Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~ 207~773
METHODS AND APPARATUS k
RELATING TO MICROPROPAGATION ;.`
The present invention relates to methods and
~5 apparatus ~or use in micropropagation. The invention
is concerned in particular, but not exclusively, with
micropropagation of seed potatoes, and harvesting of
potato microtubers from plantlets which have been grown
by micropropagation. I
.
Micropropagation of plants involves the use of the .
techniques of plant tissue culture and the application
of these to the propagation of plants. At its
simplest, micropropagation consists initially of
surface sterilising and excising small pieces of
actively growing tissue, normally shoot tips or nodes
cut from the stems of plants. Then, under aseptic
conditions, the piece3 of t~ssue are trans~erred to a
nutrient metiu~ which sùppor~s plant growth. The plant
ma~eri~l will ~lnally develop into entire plantiets.
These plantlets must then be weaned from the axenic
conditions in which they have existed within the
laboratory into viable, rooted plants capable of
survival in conventional horticultural or agricultural
env~ronments.
I ,
Normally the step of cutting a plantlet into small
pieces, referred to as explants, for regrowth ~the
multiplication stage) is repeated several times before
a batch of plantlets is grown to viable plants. Where
the plants propagate by propagules such as tubers, a
further stage is introduced. The plantlets at the end
of the multiplication process are grown until small
tubers, called microtubers, are formed, which are then
_ 1 35 removed for planting to p.oduce viable plants.
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The growt~ of plants from tissue culture is a
technique which can produce large numbers of
genetically identical plants, perhaps possessing a
desirable quality such as disease resistance, in a ¦
S short time. The tasks of dissecting and transplantins
such plants are labour-intensive and repetitive, and
the gains in speed, sterility and labour costs which
could be achieved by the use of automation make it an ¦
attractive prospect for the fast-expanding
micropropagation industry. However, robotic automation
is difficult, since it requires methods of handling and
robot guidance which are able to deal with the natural
variability of biological objects. 1
lS In known, manual, methods of micropropagation,
four of the particularly ~mportant operations which are
repeated freguently are (i) removing a plant from a
container, (ii) cutting a reguiret portion of plant
tissue from the donor plant, ~iii) transferring the cut ;
~ portion of plant ~aterial, and (iv) placing the plant
portion in a sot nutrient medium in such a manner that
it stands upright. As performed at present, the
cutting operation normally consists of an operator
holding the plant material by forceps on a sterilised
surface by one hand, and cutting the reguired portion
of the plant by strokes of a scalpel, by the other
hand. Commonly, the cuts are required to cut from the
donor a shoot tip, or a node, from which a side shoot
will develop from an axillary bud. The cut portion is
30 then transferred by the forceps to the soft nutrient ,
medium, which is in the nature of a gel, and the i~i
cutting is then placed upright with the stem part of ;'i
the cutting in the soft nutrient medium. The
positioning is normally carried out with the use of
forceps.
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It is an object of the invention, in-a number of
different aspects, to provide techniques for automating
the micropropagation of plantlets, particularly of the
kind normally propagating by tubers, in which very
OS simple methods of cutting, transfer, planting and
harvesting can be used which do not depend upon robotic
selection of particular plantlets for processins, nor
individual planting of cut portions of plant material.
Although the multiplication rate may be reduced by such
techniques compared with individual micropropagation
cutting and planting, the techniques of the invention
provide for easy automation, and are therefore
particularly suitable for bulk micropropagation of
relatively low value plants such as seed potatoes,
lS thus leading to lower labour costs and reduction in the
cost of the final product.
In accordance with the invention in one aspect,
there is pro~ided a ~ethod of micropropagation
including tho steps of growing a plurality of plantlets
in a nutrient mediu3, the plantlets having stems
bearing tips and/or nodes, cutting through a plurality
of stem~ of the plantlets at a predetermined distance
from the nutrient medium to provide a plurality of
plant portions containing tips and/or nodes, repeating
the cutting step at a further, smaller, predetermined
distance from the nutrient medium, to produce frcm the
remaining cut stems before regrowth a further plurality
of cut plant portions, and transporting the cut plant
portions under aseptic conditions in bulk to a fresh
nutrient medium.
Most preferably the method includes repeating the
cutting step at a series of different distances from
the nutrient medium to produce a series o pluralities
~ of cut plant portions. 207~ ~73
Conveniently the step of transporting cut plant
portions includes depositing the cut plant portions at
S random on the surface of the fresh nutrient medium,
where it is found such explants will grow
satisfactorily without individual siting and
positioning. The step of transporting the cut plant
portions to the fresh nutrient medium can be achieved
conveniently at least partly by gravity, and/or by a
forced airflow carrying the cut plant portions in the
airflow.
!
As a final step in the micropropagation (for
example where the plants are of the kind normally
propagating by propagules such as tubers), the method
may include the step of harvesting propagules from the
plantlets by combing through the plantlets with a comb
and separating the propagules from the plantlets.
In general, in all the different aspects of the
invention, where features are set out in connection
with a method accorting to the invsntion, these
features may ~lso be provided in apparatus according to
the invention.
In particular, there may be provided in accordance
with the invention, apparatus for use in micropropag-
ation of plants comprising a first support means for
supporting a nutrient medium for growing plantlets
having stems bearing tips and/or nodes, cutting means
adapted for cutting at least twice through a plurality
of stems of the plantlets at decreasing predeter~ined
distances from the nutrient medium to remove before
regrowth at least two pluralities of plant portions
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con~alning tips and/or nodes, and a second support
means for sùpporting a second nutrient medium in which
plantlets may be grown, the second support means being
positioned to receive CUt plant portions in bulk under
05 aseptic conditions, cut from the said plantlets.
Conveniently the cutting means may comprise shear
cutting means, for example by relative reciprocatory
motion of sets of cutting teeth, generally in the
manner of a clipper for pet animals.
i
In some arrangements, the plantlets may be
presented at a workstation for cutting, in a
conventional configuration with the stems growing
substantially vertically from a horizontal nutrient
medium. However, in other arrangements the nutrient
medium may conveniently be presented with its normal
upper growing surface oriented away from the normal
horizontal pO5 ition. For example the ~upporting
container ~ay be inverted so that the plantlets are
d,rected downwardly from a horizontal surface of the
nutrient medium, or the surface may be vertical with a
cutter moving upwardly to release material sequentially
onto a second moving nutrient medium in a gensrally
regular manner.
The orientation of the nutrient medium may provide
a further independent aspect of the present invention.
$hus there Day also be provided in accordance with the
invention a method of micropropagation including the
steps of presenting at a workstation a plurality of
plantlets held in a nutrient medium, the medium being
presented in an orientation such that a portion of a
plantlet which is cut from the plantlet falls free from
the plantlets held in the nutrient medium, cutting
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through a plurality of the growing plantlets to release
in bulk cut-away plant portions large enough to contain
tips and/or nodes, and receiving the released plant
portions on a second nutrient medium.
05
There may be provided correspondingly, in
accordance with this aspect, apparatus for use in
micropropagation of plants comprising a first support
means for supporting a nutrient medium with plantlets
growing in the medium, means for presenting the
nutrient medium and plantlets in an orientation such
that a portion of a plantlet which is cut from the
.plantlet alls free of the plantlets held in the
nutrient medium, cutting means for cutting through a
plurality of growing plantlets to release cut-away
plant portions large enough to contain tips and/or
- nodes, and second support means for supporting a second
nutrie~t medium, the second support means being
arranged in a position such as to receive in bulk the
rsleased plant portions cut by the cutting means.
As has been mentioned, a later step in the method
may comprise harvesting propagules from plantlets
growing in a nutrient medium. This step may be
utilised independently of the other steps of the
invention, and there is therefore provided in
accordance with another independent aspect of the
invention a method of harvesting propagules from
plantlets growing in a nutrient medium comprising
producing rel~tive movement between the plantlets and a
comb, the relative movement being such as to cause the
teeth of the comb to enter into an array of the stems
. of the plantlets, and also being such as to cause the
comb to move away from the bases o~ the stems, in such
a manner that the teeth comb through the plantlets and
remove propagules Erom the plantlets.
Correspondingly there may be provided with this
aspect apparatus for harvesting propagules from
05 plantlets growing in a nutrient medium comprising
a comb, support means for supporting a nutrient medium
having plantlets growing therein and means for
producing relative movement between the support means
and the comb, the relative movement being such as to
cause, in operation, the teeth of the comb to enter
into an array of stems of plantlets growing in a
nutrient medium supported by the support means, and
also being such as to cause the comb to move away from
the bases of the stems, in such a manner that the teeth
comb through the plantlets and remove propagules from
the plantlets.
In accordance with a yet further aspect of the
invention the~e may bo pxovided an apparatus for use in
particular, but not exclu~ively, in the methods set out
above concerned with ~oving cut plant portions by
gravity and concerned with combing propagules. In this
aspect there may be provided apparatus for use in
micropropagation of plants comprising a main support
structure, engaging means extending from the support
structure and adapted to engage a container containing
a nutrient medium with a plurality of plantlets growing
therein, and a plurality of fingers extending from the
support structure in a region spaced from the engaging
means, the fingers being adapted to be positioned
between plantlets and in contact with the surface of
the nutrient medium to retain the nutrient medium in
the container.
There may further be provided a method of operati~g
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on a plurality of plantlets during micropropagation,
including the steps of inserting between plantlets
growing in a nutrient medium a ~lurality of fingers,
and contacting the sur.ace o the medium by the fingers
05 to retain the nutrient medium during an operating step '
being carried out on the plantlets during
micropropagation. t
11
The invention extends to microplants and 1'
microtubers whenever produced by any method set out in
the previous paragraphs in accordance with the
invention.
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The term microtuber refers to a small tuber grown
by the process of micropropagation, and is to be
distinguished from the term minituber which indicates !;
merely the small size of the tuber rather than the
method of production. With regard to potatoes, a
microtuber is a small (l-lOmm, usually 3-8mm diameter)
pot~to tuber produced on a potato microplant, or part
thereof, in vitro, which may be maintained in an
aseptic state after harvest until planting. It is to ,l
be differentiated from a minituber which is a small
(5-45mm usually 20-30mm diameter) potato tuber produced
25 on a potato microplant, or part thereof, in vivo, i,
usually under protected, clean, though not aseptic
conditions, e.g. in sterilized peat in an aphid proof
glasshouse.
. .
1 30 It may be hard to distinguish by the naked eye
I between microtubers at the higher end of their size
¦ range and minitubers at the lower end of theirs.
However, histologically differences may be seen between
tubers produced by the two different methods in the
thickness of the periderm, also in pore size.
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WO91/15110 PCTtGB90/01001 1 ~
2~7~3
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In this speci ication, by plantlet is meant a
small plant grown in aseptic conditions, in a nutrient
medium. 8y an explant is meant a portion of plant
material cut from a plant or plantlet and suitable to
OS be transferred to a sterile nutrient medium ~or growth
into a plantlet. An example of a propagule is a ,~
! microtuber. 8y a microtuber is meant an enlarged part
of a stem of a plantlet, which can be removed from the
plantlet and used for subsequent growth of a plant.
Typically the microtubers of potatoes are very small,
¦ for example approximately 5mm in diameter.
Embodiments of the invention will now be described
by way of example with reference to the accompanying
drawings in which:-
Figures la and lb illustrate diagrammatically
i steps in a method o~ micropropagation embodying
invention;
Figures 2a, 2b and 2c illustrate diagrammaticallyfurther steps in a method of micropropagation embodying
the invention, and Figures 2d and 2e illustrate
diagrammatically a tool for holding a container in the
step shown in Figure 2c during a method of
micropropagation embodying the invention:
Figure 3 is a diagrammatic perspective view of a
reciprocatory cutter for use in embodiments of the
invention;
Figure 4 is a diagrammatic perspective view of
apparatus embodying the invention for use in
micropropagation;
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Figure S is a diagrammatic schematic view of a
comb for use in a method of harvesting propagules for
use in micropropagation embodying the invention; and
05 Figure 6 is a diagrammatic side view of apparatus
for harvesting propagules for use in micropropagation,
embodying the invention, Figure 6a showing a
diagrammatic plan view of a comb shown in ?igure 6.
Embodiments of the invention will now be described
by way of example with reference to micropropagation of
seed potatoes. In summary, the method is as follows.
Small potato plantlets (up to 75mm tall) are grown in
aseptic conditions in sterile containers containing
gelled nutrient medium. The gelling agent, such as
agar, is mixed with specific nutrient solution which
may consist of Murashige and Skoog medium without Agar,
IAA, Kinetin or Sucrose u~ed at 4.71g 1-1, with the
addition o extra iron to double that in the Murashige
and Skoog medium and sucrose at 4% w/v. The agar may
consist of Oxoid Technical Agar No. 3, used at 0.7 -
0.8% w/v. The plants should be grown in growth rooms at
a temperature of between 17'C and 21-C, preferably
l9-C. The surface of the nutrient medium should be
between 30cm and 40cm from the light source, which may
be provided by, for example, Philips 84HF tubes. After
one month of sixteen hour ~days~ in a temperature-
growth room the stems of the plants need to be cut up
into nodes and tips, and the cut p}ant portions
transferred to new containers for regrowth. These
nodes and tips may now be regrown as before to produce
more nodes, or used to produce a harvest of
microtubers. This latter stage uses a slightly
different nutrient solution ~as above but with 8%
sucrose instead of 4%), and shorter, eight hour light
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periods are given for two months followins one month of
sixteen hour days again with the same type of light
source and similar temperature regime; thus this part
of the process takes three months. The microtubers, 3
05 to lOmm in diameter, are harvested and replanted
conventionally after a three month dormancy period, te '.
produce conventional seed potatoes. ¦
At the end of the first growth stage, the
plantlets would, in accordance with known techniques,
be cut up using a scalpel and forceps, (frequently
sterilised), and the whole operation would occur within
a laminar air-flow cabinet in order to maintain
aseptic conditions. The process of plant transfer is
lS time consuming, and the labour costs form a substantial
proportion of the overall cost of a microtuber. A
¦ reduction in labour cos~s of this stage would lead to a
lower production cost for potato microtubers.
In accordance with th~ present invention the
cutting step at the end of growth stage, is carried out
as shown for example in Figures la and lb. A plurality
of plantlets 11 are grown in a gelled nutrient medium
12 placed in a container 13. The first concept of the
! 25 invention is that, instead of cutting each individual
stem of a plantlet 11 at a position dictated by the
¦ required nodes 14, a single cut, indicated at Cl is
! made at a predetermined distance Ll from the growing
! surface 16 of the gel 12. This distance Ll is set to
¦ 30 be such as to be likely to give in portions 17 cut from
.he plantlets, on average, one tip or one node in each
cut plant portlon. The cut plant portlons are then
removed, as will be descrlbed hereinafter, and a second
cut ls made at the llne lndlcated at C2. The cut C2 ls
set at a level L2 above the growing surface 16 of the
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agar, anc is again set tO be such that the cu~ portions
:ontain on average one node. The levels Ll and L2 may
be varied according to the characteristics of the plant
species or cultivar being handled.
05
As an alternative, mixed depth cuts may be made,
as shown in Figure lb at lines C3, C4 and C5, and the
tips are shared equally between all the cuts. In the
arrangement of Figure la, cut Cl contains principally
the tips of the shoots.
By way of example, using a wide range of plant
sizes, incorporating, on average one tip and 4.3 nodes
per plantlet, it was found that a total tip and node
harvest greater than three per plant could be achieved
by severing all stems at the heights shown. Whereas
manually virtually all nodes may be identified and
separated (over 95~ y this method usually 75% u~eful
plant portions may be obtained. This however varies
not only between plant species, but also between
varieties, as is the case with potato cultivars.
Though significantly less than the maximum, this is
sufficient for continuous production and completely
eliminates vision systems or robot arms for cutting
individual nodes, etc.
A major difficulty in a mass tissue culture is
sterility. Provision of sterility is eased by reducing
the number of items contacting the plantlets and
explants, by reducing access and operator contact and
by keeping the enclosed volume small. In Figures 2a, b
and c are shown three alternative methods of
transporting cut plant portions to fresh gelled medium,
whilst minimising contact with operational elements and
working in sterile conditions. In Figure 2a the cut
portions 1~ of the plantlets are removed by suction and
~ - 13 - ~ ~`7g~73 ' ll
transported`along a conduit 18 and deposited on the
growing surface of fresh gelled medium 12A in a second
container 13A.
I
S In Figure 2b the container 13 of gelled medium 12
is ~resented at the work statlon with the growing
surface 16 of the gelled medium vertical. As the cut
portions 17 are released, these fall onto a conveyor
belt 19 which transports them to fresh gelled medium
12A in a further container 13A.
In Figure 2c the container 13 is suspended upside
down so that the plantlets 11 protrude downwardly from
the gelled medium. Conveniently the container 13 is
positioned above a second container 13A containing
gelled medium 12A, and both containers 13 and 13A are
~oved in unison ~to the right in Figure 2c) to meet a
5ta~0nary reciprocatory cutter 20. The cut partions
17 fall onto the growing surface of the gelled mediu~
12a.
With regard to the method shown in Figure 2c,
there may be a difficulty with a large container that
the gelled nutrient medium may not stay in the
container when it is inverted. In Figures 2(d) and
2(e) there is shown a tool for overcoming this
difficulty. In Figure 2(d) there is shown a hand tool
60 comprising a handle 61, a transverse arm 62, a set
of fingers 63 extending from the arm 62 and a backing
plate 64 with sides 65. The fingers 63 and transverse
arm 62 are arranged generally in the shape of a garden
hand fork, but the fingers 63 are set at the top of the
transverse arm 62, so as to be spaced from the backing
plate 64 by approximately the depth of the gelled
medium 12 plus the base of the container 13. In use
the tool 60 is slid over the container 13, as shovn in
- 14 - 2~ 73 1-
Figure 2(e), so that the fingers 63 pass between plants
il and backing plate 64 passes under the container 13.
The assembly is then inverted and the fingers 63 hold
the gelled medium in place. The tool 60 will of course
S also be usèable in an automatic system.
Figure 3 shows a suitable cutting means 20 for
cutting plantlets. The cutting means has an upper set
of cutting teeth 21 and a lower set of cutting teeth
22. The set 22 is maintained stationary and the other
set 21 is reciprocated, although in other arrangements
both sets may be reciprocated. The teeth cut by shear
between the upper and lower teeth 21 and 22. The arrow
C indicates the direction of cut of the cutting means.
Shear cutting is preferred so as to avoid the cutter
imparting a translational motion to the cut plant
portions.
The cutter itself will need frequent cleaning and
sterilisation, and must have height adjustment to
perform the different cuts. It may therefore have
détachable, sterilisable blades (or the whole motor
unit and blades may be removed from position for
cleaning)~ and preset positions which are easy to
select.
Figure 4 shows schematically apparatus for
carrying out the cutting and transfer steps shown
diagrammatically in Figures la and 2. A main frame 23
has upper ledges 24 and 25 on which can rest an
inverted container 13 containing a gelled nutrient
medium 12 in which are growing plantlets 11. At this
stage the container is preferably held by the tool 60,
which is omitted for simplicity. The nature of the
gelled nutrient medium, is such that the plantlets are
held in the gel and do not fall when the container is
`^ WO91ilsllo PCT/GB9O~OlOOl I:t
~ - 1S - 2~ 73
inverted. ~he ~rame 23 has secona ledges 26 and 27 on
which may be rested and held, by means not shown,a -
second container 13A containing fresh gelled nutrient
medium 12A, this container 13A being positioned in the
05 normal upright position. The frame 23 is moveable on
wheels 28 resting on rails 30. Next to the frame 23 is
a cutter driver assembly 31 from which protrudes a
cutter means 20, for example as shown in ~igure 3. The
cutter 20 is mounted on an support 32 which may be
raised and lowersd by passing along a vertical slot 33
in the side of the cutter driver assembly 31. A manual
adjustment of the height of the arm 32 is conveniently
provided by a cranked handle 34. In operation the !
reciprocatory cutter 20 is driven, by an electric motor
lS within the cutter driver assembly 31.
In operation, the container 13 and container 13A
are placed on the frame 23 and the cutter 20 is set at
a height equivalent to the line Cl in Figure la. The
frame 23 is then moved either manually or for examole
by electrical drive means not shown, from left to right
in Figure 4, so that the plantlets 11 are cut by the
cutter 20. The cut portions then fall by gravity onto
the fresh gelled medium 12A. The entire operation
takes place within a sterilised aseptic environment.
It is to be appreciated that the methods of
cutting and transport which have been described are
applicable to a wide variety of plantlets. There will
now be described a further step which is appropriate
where the plants are normally propagated by tubers, for
example seed potatoes. In such plants, the final step
of micropropagation is to allow the plantlets to grow
for a longer period, so that microtubers grow on the
stems. The micro tubers are tough and can be extracted
by a combing action using a comb 39 such as that shown
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in Figure 5. The tool of Figure S is a manual tool,
and has a handle 35 supporting a tray-like structure 34
having side walls 36, 37 and 38, and being open at the
end remote from the handle 35. The base 39 of the
S tray-like structure 34 is divided to form a comb having
teeth 40 projecting away from the handle 35. Each
tooth has a rounded distal end 41, tapered sides for
part of its length, and a main portion 42 with parallel
sides. The end of each s}ot formed by the parallel
sides is rounded at the base at the region 43. It is a
feature o~ the system of harvesting by combing that the
tubers are in effect being graded by the combing
action, since tubers smaller than the spacing of the
parallel sided parts of the teeth, are not removed by
the combing action.
The gap between the parallel sided parts 42 of the
teeth 40 indicated at G is set to be smaller than the
smallest usable microSuber, but large enough to allow
passage o stem~, leavos, etc. through between the
te~th. In a manual system, the tool i5 co~bed through
the plantlets growing in the agar, and lifted gently
away from the agar to pluck the microtubers. The tool
illustrated in Figures 2d and 2e may be employed also
to prevent removal of plants from the agar whilst
combing i5 carried out.
Figure 6 illustrates an automated version of the
combing tool of Figure S. The teeth 40 of the comb 39
are shown in Figure 6a, the region indicated at Rl
being for entrainment of tubers, and the region
indicated at R2 being for plucking. As shown in Figure
6, containers 13 of plantlets ready for harvesting are
fed as indicated by arrow F onto a conveyor system S0
which carries the containers 13 downwardly and to the
right in Figure 6. Conveniently the conveyor system 50
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. consists of a conveyor belt Sl entrained about three
pulleys 52, 53 and 54, the conveyor belt Sl having, for
example, apertures into which the container 13 can fit.
Alternatively, two conveyor belts can be positioned
OS side by side, with the container 13 resting between the
two conveyor belts.
The comb 39 is positioned without net 1-
translational movement relative to the pulleys 52, 53 t~
and 54 but is vibrated by a vibration drive system SS.
The comb 39 is set at an angle to the surface of the
conveyor belt Sl, such that the teeth 40 of the comb 39
diverge from the surface of the conveyor belt Sl in a
direction away from the distal ends of the teeth 40. ,'
lS The effect of this is that as the container 13 is .,
carried downwardly on the conveyor belt Sl, the distal '~
tips of the teeth 40 intrude into the stems of the
plantlets 11, and progressively comb upwardly through
the plantlets 11, ~hus over the region R2 of the teeth ~'
40, tubers 56 are plucked from the plantlets 11 and
then roll down the comb 39 onto a conveying chute 57
contiguous with the comb 39. The stripped plantlets 11
in the waste container 13 are then ejected from the
conveyor system S0 over the pulley 53 as indicated at
the arrow W.
In the methods of micro processing described
above, it will be seen that in two aspects automation
has been achieved by accepting a deqree of randomness
in the operation. With regard to the cutting, it is
appreciated that some wastage of nodes occurs because
of the irregular, unpredictable internode spacings.
Though the multiplication rate is less than the
maximum, being reduced for example from a multiple of
35 iour to a multiple oi three, it is suif icien~ ior
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continuous production, and completely eliminates vision ,
systems or robot arms for cutting individual nodes
etc.
S With regard to the planting of explants by simple
scatter technique, as opposed to individually planting
each explant, the random system has been found to be
successful. For example, tests by literally dropping
cut nodes onto the gelled surface have shown that
resulting microplants rooted and grew normally. A
slightly different shape is usually obtained compared
with an individually planted explant, but this presents
no problems where mass production of plants is required
for example with seed potatoes. The scatter planting
technique has substantial ad~antage because no
components, or fewer components, touch the explants
before they reach the gelled medium, leaving less
chance of infection.
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