EP0523040A1 - Methods and apparatus relating to micropropagation - Google Patents

Abstract

In a micropropagation process, a series of horizontal cuts is made through a mass of seedlings (11) with practically straight stems and growing in a nutritious gelled medium (12), in order to obtain cut parts (17) each containing at least one termination or node. After each cut, the cut parts (17) are transferred in bulk, by means of an air stream or by gravity, to a new gelled nutritive medium (12A), in which they are planted by scattering them. In the final step, the propagules (56) of the seedlings (11) are harvested by passing a comb (39) through the seedling and by separating the propagules by relative movement of the comb (39) carried out from the gelled medium (12). .

Description

METHODS AND APPARATUS RELATING TO MICROPROPAGATION

The present invention relates to methods and apparatus for 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.

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 pieces of tissue are transferred to a nutrient medium which supports plant growth. The plant material will finally develop into entire plantlets. 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 environments.

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 removed for planting to produce viable plants. The growth 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 short time. The tasks of dissecting and transplanting 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.

In known, manual, methods of micropropagation, four of the particularly .important operations which are repeated frequently are (i) removing a plant from a container, (ii) cutting a required portion of plant tissue from the donor plant, (iii) transferring the cut portion of plant material, and (iv) placing the plant portion in a soft 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 required 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 then transferred by the forceps to the soft nutrient medium, which is in the nature of a gel, and the cutting is then placed upright with the stem part of the cutting in the soft nutrient medium. The positioning is normally carried out with the use of forceps. 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 simple methods of cutting, transfer, planting and harvesting can be used which do not depend upon robotic selection of particular plantlets for processing, 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, 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 provided a method of micropropagation including the steps of growing a plurality of plantlets in a nutrient medium, the plantlets having stems bearing tips and/or nodes, cutting through a plurality of stems 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 from 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 of pluralities of cut plant portions.

Conveniently the step of transporting cut plant portions includes depositing the cut plant portions at 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 according to the invention, these features may also 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 predetermined distances from the nutrient medium to remove before regrowth at least two pluralities of plant portions containing tips and/or nodes, and a second support means for supporting a second nutrient medium in which plantlets may be grown, the second support means being positioned to receive cut plant portions in bulk under 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.

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 position. For example the supporting container may be inverted so that the plantlets are directed 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 generally regular manner.

The orientation of the nutrient medium may provide a further independent aspect of the present invention. Thus there may 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 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.

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 falls 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 nutrient medium, the second support means being arranged in a position such as to receive in bulk the released 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 relative 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 of the stems, in such a manner that the teeth comb through the plantlets and remove propagules from the plantlets.

Correspondingly there may be provided with this aspect apparatus for harvesting propagules from 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 there may be provided an apparatus for use in particular, but not exclusively, in the methods set out above concerned with moving 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 operating on a plurality" of plantlets during micropropagation, including the steps of inserting between plantlets growing in a nutrient medium a plurality of fingers, and contacting the surface of the medium by the fingers to retain the nutrient medium during an operating step being carried out on the plantlets during micropropagation.

The invention extends to icroplants and microtubers whenever produced by any method set out in the previous paragraphs in accordance with the invention.

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 (1-lOmm, usually 3-8mm diameter) potato 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 be differentiated from a minituber which is a small (5-45mm usually 20-30mm diameter) potato tuber produced on a potato microplant, or part thereof, in vivo, usually under protected, clean, though not aseptic conditions, e.g. in sterilized peat in an aphid proof glasshouse.

It may be hard to distinguish by the naked eye 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. In this specification, by plantlet is meant a small plant grown in aseptic conditions, in a nutrient medium. By an explant is meant a portion of plant material cut from a plant or plantlet and suitable to be transferred to a sterile nutrient medium for growth into a plantlet. An example of a propagule is a microtuber. By 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 steps in a method of micropropagation embodying invention;

Figures 2a, 2b and 2c illustrate diagrammatically further 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; - 10 - Figure 5" is a diagrammatic schematic view of a comb for use in a method of harvesting propagules for use in micropropagation embodying the invention; and

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 Figure 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 used at 4.71g 1~1, with the addition of 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 19"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 plant 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 periods are given for two months following 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 to 10mm in diameter, are harvested and replanted conventionally after a three month dormancy period, to 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 time consuming, and the labour costs form a substantial proportion of the overall cost of a microtuber. A reduction in labour costs of this stage would lead to a lower production cost for potato microtubers.

in accordance with the 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 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 LI from the growing surface 16 of the gel 12. This distance Ll is set to be such as to be likely to give in portions 17 cut from the plantlets, on average, one tip or one node in each cut plant portion. The cut plant portions are then removed, as will be described hereinafter, and a second cut is made at the line indicated at C2. The cut C2 is set at a level L2 above the growing surface 16 of the agar, and is again set to be such that the cut portions contain on average one node. The levels Ll and L2 may be varied according to the characteristics of the plant species or cultivar being handled.

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%), by this method usually 75% useful 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 17 of the plantlets are removed by suction and transported along a conduit 18 and deposited on the growing surface of fresh gelled medium 12A in a second container 13A.

in Figure 2b the container 13 of gelled medium 12 is presented at the work station 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 moved in unison (to the right in Figure 2c) to meet a stationary reciprocatory cutter 20. The cut portions 17 fall onto the growing surface of the gelled medium 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 shown in Figure 2(e), so"that the fingers 63 pass between plants 11 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 also be useable 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 detachable, 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 inverted. The frame 23 has second 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 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 Figure 3. The cutter 20 is mounted on an support 32 which may be raised and lowered 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 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 example 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 in Figure 5. The tool of Figure 5 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 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 slot formed by the parallel sides is rounded at the base at the region 43. It is a feature of 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 microtuber, but large enough to allow passage of stems, leaves, etc. through between the teeth. In a manual system, the tool is combed 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 is carried out.

Figure 6 illustrates an automated version of the combing tool of Figure 5. 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 50 which carries the containers 13 downwardly and to the right in Figure 6. Conveniently the conveyor system 50 consists of a conveyor belt 51 entrained about three pulleys 52, 53 and 54, the conveyor belt 51 having, for example, apertures into which the container 13 can fit. Alternatively, two conveyor belts can be positioned side by side, with the container 13 resting between the two conveyor belts.

The comb 39 is positioned without net translational movement relative to the pulleys 52, 53 and 54 but is vibrated by a vibration drive system 55. The comb 39 is set at an angle to the surface of the conveyor belt 51, such that the teeth 40 of the comb 39 diverge from the surface of the conveyor belt 51 in a direction away from the distal ends of the teeth 40. The effect of this is that as the container 13 is carried downwardly on the conveyor belt 51, the distal tips of the teeth 40 intrude into the stems of the plantlets 11, and progressively comb upwardly through the plantlets 11. Thus 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 50 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 degree 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 four to a multiple of three, it is sufficient for continuous production, and completely eliminates vision systems or robot arms for cutting individual nodes etc.

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 advantage because no components, or fewer components, touch the explants before they reach the gelled medium, leaving less chance of infection.

Claims

1. A method of micropropagation including the steps of growing a plurality of plantlets in a nutrient medium, the plantlets having stems bearing tips and/or nodes, cutting through a plurality of stems 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 from 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.

2. A method according to claim 1 including repeating the cutting step at a series of different distances from the nutrient medium to produce a series of pluralities of cut plant portions.

3. A method according to claim 1 in which the step of transporting the cut plant portions includes depositing the cut plant portions at random on the surface of the fresh nutrient medium.

4. A method according to claim 1 in which the step of transporting the cut plant portions is achieved at least partly by gravity.

5. A method according to claim 1 including a further step of harvesting propagules from plantlets grown from the said plant portions, by the step of combing through the plantlets with a comb and separating the propagules from the plantlets.

6. Apparatus for use in micropropagation 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 predetermined distances from the nutrient medium to remove before regrowth at least two pluralities of plant portions containing tips and/or nodes, and a second support means for supporting a second nutrient medium in which plantlets may be grown, the second support means being positioned to receive cut plant portions in bulk under aseptic conditions, cut from the said plantlets.

7. A method of harvesting propagules from plantlets growing in a nutrient medium comprising producing relative 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 of the stems, in such a manner that the teeth comb through the plantlets and remove propagules from the plantlets.

8. Apparatus for harvesting propagules from 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.

9. 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 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.

10. 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 falls 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 nutrient medium, the second support means being arranged in a position such as to receive in bulk the released plant portions cut by the cutting means.

11. 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.

12. A method of operating on a plurality of plantlets during micropropagation, including the steps of inserting between plantlets growing in a nutrient medium a plurality of fingers, and contacting the surface of the medium by the fingers to retain the nutrient medium during an operating step being carried out on the plantlets during micropropagation.

13. Micropropagated plants whenever produced by a method according to claim 1.

14. Microtubers whenever produced by a method according to claim 1.