liquid tissue

culture medium in the vessel via the at least one select¬

ably openable port with a nutrient solution suitable for

plant tissue culture hardening, and hardening the plant

material inside the vessel.

Additionally in accordance with a preferred

embodiment of the present invention, the method includes

placing a plurality of vessels in a tray in communication

with the nutrient solution suitable for plant tissue

culture hardening following opening of the selectably

openable ports thereof.

The present invention will be better understood

from the following detailed description of a preferred

embodiment, taken in conjunction with the following

drawings in which:

Fig. 1 is a simplified pictorial view illustra¬

tion of a plant tissue culture vessel constructed and

operative in accordance with a preferred embodiment of

the present invention;

Fig. 2 is a simplified side sectional view

illustration of the apparatus of Fig. 1;

Fig. 3 is a simplified side sectional view

illustration of sealing apparatus useful in the inven¬

tion;

Fig. 4 is flow diagram of a method for culture

of plant cells, tissues and organs in accordance with a preferred embodiment of the present invention;

Fig. 5 is a simplified pictorial illustration

of apparatus for plant tissue culture and hardening

constructed and operative in accordance with a preferred

embodiment of the present invention;

Fig. 6 is a side sectional illustration of the

apparatus of Fig. 5;

Fig. 7 is an illustration of the apparatus of

Fig. 6 containing plant material following initial plant

growth and prior to plant hardening, following prior to

opening of selectably openable ports therein;

Fig. 8 is an illustration of a plurality of

plant containing vessels during hardening;

Fig. 9 is a flow chart illustrating a method of

plant tissue culture and hardening, in accordance with

a preferred embodiment of the present invention;

Fig. 10 is a simplified pictorial view illus¬

tration of a plant tissue culture vessel constructed and operative in accordance with yet another preferred embod¬

iment of the present invention;

Fig. 11 is a simplified pictorial view illus¬

tration of a plurality of plant tissue culture vessels,

with a sterile exchange of at least a portion of liquid

tissue culture medium therein, in accordance with a

preferred embodiment of the present invention; and

Fig. 12 is flow diagram of a method for culture

of plant cells with a sterile exchange of at least a

portion of liquid tissue culture medium, in accordance

with a preferred embodiment of the present invention.

Reference is now made to Figs. 1 and 2 which

illustrate a vessel 10 for culture of plant cells, tis¬

sues and organs constructed and operative in accordance

with a preferred embodiment of the present invention.

Vessel 10, which is typically fabricated from a

generally rigid, light transmissive plastic such as

polypropylene, includes a container 12 having a generally

planar base 14. Walls 18 extend in a direction nearly

perpendicular to base 14 to a location 20 and then con¬

tinue in a direction substantially perpendicular to base

14 to a container opening 22, where they terminate in a

peripheral flange 24. The flange 24 includes a top sur¬

face 26 disposed in a plane which lies substantially

perpendicular to the portion of walls 12 adjacent there¬

to. Flange 24 terminates in a depending peripheral lip

28, which extends downward from an outer peripheral edge

30 of top surface 26 in a direction generally perpendic- ular to top surface 26. Flange reinforcing members 31 may

be provided underlying top surface 26 and outside of the

adjacent portion of wall 12, peripherally of the contain¬

er, to prevent deformation of the top surface 26.

Vessel 10 also includes a lid 32 which is

removably or non-removably securable to opening 22. The

lid 32 preferably includes a substantially flat portion

34 sized to occlude the container opening 22 when the lid

32 is secured to the opening 22, thereby forming an

enclosure for the culture of plant cells, tissues and

organs.

The flat portion 34 preferably includes an

inner-facing surface 36, an outer-facing surface 38 and a

rim portion 40 surrounding the flat portion 34. Depending

downwardly from the rim portion 40 there is preferably

provided an outer edge wall 46 which extends substantial¬

ly perpendicularly to the flat portion 34 and an inner

edge wall 48 which is spaced inwardly from the outer edge wall 46 and extends generally parallel thereto, but has a

greater depth than outer edge wall 46.

The space bounded by the inner edge wall 48,

the outer edge wall 46 and the rim portion 40 defines a

flange receiving channel 50. The flange receiving

channel 50 is sized to grippingly and slidingly

receive the flange 24 thereby providing a mechanical

closure for removably securing the lid 32 to the

container 12.

Typically the flange 24 is slid in the flange

receiving channel 50 until rim portion 40 of the lid 32

abuts top surface 26 of flange 24. The fit of the flange

24 in the flange receiving channel 50 is sufficiently

tight to provide secure attachment of the lid 32 to the

container 12 while still permitting manual removal and

replacement of the lid 32.

A sealing closure may also be provided by

sealing the rim portion 40 to top surface 26. Sealing may be accomplished by any suitable convenient method,

such as heat sealing, which may be employed when both

the rim portion 40 and top surface 26 are fabricated

from a heat sealable plastic such as polypropylene.

Typically, sealing the vessel 10 is accomplished by

introducing the vessel 10 to sealing apparatus 52 shown

in Fig. 3 and welding rim portion 40 to top surface 26.

Reference is now made to Fig. 3 which illus¬

trates sealing apparatus 52 which typically includes a

stand 54, a vessel support 56 and a heat sealer general¬

ly referenced 58. The vessel 10 with the lid 32 removably

secured to the opening 22 of the container 12 is remova¬

bly supported by the vessel support 56.

The heat sealer 58 preferably includes a pneu¬

matic piston 60 having a piston head (not shown) , and a

shaft 62 having a first end 64 and a second end 66. The

first end 64 of shaft 62 is fixed to the piston head and

the second end 66 is fixed to a heated die 68. The vessel support 56 includes walls 70 which

at a first end 72 are fixed to the stand 54 and at a

second end 74 form a flange support 76. When vessel 10

is disposed in vessel support 56, flange support 76 abuts

top surface 26 of container 12 between flange reinforcing

members 31, thereby supporting flange 24.

Sealing apparatus 52 may also include a spacer

78 removably disposed between the heated die 68 and

the lid 32 to facilitate even sealing. The spacer 78

typically may be made of fiberglass reinforced Teflon.

The spacer 78 is typically placed between the heated die

68 and the lid 32 prior to sealing and removed after

sealing.

In a first position the heated die 68 of the

heat sealer 58 is spaced from the vessel support 56

to permit removable positioning of the vessel 10 in

the vessel support 56. To seal vessel 10, compressed

air is admitted to the pneumatic piston 60 forcing the piston head, the shaft 62 and the heated die 68 to a

second position wherein the heated die 68 is in heat

transferring contact with rim portion 40 of lid 32

through the spacer 78.

The heated die 68 is maintained in heat trans¬

ferring contact with rim portion 40 until rim portion

40 is heat welded to top surface 26 thereby sealing the

vessel 10. After vessel 10 is sealed, compressed air is

used to force the piston head of the pneumatic piston 60

and the connected heated die 68 to return to the first

position. The sealed vessel 10 is then removed and anoth¬

er vessel 10 may be disposed in sealing apparatus 52

for sealing.

Vessel 10 preferably also includes gas

exchange apparatus 80 typically disposed in the lid

32, as best seen in Fig. 1. Gas exchange apparatus 80

typically includes a port 82, a port wall 84 and a

sterilizing filtration pad 86. The sterilizing filtra- tion pad 86 is typically a nominal 0.2 - 0.5 micron

pore size membrane filter sealed to the port wall 84

either by heat sealing or sonic welding. Gas exchange

apparatus 80 provides gas exchange between • the

interior of the vessel 10 and the exterior envi¬

ronment when the lid 32 is secured to container 12.

Gas exchange apparatus 80 also includes gas

exchange control apparatus, generally referenced 88,

including a gas impermeable plastic film 90 secured to

the outer surface 38 of the flat area 34. The plastic

film 90 includes weakened regions 92 which permit

removal of sections of the plastic film 90 to provide

selectable control over the rate of gas exchange be¬

tween the interior of vessel 10 and the external envi¬

ronment.

Vessel 10 may be used to provide an aseptic

microenvironment for the culture of plant cells, tissues

and organs. Typically, a sterile vessel 10 is filled with either sterile liquid or semisolid medium

and plant tissue culture aseptically introduced into

or on the surface of the medium.

The method of the present invention will now

be described with reference to Fig. 4. In one embod¬

iment of the method, the vessel 10 is cold sterilized in

step 94. Methods for cold sterilization include gamma

radiation and ethylene oxide treatment.

In step 96, the vessel 10 is aseptically filled

with a sterile medium and the lid 32 is removably secured

to the container 12. Then in step 98, the vessel 10

containing the medium is stored until use, with the

contents of the vessel 10 being under aseptic conditions.

Alternatively, in step 100, the vessel 10 may

be non-aseptically filled with a medium, and then in

step 102, the lid 32 may be mechanically secured to the

container 12 and the vessel 10 containing the medium may

be autoclaved. Once the vessel 10 is sterile and filled with

sterile medium, the interior of the vessel 10 must

remain free of contamination during storage prior to

use. Under relatively clean environments the mechanical

closure can maintain the sterility of the medium since

the sterile filtration pad 58 which substantially pre¬

cludes the passage of contamination provides little

barrier to gas exchange and the mechanical closure

provides significant resistance to gas exchange.

However, in relatively contaminated environments the

vessel 10 when filled with medium, should be stored in

clean sealed containers.

Once vessel 10 is sterile and filled with

sterile medium, plant tissue culture may be aseptical¬

ly introduced into the vessel for growth and develop¬

ment, in step 104. The vessel 10 is then heat sealed in

step 106 as described hereinabove. If the culture is to

be incubated in a low cost growth room where airborne contamination is poorly controlled, sealing the vessel

10 while providing sterile gas exchange to the interior

of the vessel 10 through sterile filtration pad 86 is

a cost effective method of maintaining sterility in the

vessel. If desired, the rate of gas exchange may be

controlled either by the size of the port 82 or the

amount of plastic film 90 removed.

Typically, the vessel 10 is fabricated at a cost

which would permit cost effective single use in a

plant tissue culture laboratory. Therefore, cutting

open the vessel to harvest the culture after growth, is

not precluded on economic grounds.

The tissue culturist thus can obtain the

advantage of a vessel which can be opened and closed

to permit filling and storage of medium and manipu¬

lation of the plant culture and which can be subsequent¬

ly heat sealed while still providing gas exchange with

the interior of the vessel. Consequently, superior re- 96/29856 PCMJS96/04216

24

suits can be obtained in low cost tissue culture labo¬

ratories in which there is little or no control of

airborne contamination in at least part of the growth

rooms.

Reference is now made to Figs. 5 and 6 which

illustrate apparatus for culture of plant cells, tissues

and organs constructed and operative in accordance with a

preferred embodiment of the present invention.

The apparatus includes a vessel 210, which is

typically fabricated from a generally rigid, light trans-

missive plastic such as polypropylene, comprising a

container 212 having a base 214 at a first end 216.

Walls 218 extend vertically from the base 214 to a second

end 220. The walls 218 at second end 220 define a con¬

tainer opening 222 and include a flange 224. The flange

224 includes a rim wall 226 extending at substantially

right angles to the walls 218 to a terminal end 228 and

a lip wall 230 extending from the terminal end 228 to- wards base 214. Flange reinforcing members 231 extend

from the walls 218 to the rim wall 226 to prevent defor¬

mation of the rim wall 226.

Vessel 210 also includes a lid 232 securable to

the second end 220. The lid 232 includes a substantially

flat area 234 sized to occlude the container opening 222

when the lid 232 is secured to the second end 220 to form

an enclosure for the culture of plant cells, tissues and

organs.

The flat area 234 includes an inner surface 236,

an outer surface 238 and a rim portion 240 surrounding

the flat area 234. The rim portion 240 includes an outer

edge 242, and an inner edge 244. An outer edge wall 246

extends at substantially right angles to the flat area

234 from the outer edge 242 at the inner surface 236. An

inner edge wall 248 extends at substantially right angles

to the flat area 234 from the inner edge 244 at the inner

surface 236. The space bounded by the inner edge wall 248, the

outer edge wall 246 and the rim portion 240 is a flange

receiving channel 250. The flange receiving channel

250 is sized to grippingly and slidingly receive the

flange 224 thereby providing a mechanical closure for

removably securing the lid 232 to the container 212.

Typically the flange 224 is slid in the flange

receiving channel 250 until rim portion 240 of the lid

232 abuts rim wall 226 of flange 224. The fit of the

flange 224 in the flange receiving channel 250 is suffi¬

ciently tight to provide secure attachment of the lid

232 to the container 212 while still permitting manual

removal and replacement of the lid 232.

A permanent sealing closure may also be provided

by sealing the rim portion 240 to rim wall 226. Sealing

may be accomplished by any convenient method such as

heat sealing which may be employed when both the rim

portion 240 and rim wall 226 are fabricated from a heat sealable plastic such as polypropylene. Typically,

sealing the vessel 210 is accomplished by introducing

the vessel 210 to a sealing apparatus and welding rim

portion 240 to rim wall 226.

Vessel 210 preferably also includes venting

apparatus 251 typically disposed in the lid 232, as best

seen in Fig. 5. The venting apparatus 251 typically

includes a port 252, a port wall 254 and a sterilizing

filtration pad 256. The sterilizing filtration pad 256 is

typically a nominal 0.2 - 0.5 micron pore size membrane

filter sealed to the port wall 254 either by heat sealing

or sonic welding. The venting apparatus 251 provides

aseptic gas exchange between the interior of the vessel

210 and the external environment when the lid 232 is

secured to container 212.

Venting apparatus 251 also includes venting

control apparatus, generally referenced 258, including a

gas impermeable plastic film 260 secured to the outer surface 238 of the flat area 234. The plastic film 260

includes weakened areas 262 which permit removal of

section of the plastic film 260 to provide selectable

control over the rate of ventilation in the vessel 210.

Reference is now made to Figs. 7 and 8 which

illustrates a raft assembly 264 disposed within container

212 at or near the surface of a plant tissue culture

medium 266. The raft assembly 264 typically includes a

raft 268 including an at least partially liquid permeable

membrane, relatively flat, support substrate 270, a frame

wall 272 surrounding the membrane support substrate 270,

struts 274 and a float 275.

The membrane support substrate 270 is secured

to a bottom portion 276 of the frame wall 272. The struts

274 are fixed at a first end 278 to the bottom portion

276 of the frame wall 272 and at a second end 280 are

typically in contact with an upper surface 282 of the

float 275. The float 275 typically provides sufficient buoyancy to support the raft 268 at or near the surface

of the medium 266.

Plant tissue culture material 284 is typically

disposed on an upper surface 286 of the membrane support

substrate 270 with at least a portion of the plant tissue

culture 284 extending above the level of the medium 266.

In accordance with a preferred embodiment of

the present invention, the container 212 is formed with

at least one and preferably multiple selectable openable

ports 288, best seen in their opened configuration in

Fig. 5. The ports 288 are typically weakened sections of

the container wall at the lower one-third of the height

of the container. These ports may be opened prior to

plant hardening to permit replacement of the plant tissue

culture medium 266, which typically includes sugar, by a

plant nutrient medium which is suitable for plant harden¬

ing. Such replacement typically takes place by circulat¬

ing the plant nutrient medium via opened ports 288 of a plurality of vessels 210 when they are supported on a

tray.

Fig. 8 illustrates plant hardening wherein a

plurality of vessels 210 each containing plant material

and having opened ports 288 in fluid communication with a

plant nutrient medium 266 which is suitable for plant

hardening. The plant nutrient medium 266 may be replaced

by circulation of fresh or treated plant nutrient medium

266 through ports 290 to control the growth of algae or

other contaminants. It is appreciated that the vessels

210 allow for partial hardening of plant tissue culture

material 284 while it is still floating on tissue culture

medium 266 so that culture material comes to the green¬

house at least partially hardened. The atmosphere in

the greenhouse further lowers the humidity in the

vessel, depending on the air flow over the vessel. For

some crops air flow over the vessel will have to be

adjusted with the fans already in the greenhouse to obtain an optimal hardening environment suitable for

plant hardening, thus obviating the need for transplant¬

ing the plants from vessels 210 to plant hardening trays

in the greenhouse, as in the prior art.

A method of plant tissue culture and hardening,

in accordance with a preferred embodiment of the present

invention, will now be described with reference to Fig.

9. In one embodiment of the method, a sterile sealable

vented vessel including selectably openable ports, such

as vessel 210 described above, is provided in step 292.

The vessel 210 is typically brought to a sterile working

surface such as by a laminar flow bench. Sterile liquid

medium is then added to the vessel 210 typically up to

1/3 the volume of the vessel in step 294. Multiplying

plant tissue culture is then aseptically divided by

any convenient means such as dissection with a sterile

scalpel and forceps. The plant tissue culture material

is then aseptically placed on support substrate such as the membrane support substrate 270 of the raft assembly

264.

In step 296 the plant tissue culture material

on a support substrate in a raft assembly 264 is then

aseptically added to the vessel. The lid 232 including

venting apparatus 251 is then placed over opening 222 to

close the vessel 210 and in step 298 vessel 210 is sealed

by sealing rim portion 240 to top surface 226.

In step 300 the sealed vessel 210 is placed in

a tissue culture growth room for a period of time. Typi¬

cally the tissue culture growth room provides controlled

temperature, humidity and light conditions to support

growth of plant tissue culture and the development of at

least partial photosynthetic competence. The length of

this period of time in the growth room will vary, and

depends on the culture conditions and the species.

After a period of time in the growth room, the

vessel 210 is transferred to a greenhouse environment in step 302. In step 304 the vessel 210 is placed in a tray

in communication with a nutrient solution and the select¬

ably openable ports 288 are then opened in step 306. The

nutrient solution may be exchanged or treated, typically

by filtration, to prevent the buildup of microbiological

of contaminants in the nutrient solution.

The vessel 210 remains in communication with a

nutrient solution during step 308, the hardening of the

plant material. Sections of the plastic film 260 of

venting control apparatus 258 may be removed to increase

ventilation.

Reference is now made to Fig. 10 which illus¬

trates a plant tissue culture vessel 410, constructed and

operative in accordance with yet another preferred embod¬

iment of the present invention.

Vessel 410 includes a container 412 which is

formed with at least one and preferably multiple sealable

ports. Typically a first sealable port 414 is located near the middle of a first wall 416 of container 412 and

a second sealable port 418 is located near the base of a

second wall 420, typically opposite to first wall 416.

The sealable ports 414 and 418 typically comprise sili¬

cone plugs 422 and 424 sealingly fixed to openings 426

and 428 in walls 416 and 420, respectively. A sterile

hollow needle cannula 430 sealingly attached to and in

fluid communication with sterile tubing 432, typically

flexible silicone tubing, may be inserted through the

silicone plugs 422 and 424 to provide a sterile medium

exchange path.

In one preferred embodiment of the invention,

vessel 410 serves as a medium exchange system which

includes a medium exchange inlet path 434 passing

through the first sealable port 416 and a medium ex¬

change outlet path 436 passing through the second seal¬

able port 418. Typically medium exchange inlet path 434

is connected to and in fluid communication with a ster- ile medium storage tank 438 while medium exchange outlet

path 436 is connected to and in fluid communication with

a spent medium storage tank 440.

During medium exchange, a medium (not shown) is

preferably pumped from sterile medium storage tank 438 by

a peristaltic pump 442 through a sterilizing filter 444,

typically a 0.20 micron membrane filter, into container

412. When the medium reaches the height of the second

sealable port 418, medium from container 412 flows

through medium exchange outlet path 436 through to spent

medium storage tank 440. Medium exchange may be continu¬

ous or intermittent and a second peristaltic pump and

filter may be used to pump medium through the medium

outlet path 436.

Reference is now made to Fig. 11 which is a

simplified pictorial view illustration of a plurality of

plant tissue culture vessels 410, with a sterile exchange

of at least a portion of a liquid tissue culture medium 450 therein, in accordance with a preferred embodiment of

the present invention. Medium 450 is pumped by peristal¬

tic pump 442 from sterile medium storage tank 438 through

sterilizing filter 444 and a medium exchange inlet path

452 to one of vessels 410. Medium 450 flows through the

series of vessels 410, eventually out a medium exchange

outlet path 454 to spent medium storage tank 440. Another

peristaltic pump and filter may be associated with medium

exchange outlet path 454, if desired.

Medium may be exchanged continuously or inter¬

mittently at a rate controlled by the rate of pumping.

The medium exchange system permits continuous maintenance

of optimal medial composition during the entire tissue

culture process.

Reference is now made to Fig. 12 which is a

flow diagram of a method for culture of plant cells with

a sterile exchange of at least a portion of liquid tissue

culture medium, in accordance with a preferred embodiment of the present invention. In one embodiment of the meth¬

od, a sterile sealable vented vessel including at least

one sealable port, such as the vessel 410 described

above, is provided in step 460. The vessel is typically

brought to a sterile working surface such as a laminar

flow bench. Sterile liquid medium is the added to the

vessel typically up to 1/3 to 1/2 of the volume of the

vessel in step 462. In step 464, multiplying plant tissue

culture is aseptically divided by any convenient means

such as dissection with a sterile scalpel and forceps.

The plant tissue culture is then aseptically placed on a

support substrate such as a membrane support substrate

of a raft assembly, as described hereinabove with refer¬

ence to Figs. 7, 8 and 9.

In step 466, the plant tissue culture material

on a support substrate in a raft assembly is then asepti¬

cally added to the vessel and the vessel is sealed.

In step 468, the sealed vessel is placed in a tissue culture growth room and in step 470, at least one

sealed vessel is aseptically connected to the medium

exchange system described above with reference to Fig.

11. In step 472, either intermittent or continuous medium

flow is initiated and maintained during the tissue cul¬

ture growth period.

It will be appreciated by persons skilled in the

art that the present invention is not limited to what has

been particularly shown and described hereinabove. Rather

the scope of the present invention is defined by the

claims which follow:

Claims

C L A I M S

1. Apparatus for tissue culture comprising:

a vessel including:

a container adapted for use in tissue

culture;

a lid removably securable and sealable to

said container; and

gas exchange apparatus for providing gas

exchange between said vessel and an external environment,

said gas exchange apparatus operative to substantially

prevent passage of bacterial and fungal contamination.

2. Apparatus according to claim 1 and wherein said

vessel is autoclavable.

3. Apparatus according to claim 1 and wherein said

lid is heat sealable to said container.

4. Apparatus according to claim 1 and wherein said

container and said lid are fabricated from a relatively

rigid, relatively clear plastic.

5. Apparatus according to claim 1 and wherein said

gas exchange apparatus is disposed in said lid.

6. Apparatus according to claim 1 and wherein said

gas exchange apparatus comprises a sterilizing membrane

filter.

7. Apparatus according to claim 1 and wherein said

gas exchange apparatus further comprises gas exchange

control apparatus operative to control a rate of gas

exchange through said gas exchange apparatus.

8. Apparatus according to claim 1 and wherein said

gas exchange apparatus further comprises: a sterilizing filter;

a film substantially impermeable to passage of

gases therethrough, said film being fixed to said lid and

covering said filter; and

apparatus for selectively removing a portion of

said film.

9. A method of tissue culture comprising:

providing a container adapted for tissue cul¬

ture therein;

providing a lid sealably and removably secura¬

ble to said container, thereby to define therewith a

vessel;

providing gas exchange apparatus for providing

gas exchange between said vessel and an external environ¬

ment, said gas exchange apparatus operative to substan¬

tially prevent passage of bacterial and fungal contamina¬

tion; sterilizing said vessel;

aseptically placing a medium suitable for a

tissue culture in said vessel;

removably securing said lid to said container;

aseptically placing said tissue culture in said

vessel; and

sealing said lid to said container.

10. A method of tissue culture comprising:

providing a vessel adapted for tissue culture

therein including a container and a lid sealably and

removably securable to said container;

providing gas exchange apparatus for providing

gas exchange between said vessel and an external environ¬

ment, said gas exchange apparatus operative to substan¬

tially prevent passage of bacterial and fungal contamina¬

tion;

placing a medium suitable for a tissue culture in said vessel;

removably securing said lid to said container;

autoclaving said vessel with said medium there¬

in;

aseptically placing said tissue culture in said

vessel; and

sealing said lid to said container.

11. A method according to either of claims 9 and

10 and wherein said sealing of said lid to said container

is performed by heat sealing.

12. A method according to either of claims 9 and 10

and wherein said medium is stored in said vessel before

aseptically placing said tissue culture in said vessel.

13. Apparatus for plant propagation comprising:

a sealable vented vessel for plant tissue culture which is adapted to contain a liquid tissue

culture medium; and

a support substrate for supporting growing

plant material above and in contact with the surface of

the liquid tissue culture medium, wherein the vessel is

formed with at least one sealable port therein at a

height suitable for providing liquid communication with

the interior thereof under the support substrate, thereby

to permit sterile exchange of at least a portion of the

liquid tissue culture medium therein.

14. Apparatus according to claim 13 and wherein

said at least one sealable port is at a height suitable

for providing liquid communication with the interior

thereof under the support substrate, thereby to permit

exchange of the liquid tissue culture medium therein with

a different nutrient solution suitable for plant tissue

culture hardening.

15. Apparatus according to claim 14 and also com¬

prising:

a tray for supporting a plurality of vessels in

communication with the nutrient solution suitable for

plant tissue culture hardening following opening of the

selectably openable ports thereof.

16. A method for tissue culture comprising:

providing a sealable vented vessel for plant

tissue culture, the vessel being formed with at least one

selectably openable port;

supplying a liquid tissue culture medium to the

vessel;

placing a support substrate for supporting

growing plant material above and in contact with the

liquid tissue culture medium;

placing plant material on the support substrate sealing the vessel closed and allowing the plant material

to grow; and

sterilely exchanging at least a portion of the

liquid tissue culture medium within the vessel during

growth of the plant material.

17. A method according to claim 16 and further

comprising the steps of:

following initial plant growth, opening the at

least one selectably openable port;

replacing the liquid tissue culture medium in

the vessel via the at least one selectably openable port

with a nutrient solution suitable for plant tissue cul¬

ture hardening; and

hardening the plant material inside the vessel.

18. A method according to claim 17 also comprising:

placing a plurality of vessels in a tray in communication with the nutrient solution suitable for

plant tissue culture hardening following opening of the

selectably openable ports thereof.