WO1986006576A1

WO1986006576A1 - Method and apparatus for dividing plant materials

Info

Publication number: WO1986006576A1
Application number: PCT/AU1986/000136
Authority: WO
Inventors: David Norman Schonstein; Brian Jordayne Johnson
Original assignee: Commonwealth Industrial Gases Ltd
Current assignee: Commonwealth Industrial Gases Ltd
Priority date: 1985-05-15
Filing date: 1986-05-14
Publication date: 1986-11-20
Anticipated expiration: 1987-11-15
Also published as: EP0222836A1; CN1011377B; BR8606674A; ZA863613B; CN86103942A; NZ216187A; JPS62502840A; EP0222836A4

Abstract

A method and apparatus for dividing plant materials with an optically detectable structure according to rules related to the structure. The plant material is scanned with a CCD camera interfaced via a video capture board with a microcomputer. An image signal from the CCD camera represents a two tone image of the plant material in a two dimensional array of pixels in which each pixel is assigned a background tone or a plant tone. The image signal is processed by systematically determining the tone assigned to each pixel in the array and the co-ordinates of plant representing pixels are recorded in a data base to form a co-ordinate map of the plant material. Selected features of the plant, for example tips of branches and nodes where branches join are identified and the co-ordinates corresponding to these features stored for use in determining division locations. The microcomputer is used to control a selectively operable cutting blade having a circular cross section which is mounted for movement over the plant material. The cutter is moved to a position over nodes adjacent tips of branches and actuated by the microcomputer to cut a selected quantity of plant material.

Description

"METHOD AND APPARATUS FOR DIVIDING PLANT MATERIALS"

TECHNICAL FIELD

This invention relates to improvements in the micropropogation of plants and in particular to a method and apparatus for dividing plant materials.

BACKGROUND ART

The advantages of micropropagation are well known: (i) It offers a convenient and effective method of disease control with consequent improvement in plant quality. Freedom from disease is becoming an increasingly important attribute in relation to quarantine requirements imposed in promising export markets such as the Middle East.

(ii) Micropropagation provides a method of rapid multiplication. In addition to its general significance as a means of achieving dramatic increases in quantity it confers specific benefits. Production of commercial quantities of a new variety can be achieved by manual micropropagation in approximately 50% of the time required by conventional methods. The facility to rapidly build up the numbers of new varieties is important even for species, such as vines, that are easily propagated by cutting.

(iii) The micropropagation technique enables the more productive use of space. Subculturing is conducive to the achievement of a pleasingly high rate of utilisation of laboratory space. Better utilization of glasshouses and plant "hardening" space is also encouraged. Where seasonal markets are being served, rapid multiplication of varieties avoids the necessity to leave unoccupied space in stock houses for protracted periods while numbers are being built up. Operation can be continued at any time of year under controlled conditions of temperature, light cycle and nutrient balance. Culture storage or reduced temperature can be used to reduce growth rate to produce synchronised high output for seasonal demand.

Application of micropropagation to a species requires the identification of a satisfactory relationship between the plant material, culture medium and incubation conditions. The range of species for which a standard procedure for micropropagation has been devised is rapidly increasing with a consequent growth in the potential area in commercial application. Involvement of Australian and overseas nurserymen in micropropagation has greatly increased in the last six years. Two main approaches have been adopted. The first involves integration of a laboratory into an existing nursery followed by enormous expansion of the nursery. Alternatively, a laboratory is established and plants in culture are sold to other nurseries. After a slow start the demand from nurseries and growers for micropropagated plants is rapidly increasing.

-The sequence of operations involved in micropropagation by organ culture is briefly as follows: (i) Initial culture; shoots are taken from the selected plant, surface sterilized and placed onto a sterile medium, (ii) The buds on these shoots eventually develop and these are subcultured to shoot multiplication medium (SM). (iii) The shoots grow rapidly on SM producing a large number of clonal shoots. To increase the number of shoots and to maintain the line, the shoots are subcultured to fresh SM every 3 to 4 weeks.

(iv) Roots develop when a sample of the shoots are subcultured to a rooting medium (RM) in a petri dish or similar container.

The plants are usually despatched in stacks of containers. The recipient removes them from the containers under non-sterile conditions and 'hardens* them to outside conditions in a high humidity environment.

Subculturing is usually performed in a transfer chamber where the shoots are removed from their containers with sterile forceps, dissected, usually quite roughly, with a sterile scalpel into small clumps of shoots or single shoots and then placed onto fresh medium. Pieces cut so as to not contain sufficient whole meristem cells will grow slowly or not at all. Plants containing few or no whole fully differentiated cells are liable to not regenerate as "true to type" or clonal plants.

The largest cost in the procedure is the labor involved in sub-culturing the shoots from one medium to another. The cost of sub-culturing is at least three times the cost of all the other procedures. The reason for this can be illustrated in terms of the sub-culturing of trees. Here the performance of 5,000 transfer operations per day is an absolute maximum for a technician. Similar productivity constraints apply to these operations whether conducted at macroscopic or microscopic scales. In addition in many laboratories sterility control is incomplete, so that contamination losses can be very substantial. Cleaning, preparation, control and movement of plant containers between cutting, storage and hardening areas and transfers to hardening medium are also labour intense.

Thus, the present ability of the Australian and overseas nursery industry to satisfy the demand for micropropagated plants is severely constrained by the cost of the manual cutting process presently employed in the micropropagation process and the maximum number of operations per day which may be performed by even the most skilled operator without deleterious side affects.

It is therefore an object of this invention to provide a method and apparatus for dividing plant materials that will overcome, or at least ameliorate, the above disadvantages.

DISCLOSURE OF INVENTION

Accordingly, in one aspect this invention consists in a method for dividing plant materials with an optically detectable structure according to rules related to the structure of the plant material, said method comprising the steps of scanning the plant material to generate an image signal representative of said structure; determining division locations from the image signal according to said rules; and generating a division signal indicative of said division locations.

For preference, the method further comprises the steps of processing the image signal to produce a co-ordinate map of the structure; identifying co-ordinates corresponding to selected features of said structure; and determining said division locations from said co-ordinates corresponding to selected features.

The image signal can represent a two tone image of the plant material in a two dimensional array of pixels, each pixel being assigned a background tone or a plant tone. The image signal is processed by systematically determining the tone assigned to each pixel in the array and recording the co-ordinates of plant tones to form the co-ordinate map. Co-ordinates corresponding to selected features, for example branch tips and nodes, are identified by determining whether previously examined adjacent pixels represent background or plant.

In accordance with the preferred method the systematic determination of the tone assigned to each image is conducted sequentially in each of two different directions and the co-ordinates of selected features identified in each direction are averaged. In this way account can be taken of branch tips that may not be otherwise identified because the branches slope away from the direction that the pixels are scanned.

In the preferred method a tip of a branch is identified when the branch is formed by a minimum of 20 pixels. This prevents identification of the tips of very small branches or the identification of a small piece of foreign matter as a branch tip and in most angiosperms ensure that each piece subsequently divided from the plant both meristem and fully differentiated cells.

In a second aspect this invention consists in an apparatus for dividing plant materials with an optically detectable structure according to rules related to the structure of the plant material, said apparatus comprising: image signal generating means to scan the plant material and generate a signal representative of the plant structure; processing means to determine division locations from the image signal according to said rules and to generate a division signal indicative of said division locations; and cutting means responsive to said division signal to divide the plant material at said locations.

For preference the processing means produces a co-ordinate map of the structure from the image signal and identifies co-ordinates corresponding to selected features of the structure.

In the preferred embodiment the image signal is generated by a Charge Coupled Device interfaced to processing means comprising a microcomputer.

The cutting means is preferably a selectively operable cutting blade mounted on a robot arm for movement over the plant material. The cutting blade can have a circular or oval cross sectional shape to cut a selected quantity of plant material at each actuation.

An embodiment of the invention will now be described, by way of example only, with reference to the accompanying drawings.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 is a schematic perspective view of an apparatus according to the invention;

Figure 2 is a flow chart of a Down Scan routine used in the apparatus of Figure 1; and

Figure 3 is a flow chart of a Process Pixel routine forming part of the routine shown in Figure 2. BEST MODE FOR CARRYING OUT THE INVENTION

Referring to Figure 1 the apparatus of this invention comprises a charge coupled device camera (CCD) 11 forming an image signal generating means. The CCD 11 is a Thomson-CSF CCD camera with a standard RSI70 video interface. The CCD 11 is connected via a Chorus Data Systems series 1000 video capture board 15 to an Olivetti M24 microcomputer 16. A signal representative of the structure of a plant 12 positioned on a continuous belt 13 and scanned by CCD 11 is captured by board 15 and the signal is digitised before being down loaded into the main memory of microcomputer 16. The plant is scanned against a background of contrasting colour and scanning of the plant 12 by CCD 11 is controlled by the microcomputer 16. In this way CCD 11 is actuated to provide a single image of plant 12.

The digitised signal stored in microcomputer 12 is representative of a two tone image of the plant in a two dimensional pixel array of 640 pixels horizontally and 400 pixels vertically. The tone assigned to each pixel corresponds to either background or plant. The digitised image signal is processed by systematically determining the tone assigned to each pixel in the array and recording the co-ordinates of plant tones in a data base to form a co-ordinate map.

In addition, co-ordinates corresponding to the tips of branches and nodes where branches join are identified and stored in a data base. The systematic processing of each pixel in the array is conducted twice, once using a Down Scan routine and once using an Up Scan routine. The Downscan and Upscan routines process the pixels sequentially in different directions and the co-ordinates representing branch tips and nodes obtained by each routine are averaged.

Figure 2 is a flow chart of the Down Scan routine which shows that the image is processed pixel by pixel starting from the upper left hand corner and working downwardly row by row. The Up Scan routine (not shown) is substantially identical but processes each pixel working upwardly row by row.

Figure 3 is a flow chart of a Process Pixel routine which is a sub routine of the Down Scan routine.

The first step in the Process Pixel routine is a backscan for an upwardly sloping branch. This is required because the same branch identifying technique used in the process pixel routine is based on determining whether background or branch is represented by the pixel to left, the pixel above and to the left and the pixel immediately above the pixel being processed. A branch sloping upwardly to the left can therefore intially be identified as a new branch until a pixel immediately above the pixel being processed is discovered to be a pixel representing plant. When this is determined the backscan is implemented to relabel plant pixels immediately to the left as being in the same branch as the plant pixel identified immediately above the pixel being processed.

The remaining steps in the routine will be apparent to persons skilled in the art from Figure 3.

The apparatus further comprises cutting means in the form of a cutter mechanism 17 operable by the microcomputer 16 to cut the plant 12 at the determined cutting locations. The cutter mechanism 17 includes tubular cutting blade 18 of circular cross section mounted in a double acting solenoid 19. Cutter 18 is moved to cut the plant 12 by selective actuation of solenoid 19 under control of microcomputer 16. The solenoid 19 is mounted on a frame 20 for movement over the plant 12 by two step motors 21. The step motors 21 are independently operable by dimension signals indicative of division locations which are generated by microcomputer 16 and each move the solenoid 19 in perpendicular directions. In this way cutter 18 can be moved to any position in a cutting area bounded by the limits of travel of the frame 20. The position of the cutter 18 at each instant is determined by microcomputer 16 by storage of the number and direction of actuating pulses supplied to step motors 21. A sensor 22 is provided to allow periodic checking of actual and calculated cutter position.

The alignment of plants 12 under cutter mechanism 19 can be achieved by means of reference holes 14 in continuous belt 13 on which the plant 12 is positioned.

The cutting locations determined by microcomputer 16 are the centres of nodes and a point 4 mm from the tips of branches. The cutting blade has a diameter of 5 mm and is centred on these cutting locations so that a selected quantity of plant material is cut at each actuation. In the case of most angiosperms this ensures that the divided plant contains sufficient meristem and differentiated cell growth to ensure monclonal propogation.

Claims

1. A method for dividing plant materials with an optically detectable structure according to rules related to the structure of the plant material, said method comprising the steps of scanning the plant material to generate an image signal representative of said structure; determining division locations from the image signal according to said rules; and generating a division signal indicative of said division locations.

2. A method as claimed in claim 1 further comprising the steps of processing the image signal to produce a co-ordinate map of said structure; identifying co-ordinates corresponding to selected features of said structure; and determining said division locations from said co-ordinates corresponding to selected features.

3. A method as claimed in claim 2 wherein said image signal represents a two tone image of the plant material in a two dimensional array of pixels, each pixel being assigned a background tone or a plant tone.

4. A method as claimed in claim 3 wherein the image signal is processed by systematically determining the tone assigned to each pixel in said array and recording the co-ordinates of plant tones to form said co-ordinate map.

5. A method as claimed in claim 4 wherein co-ordinates corresponding to said selected features are identified by determining whether previously examined adjacent pixels represent background or plant.

6. A method as claimed in claim 5 wherein the systematic determination of the tone assigned to each pixel is conducted sequentially in each of two different directions and the co-ordinates of said selected features identified in each direction are averaged.

7. -A method as claimed in any one of claims 2 to 5 wherein said features are the tips of branches of the plant material and nodes where the branches join.

8. A method as claimed in claim 7 wherein wherein a tip of a branch is identified when the branch is formed by a selected minimum number of pixels.

9. A method as claimed in claim 7 wherein said selected minimum number is 20.

10. A method as claimed in any one of claims 2 to 9 wherein said division locations are a selected distance from said selected features.

11. A method for dividing plant materials with an optically detectable structure according to rules related to the structure of the plant material, said method being substantially as herein described with reference to the accompanying drawings.

12. An apparatus for dividing plant materials with an optically detectable structure according to rules related to the structure of the plant material, said apparatus comprising: image signal generating means to scan the plant material and generate a signal representative of the plant structure; processing means to determine division locations from the image signal according to said rules and to generate a division signal indicative of said division locations; and cutting means responsive to said division signal to divide the plant material at said locations.

13. An apparatus as claimed in claim 12 wherein said processing means produces a co-ordinate map of the structure from said image signal and identifies co-ordinates corresponding to selected features of the structure.

14. An apparatus as claimed in claim 13 wherein said processing means includes a data base for storage of said co-ordinate map and said co-ordinates corresponding to selected features.

15. An apparatus as claimed in any one of claims 12 to 14 wherein said image signal is generated by a Charge Coupled Device.

16. An apparatus as claimed in claim 15 wherein said Charge Coupled Device is interfaced to processing means comprising a microcomputer.

17. An apparatus as claimed in any one of claims 12 to 16 wherein the plant material is scanned against a background of contrasting colour.

18. An apparatus as claimed in any one of claims 12 to 17 wherein said cutting means comprises a selectively operable cutting blade mounted for movement over the plant material .

19. An apparatus as claimed in claim 18 wherein the cutting blade has a circular cross section to cut a selected quantity of plant material at each actuation.

20. An apparatus for dividing plant materials with an optically detectable structure according to rules related to the structure of the plant material, said apparatus being substantially as herein described with reference to the accompanying drawings.