Plant tissue culture device

A plant tissue culture device is disclosed for the culturing of a plurality of plant cell tissue cultures or callus cultures on a liquid medium. The cultures are maintained in culture wells on a culture plate and a porous wick is used to transport nutrient medium to the cultures from a supply of medium in a medium vessel underneath the culture plate.

TECHNICAL FIELD 
The present invention relates to the general subject matter of trays or 
containers for maintaining cultures of live tissues, and relates, in 
particular, to a device for the in vitro culture of plant cell tissues or 
callus cultures. 
BACKGROUND OF THE INVENTION 
It has become common place in the technology of plant husbandry and plant 
research to cultivate plant tissues in vitro for a wide variety of 
purposes. The purposes include conducting experiments with plant tissues, 
genetically engineering plant tissues, the asexual reproduction of 
commercial flowering and decorative plants, and other similar purposes. It 
is generally known that certain nutrient components such as salts, 
vitamins and hormones are necessary to foster plant tissue growth in or on 
a culture medium and various different mechanisms are used to provide 
plant tissues grown in culture with such requirements. 
Currently, the vast majority of plant tissue culture methods employ a solid 
phase medium as a growth substrate for plant calli and plant tissue 
explants. The typical solid or semi-solid medium utilized is an agar type 
substrate, such as that described in Gamborg and Shyluk, "Nutrition, Media 
and Characteristics of Plant Cell and Tissue Cultures", Plant Tissue 
Culture: Methods and Application in Agriculture, Academic Press, 1981; 
Helgeson, "Plant Tissue and Cell Suspension Culture", Tissue Culture 
Methods for Plant Pathologists, Blackwell Scientific Publishing, 1980, 
page 19. Using an agar substrate, the plant tissues themselves are placed 
directly on the solid medium which is dosed with the nutrient requirements 
of the plant cells growing thereon. It is recognized, however, that many 
such nutrients and also cellular metabolites do not diffuse as effectively 
across a solid or semi-solid agar matrix as compared with their rates of 
diffusion in aqueous solution. Therefore, around plant cell cultures grown 
on a solid phase agar substrates, chemical gradients of both nutrient 
components and cellular metabolites tend to be generated in the agar layer 
surrounding the cultured plant tissues. These gradients can cause 
undesirable or imbalanced chemical conditions in the environment 
immediately about the growing plant callus or tissue which can sometimes 
interfere with effective continuous growth of the cultured plant tissues. 
The deleterious effects of the development of these gradients can be 
partially reduced by frequent transfer of the callus or culture tissues to 
fresh culture plates. This practice is, however, relatively expensive and 
laborious since the tissue cultures must be individually transferred by 
hand from new to old plates. Furthermore, the continuous growth of the 
cultured specimens is often disturbed, sometimes irreparably, because of 
this manipulation, and the handling of the cultures increases the chance 
of microbial contamination of the growing cultures and their media. 
One other previously used method of fostering the growth of plant cell 
cultures or calluses is to grow the cultures on filter paper bridges 
folded and inserted into a culture vessel, such as a test tube, supplied 
with a quantity of liquid nutrient medium. The liquid medium travels up 
the filter paper bridge by capillary action and thus provides nutrients to 
the plant tissues or callus cultures grown on the filter paper bridge. 
This method does provide a better exchange of nutrients between the 
culture and the medium than does the use of solid or semi-solid supports 
like agar and agarose. This method is described in the literature by 
Tokumasu and Kato "Variation of Chromosome Numbers and Essential Aid 
Components of Plants Derived from Anther Culture of the Diploid and the 
Tetraploid in Pelargonium Rozeum", Euphytica, 28; 329, 1978, and by Pillai 
and Hildebrandt, "Induced Differentiation of Geranium Plants from 
Undifferentiated Callus In Vitro", Amer. J. Bot., 56: 52, 1969. The 
problem with the use of the filter paper bridge and test tube method is 
that the bridge portion of the paper holding the callus does not have a 
firm support and therefore the filter paper bridges have to be carefully 
folded so that the legs of the filter paper wicks are positioned along the 
walls of relatively narrow culture vessels, like test tubes, to provide a 
support for the tissue culture. This design makes it impractical to 
culture a large number of samples under identical conditions in one 
vessel, since a different vessel must be provided for each of the 
cultures. A problem also occurs in that only a certain filter paper 
materials are suitable for use in this method, since when the paper bridge 
is wet, the combined weight of the absorbed liquid nutrient medium and the 
cultured plant tissue specimen can collapse the bridge. Even for commonly 
available types of filter paper, the use of the filter paper bridge method 
is limited to cultures of small size since the growing culture can often 
by itself collapse the bridge thereby dumping the culture into the 
nutrient medium. It is also common, because there is not regulatory 
mechanism for the removal of excess liquid, for the tissues cultivated 
under such a method to become overly soaked with liquid medium to thereby 
suffer from inadequate gas exchange with the atmosphere to the detriment 
of the culture. Thus while this method is useful in a laboratory on small 
scale experiments, the adaptation of such a system to the larger scale 
cultivation of plant tissue cultures is not practical. 
It has also been known previously in the technology that whole plants, as 
opposed to plant tissue cultures or callus cultures, can be cultivated in 
apparatus wherein the liquid nutrient requirements of the plant are met by 
use of a wick providing capillary action from a nutrient medium located 
beneath the plant. For example, the disclosure of U.S. Pat. No. 4,299,054, 
to Ware, describes a hydroponic assembly for growing plants which includes 
a wafer upon which the plant is grown. One or more wicks are provided 
depending from the wafer into a nutrient media provided in the apparatus 
so that liquid nutrient media travels by capillary action up the wick to 
saturate the wafer to provide liquid requirements for the plants needs. 
Such a system, while possibly satisfactory for the growth of an intact 
plant including roots, is not satisfactory for the growth of a plant 
explant tissue cultures or callus cultures since such a system requires 
root growth into the liquid culture medium for continued plant viability. 
SUMMARY OF THE INVENTION 
The present invention is summarized in that a plant tissue culture device 
includes a medium vessel holding a supply of liquid nutrient medium for 
the plant tissue culture; a culture plate mounted in the culture vessel 
above the liquid medium, the culture plate having at least one pair of 
openings therein; at least one culture well formed on the culture plate to 
contain a plant tissue culture therein; and at least one continuous wick 
of porous material folded so as to have a central culture host portion and 
a pair of depending legs, the culture host portion located in the culture 
well and receiving the plant tissue culture thereon, supported firmly on 
the culture plate, while the legs extend through the openings in the 
culture plate to depend into the liquid medium in the vessel so that 
liquid medium will be transported to the culture medium portion in the 
culture well by capillary action to sustain any plant tissue on the 
culture portion. 
It is an object of the present invention to provide a plant tissue culture 
device which is specifically adapted to facilitate the cultivation and 
maintenance of plant tissue cultures and plant callus cultures on liquid 
medium. 
It is an object of the present invention to provide such an apparatus which 
is capable of external automatic operation so as to avoid the need for 
continuous manual maintenance. 
It is yet another object of the present invention to provide such an 
apparatus which also facilitates the recovery of plant metabolites or 
exudates from the tissue cultures or plant callus cultures. 
Other objects, advantages, and features of the present invention will 
become apparent from the following specification when taken in conjunction 
with the accompanying drawings.

DESCRIPTION OF THE PREFERRED EMBODIMENT 
Shown in FIG. 1, and generally indicated at 10, is a plant tissue culture 
device constructed in accordance with the present invention. In the 
exploded view of the device as illustrated in FIG. 1, the three main 
structural components of the tissue culture device 10 are shown separated. 
The bottom of the tissue culture device 10 is a medium vessel 12. Into the 
medium vessel 12 is inserted a culture plate assembly 14, and a cover 
plate 16 is adapted to fit over and cover the medium vessel 12 with the 
culture plate assembly 14 therein. Each of these components is adapted to 
be constructed from lightweight, rigid, preferably transparent materials, 
such as polycarbonate plastics. 
The medium vessel 12 is a large rectangular receptacle adapted to receive a 
quantity of liquid culture medium therein. At each end of the medium 
vessel are a respective one of a pair of inlet and outlet connectors 18 
which are adapted to being connected to standard laboratory hosing for 
introducing media fluid into and draining fluid out of the medium vessel 
12. The medium vessel should be fluid-tight and may be constructed of any 
suitable material, but, as stated above, it is preferably constructed of 
transparent synthetic resin material. A magnetically operable stirrer 13 
is placed in the bottom of the medium vessel so that the medium in the 
vessel 12 can be stirred under external magnetic control. 
The culture plate assembly 14 is designed in size so as to fit into and to 
generally extend completely across the interior of the medium vessel 12. 
The culture plate assembly 14 includes a planar culture plate 20 which is 
largely rectangular in shape and size and adapted to be only very slightly 
smaller than the interior horizontal rectangular size of the medium vessel 
12. The four corners of the culture plate 20 are cut away to provide 
arcuate access ports 22. The access ports 22 may be of any suitable shape, 
such as the semi-circular cut-outs illustrated in the culture plate 
assembly 14 of FIG. 1, but are preferably of sufficient size to allow 
tubing, pipets, or other standard laboratory apparatus to reach past the 
culture plate assembly 14 into the bottom of the medium vessel 12, even 
when the culture plate assembly 14 rests inside of the medium vessel 12. 
Mounted on the culture plate 20 are a plurality of culture well walls 24. 
Each of the culture well walls 24 is formed as an upstanding cylinder 
defining therein a culture well, indicated at 26. The culture well walls 
24 are preferably sufficiently high so as to accommodate therein the 
entire tissue culture which it is desired to cultivate or maintain within 
the device 10. On the bottom of each of the culture wells 26 are a 
plurality of openings 28 cut entirely through the culture plate 20. In the 
embodiment of the device as illustrated in FIG. 1, there are four of the 
openings 28 each of which is in an arcuate shape so as to form an 
interrupted circle centered in the central portion of the bottom of the 
culture well 26. 
Included in the culture plate assembly 14 are a plurality of upstanding 
vertical supports 30. Each of the upstanding vertical supports 30 extends 
through suitable appropriate hole provided in the culture plate 20. Each 
of the vertical supports 30 is a hollow cylinder, preferably again formed 
of a rigid synthetic resin material, which has a plurality of spaced pairs 
of adjustment holes 32 provided extending horizontally therethrough. A peg 
34 is provided with each of the vertical supports 30 which can be inserted 
through a selected pair of adjustment holes 32 with the culture plate 20 
resting on the pegs 34 so as to support the culture plate 20 at a selected 
adjustable height relative to the vertical supports 30. Alternatively, if 
a fixed height for the culture plate 20 was acceptable, the culture plate 
20 could be fastened permanently to the medium vessel 12 at that 
appropriate fixed height. 
Also included in the culture plate assembly 14 are a plurality of wicks 36. 
In the embodiment illustrated in FIGS. 1 and 2, there is one wick 36 
provided for each of the culture wells 26 although it is possible, as 
illustrated in the embodiment of FIG. 3, to have one wick for the entire 
culture plate assembly 14. In the embodiment of FIG. 1, however, each 
culture well 26 has associated with it a single wick 36 formed of filter 
paper or other highly porous sheet material capable of fostering transport 
of liquid by capillary action when placed partially in a fluid medium. 
Each of the wicks 36 is bent twice to form a culture host portion 38 
located in the bottom of the respective culture well 26, resting firmly on 
the culture plate 20, and a pair of depending legs 40 each extending 
through one of the openings 28 and downwardly draped from the culture 
plate 20 into the liquid medium in the vessel 18. The elongated arcuate 
shape of the openings 28 allow easy passage of the legs 40 of the wick 36 
therethrough. The wick 36 is preferably sized and folded such that each of 
the legs 40 will depend downwardly near to the bottom of the medium vessel 
12 when the culture plate assembly 14 is placed inside of the medium 
vessel. To facilitate the flow and exchange of nutrient medium solution 
through the wicks 36, the medium can be stirred or the entire device can 
be shaken or swirled to maintain a homogeneous medium. 
The cover plate 16 is a plate adapted in size so as to fit over the top of 
the medium vessel 12 enclosing the culture plate assembly 14 entirely 
therein. Preferably the cover plate 16 is again constructed of transparent 
synthetic resin material, such as a polycarbonate, although in some 
circumstances it may be desirable to have the cover plate constructed of 
an opague material where it is desired to avoid photosynthesis in plant 
cultures being maintained or cultivated in the culture device 10. 
Illustrated in FIG. 2 is a cross-sectional view of the assembled tissue 
culture device 10 as used in operation. The culture plate assembly 14 is 
mounted inside the medium vessel 12 in such a fashion that the culture 
plate 20 itself is mounted parallel to and above the level of the liquid 
contained in the medium vessel 12. The culture plate 20 rests securely in 
position by resting on a series of pegs 34 which are inserted through a 
corresponding set of adjustment holes 32 in each of the vertical supports 
30. By changing the appropriate pairs of adjustment holes 32 into which 
the pegs 34 are inserted, the height at which the culture plate 20 is 
mounted in the medium vessel 12 can be adjusted. As can be clearly seen in 
FIG. 2, each of the wicks 36 is folded twice at right angles so as to form 
generally three portions. The culture host portion 38 rests on the top of 
the culture plate 20 and the bottom of the culture well 26. In this 
fashion the culture host portion 38 is firmly supported in a firm planar 
surface which forms a rigid and secure base onto which plant tissues can 
be deposited and grown. Depending downward from the culture host portion 
38 are the legs 40 which are folded at approximately perpendicularly to 
the culture host portion 38. The legs 40 depend downwardly to be immersed 
into the liquid medium contained inside of the medium vessel 12. 
In its operation, the tissue culture device the present invention is used 
to foster the growth plant tissues in a culture sustained by liquid 
medium. As can be seen in FIG. 2, plant tissues 50 are placed on top of 
the culture host portion 38 of each of the wicks 36 contained in each of 
the culture wells 26. Liquid nutrient medium from the reservoir contained 
in the medium vessel 12 as absorbed by capillary action up the legs 40 of 
each of the wicks 36. Thus the culture host portion 38 of each of the 
wicks 36 eventually dampens with culture medium and the plant tissue 
culture 50 growing on the culture host portion 38 can obtain their liquid 
nutrient requirements from the constantly replenished liquid in the 
culture host portion 38. Excess dampening of the cultures is prevented by 
the provision of the excess openings 28 provided through the culture plate 
20 in each of the culture wells 26. Should the capillary action provided 
by the wicks 36 succeed in bringing too much liquid into the culture well 
26, the excess liquid drains through the extra openings 28 back into the 
reservoir of liquid medium contained in the medium vessel 12. Thus 
oversaturation of the tissue culture 50 in each of the culture wells 26 is 
avoided. Also the level of moisture to which the cultures are exposed can 
be adjusted by changing the distance between the culture plate 20 and the 
level of the liquid in the medium vessel 12. This can be done either by 
adjusting the level of the culture plate 20 upward or downward on the 
vertical supports 30 or by raising or lowering the liquid level in the 
vessel 12 through liquid exchange through the inlet or outlet connectors 
18. 
The inlet and outlet connectors 18 allow for automatic additions to, 
removals from, adjustment of level of, or complete changing of the liquid 
nutrient medium contained in the medium vessel 12. It is envisioned in a 
large-scale facility for culturing plant tissues that the operation of 
many culture devices 10 would be under automatic control in a large-scale 
system so that periodically, on an automatically timed or on a continuous 
feedback responsive basis, additional nutrient medium would be introduced 
into or removed from the medium vessels 12 through the inlet and outlet 
connectors 18. A mariotte flask reservoir system is an example of an 
efficient system for supplying liquid to and maintaining the liquid level 
in the medium vessel 12 of the present invention independent of the rate 
of evaporation or transpiration losses. The magnetic stirrer 13 is 
provided in the bottom of the medium vessel 12 so that automatic stirring 
of the liquid medium contained in the medium vessel 12 can be accomplished 
if the nutrient medium contained substances which would otherwise stratify 
within the vessel. 
The apparatus according to the present invention provides several 
advantages of prior art devices. One of the most significant advantages 
the present device provides over the now commonly used paper filter bridge 
structure is that the carrying capacity of the culture wells 26 of the 
present invention is significantly higher than that obtainable by a paper 
filter bridge alone contained in a test tube since the culture host 
portion 38 rests firmly on a fixed and rigid surface, i.e. the culture 
plate 20. Furthermore, the provisions for the extra openings 28 allow 
excess fluid to be drained from each of the plant cell culture wells 26 so 
as to avoid oversaturation of the cultures. Since the liquid nutrient 
medium in the medium vessel 12 can be supplemented or removed or have its 
level adjusted without disturbing the tissue cultures being maintained in 
the culture wells 26, i.e. either by use of the inlet and outlet valves 16 
or by inserting appropriate liquid conduits through the access ports 22 
provided in the corners of the culture plate 20, there is no need to 
disturb the culture when adding additional nutrient medium, when changing 
the medium in accordance with the plants needs during growth phases, or 
when removing the medium to retrieve exudates or metabolites therefrom. 
It is particularly envisioned that the apparatus according to the present 
invention will be particular useful for the collection of plant exudates 
and metabolites from plant cell tissues grown in culture. Much 
experimentation is currently being undertaken to develop genetically 
engineered plant tissues which can be grown in culture and which exude or 
metabolize certain desirable substances. If the plant tissue cultures 
grown within the culture device 10 which exude desirable substances, the 
chemical substances would diffuse down the wick 36 to the liquid nutrient 
medium contained in the medium vessel 12. Since the medium in the medium 
vessel 12 can be periodically removed and processed through appropriate 
separation steps to remove the desired exudates from the medium, any 
desired exudates or metabolites which are produced by any plant cell 
tissue cultures grown on the culture device 10 can be recovered easily, 
and under automatic control, something heretofore not possible in the art. 
Furthermore the culture device of the present invention allows a large 
number of plant tissue cultures to be exposed to identical growth 
conditions both in terms of temperature and light and also in terms of 
chemical environment since all the tissues in a single culture device will 
by definition be exposed to the same conditions. This feature can never be 
completely assured in a system in which the cultures are kept in separate 
containers, i.e. when they are kept in individual test tubes. 
Shown in FIG. 4 is an alternative embodiment of a plant tissue culture 
device, generally indicated at 110, constructed in accordance with the 
present invention. Like parts in the plant tissue culture device 110 have 
been given numerals similar to those in the plant tissue culture device 
10. The plant tissue culture device 110 includes a medium vessel 12 and a 
top cover 16 which were identical to those in the tissue culture device 
10. The culture plate assembly 114 of the tissue culture device 10 is 
somewhat modified from that in the embodiment of FIGS. 1 and 2. In the 
plate assembly 114 the culture well walls 128 are raised up above the 
culture plate 120 to define a continuous longitudinal slot 115 is 
underneath each of the culture wells 126 formed inside the walls 28. A 
pair of openings 127 are provided at each end of the slot 115. The 
openings 128 provided in the culture plate 120 are numerous in character 
and are located below the longitudinal slot 115. The culture plate 
assembly 114 rests above the bottom of the medium vessel 12 on four simple 
mechanical legs 140. In the tissue culture device 110 there is only one 
wick 136 which has an extremely large culture host portion 138 which 
extends completely through the slot 115 and underneath each of the raised 
culture well walls 128 in the culture plate assembly 114. The legs 140 of 
the wick 136 are bent downward at the ends of the tissue culture plate 
assembly 14 to extend through the openings 127 to depend downwardly into 
liquid medium contained in the medium vessel 12. 
In its operation, the tissue culture device 110 of FIG. 3 functions in a 
fashion identical to the tissue culture device 10 of FIGS. 1 and 2. Liquid 
nutrient medium is absorbed by the wick 136 and travels up the legs 140 by 
capillary action to soak the culture host portion 138. The culture host 
portion 138, which extends continuously along the bottom of all of the 
culture wells 126, is drained by the openings 128 provided in the culture 
plate 120 just beneath the culture host portion 138. The culture plate 120 
still supports the culture host portion 138 so that the tissue cultures 
are firmly supported thereon. This structure provides all of the 
advantages inherent in the tissue culture device 10. For extremely large 
plates constructed according to this embodiment it would be particularly 
helpful to gently continuously swirl the culture device on top of a shaker 
to assure good fluid contact with the filter paper to facilitate even 
exchange of growth medium. 
As an additional modification of any embodiment of the present invention, 
it is possible to utilize multiple layer wicks in the device of the 
present invention in which various nutrient or non-absorbent layer 
laminations can be combined with porous layers. One advantageous 
lamination would be to cover the culture host portion of the wick or wicks 
with a thin layer of agarose gel, perhaps 1 mm thick. This layer could 
serve as an advantageous growth substrate for some plant tissue cultures 
without significantly interferring with nutrient transport. 
It is understood that the present invention is not limited to the 
particular construction and arrangement of parts disclosed herein, but 
embraces all such modified forms thereof as come within the scope of the 
following claims.