James Cock, Patricia Moreno Cadena, Mayra Alejandra Toro, y Johanna Arango

Contents 1. INTRODUCTION .................................................................................................................... 1

2. OUR GOAL .............................................................................................................................. 1

3. MATERIALS ............................................................................................................................ 1

4. FIELD MEASUREMENTS...................................................................................................... 2

4.1 Sample size: ........................................................................................................................ 2

4.2 Frequency of monitoring: ................................................................................................... 2

First sample: ........................................................................................................................... 2

Weekly Monitoring: ............................................................................................................... 2

Fortnightly Monitoring: ......................................................................................................... 2

Monthly monitoring: .............................................................................................................. 2

4.3 Procedure ............................................................................................................................ 2

Record of germination ........................................................................................................... 2

A. Germination date: ........................................................................................................... 2

First sampling: ....................................................................................................................... 3

A. Selection of shoots to monitor: ..................................................................................... 3

B. Tickets: ........................................................................................................................... 3

Weekly monitoring ................................................................................................................ 5

A. Collection of tickets: ...................................................................................................... 5

B. Rate leaf formation: ........................................................................................................ 5

C. Height of plant:............................................................................................................... 6

Biweekly monitoring ............................................................................................................. 6

A. Count of nodes: .............................................................................................................. 6

B. Determination of the length and diameter of internodes: ............................................... 7

C. Identification of first fully expanded leaf: ...................................................................... 8

D. Determination of leaf area: ............................................................................................ 9

ANNEX 1: SAMPLE OF THE WORKSHEET TO REGISTER THE SAMPLING DATA ...... 11

ANNEX 2: DEVELOPMENT OF THE INSTRUMENT TO MEASURE LEAF AREA ........... 12

ANNEX 3: IMAGE PROCESSING WITH IMAGE J SOFTWARE .......................................... 17

List of Figures Figure 1. Record of germination at 14 days after planting ............................................................. 3

Figure 2. Plants with one (left) and two (right) shoots that grew from the originally sown stake . 3

Figure 3. Example of a ticket with the plot identification and the date .......................................... 4

Figure 4. Tickets with color coded with a different color for each date ......................................... 4

Figure 5. New ticket in the youngest leaf with a petiole separated stem ........................................ 4

Figure 6. Plant with large labels in contact with the ground (left) and plants with small labels

(right) .............................................................................................................................................. 5

Figure 7. Collection of the fallen tickets on the ground ................................................................. 5

Figure 8. First branching height measurement ............................................................................... 6

Figure 9. Dead apex ........................................................................................................................ 6

Figure 10. Identification of levels of branching.............................................................................. 7

Figure 11. Identification of sections according to the length of internodes. Branch with two

different sections identified (left) and branch with only a section identified (right). ..................... 7

Figure 12. Determination of the change in the branch, of green to lignified .................................. 8

Figure 13. Measurement of the stem diameter ................................................................................ 8

Figure 14. Angle between the petiole of fully expanded leaf and stem .......................................... 9

Figure 15. Size difference between an unexpanded and fully expanded leaf ................................. 9

Figure 16. Capture process of photography to the determination of leaf area: Identification of the

fully expanded leaf, insertion between the laminae and take photography .................................. 10

Figure 17. Position of the camera to take the picture ................................................................... 10

Annex 2

Figure 18. Materials used in preparing the template to measure leaf area ................................... 12

Figure 19. Shiny side (left) and dull side (right) of the lamina of polystyrene and acrylic .......... 12

Figure 20. Hinge location and marking plates .............................................................................. 13

Figure 21. Opening holes with the drill in the plates .................................................................... 13

Figure 22. Locations of screws a hinge and plate ......................................................................... 14

Figure 23. Assurance hinges on acrylic sheet ............................................................................... 14

Figure 24. Union of acrylic and polystyrene laminas. .................................................................. 15

Figure 25. Dialing scale ................................................................................................................ 15

Figure 26. Definition of grid ......................................................................................................... 16

Figure 27. Instrument to measure leaf area ................................................................................... 16

Annex 3

Figure 28. ImageJ installation ....................................................................................................... 18

Figure 29. Working window for ImageJ ....................................................................................... 18

Figure 30. Menu to open the image and emerging window ......................................................... 19

Figure 31. Straight icon from the principal menu of ImageJ ........................................................ 19

Figure 32. Scale definition according with the image .................................................................. 20

Figure 33. Image conversion to 8 bits ........................................................................................... 20

Figure 34. Image conversion to binary ......................................................................................... 21

Figure 35. Selection of the variable to measure, area ................................................................... 21

Figure 36. Menu Analyze particles ............................................................................................... 22

Figure 37. Results visualization for the images without shade and reflection .............................. 22

Figure 38. Threshold menu ........................................................................................................... 23

Figure 39. Menu to define hide, saturation and brightness properties .......................................... 23

Figure 40. Menu Analyze particles ............................................................................................... 24

Figure 41. Results visualization for images with shades and reflection ....................................... 24

1

1. INTRODUCTION This non-destructive method provides a means of monitoring the development of the cassava

crop under a range of biophysical environments without the need to plant trials specifically

for this purpose. The plots established or commercial lots can be monitored nondestructively:

therefore any parcel can be monitored. The methodology collects data on the development of

the cassava crop and can use this data in understanding the development of the crop, and also

to generate, validate and improve the robustness of simulation models cassava. This is the

first approximation of the methodology; we encourage users to voice any concerns and

suggest ways of improving the non-destructive monitoring system.

2. OUR GOAL To improve our understanding of the physiological development of cassava through detailed

recording of cassava growth during all stages of its development under a range of conditions.

The information and understanding generated can be used to improve cassava growth and

simulation models.

3. MATERIALS a) Vernier caliper

b) Tape measure/ ruler

c) Forms for recording information (Annex 1)

d) Labels or tags with string for marking leaves

e) Pencil

f) Permanent marker

g) An instrument for measuring leaf area in field (Annex 2)

h) Camera

i) Computer

A B D E F

H I

2

4. FIELD MEASUREMENTS

4.1 Sample size: five representative plants for each treatment or variety should be monitored.

All monitored plants should be surrounded by at least one border plant.

4.2 Frequency of monitoring: After the first sampling, the plots should be monitored at

fixed intervals. When monitoring is very frequent great care must be taken not to compact the

soil, especially during the rainy season. The sampling frequency may be varied with some

weekly, biweekly or monthly intervals up to monitoring and measuring certain variables in

each of these:

First sample: For this monitoring germination date must be pre-defined, and a shoot

should be selected to be followed during the development of the plant; additionally must

identify the youngest leaf with a petiole separated stem to identify with a ticket and start

the evaluation of the rate of leaf formation.

Weekly Monitoring: The variables to be measured every week are the rate of formation

of leaves, leaf longevity through ticket collection; and plant height. These variables are

measured at this frequency because they are more dynamic and more change with the

development of the plant, with significant differences between one week and one; also do

not require much time in the field to be determined.

Fortnightly Monitoring: The variables that should be measured every two weeks are the

number of nodes, length and diameter of internodes, the identification of the first fully

expanded leaf and leaf area of this. These variables require more time in the field to be

determined.

Monthly monitoring: The variable to be measured monthly, is the total apex number,

taking into account live and dead apices

If a sampling date is missed due to unforeseen problems continue with the sampling as soon as

possible after the missed sample: the data can still be used. In this case, the variables are

measured each week can be measured biweekly and these in turn on a monthly basis.

4.3 Procedure

Record of germination

A. Germination date: Monitor the crop every 5 days after planting to observe the date

when the first shoots appear from the original planting piece or cutting (Figure 1).

Evaluate at least twenty plants if possible and when 50% of the cuttings have germinated

with new green shoots emerging, record the date of germination. Record the germination

data in the form established for the first sampling (ANNEX 1).

3

Figure 1. Record of germination at 14 days after planting

First sampling:

A. Selection of shoots to monitor: When 50 % of the plants have germinated and the first

leaves are fully expanded that the petiole is clearly visible and separates the leaf from

stem, select five representative plants in each plot or field. Count and record the number

of shoots on each of the plants selected to be monitored in ANNEX 1 (column D). For

each plant select at random one of the shoots (Figure 2).

Figure 2. Plants with one (left) and two (right) shoots that grew from the originally sown stake

B. Tickets: Label the tickets with the plot identification, and the date (Figure 3). These

tickets can be color coded with a different color for each date of placement to facility

later collection of fallen tickets (Figure 4). On the selected shoot of each plant hang a

ticket on the highest leaf on which the ticket can easily be placed; note that this leaf is

never expanded (Figure 5). Count the number of existing leaves to this leaf. When plants

are very small it is advisable to use small tickets that do not damage the plant and are not

in contact with the soil (Figure 6).

4

Figure 3. Example of a ticket with the plot identification and the date

Figure 4. Tickets with color coded with a different color for each date

Figure 5. New ticket in the youngest leaf with a petiole separated stem

5

Figure 6. Plant with large labels in contact with the ground (left) and plants with small labels (right)

Weekly monitoring

A. Collection of tickets: On entering the field for both weekly, two weekly or monthly

monitoring the fallen leaves or tickets on the ground are collected (Figure 7). The date of

collections is recorded (ANNEX 1, column E). If you cannot verify the information on

the ticket because the label is in illegible due to its poor condition, it should be possible

estimate the original placement date considering the dates on which have been monitored

plants and checking in tickets that are still in the shoot date is missing. As mentioned

earlier, color coding of the tickets also aids ticket date identification.

Figure 7. Collection of the fallen tickets on the ground

B. Rate leaf formation: After the first sample, count the number of new leaves starting

from leaf to put the ticket on the last visit to the new youngest leaf with a petiole

separated stem. At each visit, do the same process by recording the number of new leaves

(ANNEX 1, Column F).

6

C. Height of plant: With a meter stick measure the height of the plant from the ground to

the leaf that is at the plants highest point and also measure the height from the ground to

the first branching (Figure 8) (ANNEX 1, column J and K).

A.

Figure 8. First branching height measurement

Biweekly monitoring

A. Count of nodes: After the first sampling should also count the number of new nodes

starting from the node that holds the ticket that was put on the last visit. Must count up

and check if in the process find a branch point or a dead apex. If you encounter a branch

point record the number of nodes up to the branch point (from the last ticket) (ANNEX 1,

Column O), and the number of branches at the branch point. (ANNEX 1, column I).

Randomly select one of the branches and continue as previously. If you encounter a dead

apex (Figure 9), record the dead apex (ANNEX 1, Column F) and return to the last

branching point, select another branch and continue the process as before. The counting

of nodes stops at the highest point where a label can easily be hung on the youngest leaf

with a petiole separated stem. Hang a new ticket on this leaf and petiole.

Figure 9. Dead apex

7

B. Determination of the length and diameter of internodes: Identify the branching

levels of the plant according to (Figure 10) and record the number of branching levels

(ANNEX 1, column H). These branching levels should be the same as those determined

when counting the number of nodes and branch points and can be easily viewed

following down the stem from the last ticket that was placed at the plant. At each level of

branching, divide each of the shoots monitored in sections, with each section

corresponding to a distinct distance between nodes, or internode length (Figure 11).

Generally, the first internodes of each branching level have a different length to the

others and should be treated as a separate section. To facilitate identification of the

sections at later monitoring visits the lignified sections can be identified by permanent

marker.

Figure 10. Identification of levels of branching

Figure 11. Identification of sections according to the length of internodes. Branch with two different sections

identified (left) and branch with only a section identified (right).

First level of branching

2 Shoots

Second level of branching

3 Shoots

8

In each of the distinct stems sections:

Record the branch level

Record if the section is green or lignified (Figure 12) (ANNEX 1, column M)

Count and record the number of nodes in the section as the number of nodes from the

last branch (ANNEX 1, column O)

Measure and record the length of the section (ANNEX 1, column Q)

Measure the diameter in the middle of the section with the Vernier caliper (Figure 13)

(ANNEX 1, column P)

Figure 12. Determination of the change in the branch, of green to lignified

Figure 13. Measurement of the stem diameter

C. Identification of first fully expanded leaf: Try to get to the field early in the morning

so as to avoid too much sun and can easily identify in the shoot is monitored the first

fully expanded leaf. Generally as you move down the stem of the plant you will find each

successive leaf is larger than the previous leaf, until you reach a point where several

9

leaves are more or less of the same size. The first of these leaves of similar size from the

top down is taken as the first fully expanded leaf.

This leaf must be complete, must not show any damage to the laminae and can be easily

identified by considering factors such as the angle between the petiole and stem and the

angle between leaf laminae and the petiole (Figure 14), color between the petiole and leaf

laminae and size (Figure 15). Usually, close to the branching point there are one or two

small, but fully expanded leaves. Do not use these leaves for leaf area measurement.

Figure 14. Angle between the petiole of fully expanded leaf and stem

Figure 15. Size difference between an unexpanded and fully expanded leaf

D. Determination of leaf area: Once the fully expanded leaf has been identified, the leaf

area is determined using the leaf area apparatus (LAA) described in the appendix. Insert

10

the first fully expanded leaf, still attached to the plant, between the two laminae of the

LAA. Care should be taken to ensure that the leaf is not damaged (Figure 16).

Figure 16. Capture process of photography to the determination of leaf area: Identification of the fully expanded

leaf, insertion between the laminae and take photography

Once the leaf is between the laminae take a photograph of the leaf in the LAA with the

camera held vertically above the LAA (Figure 17). A label should be placed in the LAA

to identify the photograph later. We have found that one person, with practice, can place

the leaf in the apparatus and take the photograph, however this takes practice. Hence, we

strongly advise practicing this procedure before monitoring and when possible having an

assistant in the field. The instructions to determine the leaf area are in the Annex 3.

Figure 17. Position of the camera to take the picture

Monthly monitoring

A. Total Apex number: Finally count the total number of live and dead apices on each

plant and record them.

11

ANNEX 1: SAMPLE OF THE WORKSHEET TO REGISTER THE SAMPLING DATA

SAMPLING

DATE GERMINATION

DATE

LOCATION CIAT

19/05/2014

31/03/2014

A B C D E F G H I J K L M N O P Q

Cu

ltiv

ar Plant ID

No

. of

sho

ots

pe

r

cutt

ing

Leaves longevity

Rate of leaf formation

No

. of

leve

ls o

f

bra

nch

ing

No

. of

bra

nch

es

pe

r

bra

nch

po

int Height Length and diameter of internodes

Blo

ck

Pla

nt

ID

Collection date

No. new

leaves

No. dead apexes

Plant height (cm)

Shoot height (cm)

No. branch

Part of the branch

Section Branch

No. de

nodes

Stem diameter

(cm)

Stem length (cm)

HMC1 1 1 3 23/04/2014 5 0 1 2 57 36 1 Lignified 1 3 5,0 11,92

1 Lignified 2 5 7,8 12,57

1 Unlignified 3 13 14,0 13,39

1 Unlignified 4 7 20,2 10,42

1,1 Unlignified 1 1 5,5 4,17

1,1 Unlignified 2 2 2,5 3,49

HMC1 1 2 2 16/04/2014 6 0 0 3 46 -- 1 Lignified 1 7 10,0 9,68

1 Lignified 2 12 12,4 10,15

1 Unlignified 3 8 12,5 9,62

12

ANNEX 2: DEVELOPMENT OF THE INSTRUMENT TO MEASURE LEAF AREA A. MATERIALS (Figure 18)

1 Ruler

1 lamina of acrylic, antiglare, glass color, 2mm thick and measures 35 x 35 cms

1 lamina of black or red polystyrene, 2mm thick and measures 35 x 35 cms

2 Hinges

1 Drill

1 Scoreboard thin tip

1 Pencil

Figure 18. Materials used in preparing the template to measure leaf area

B. PROCEDURE

Make sure you are using the laminas on the right side. The two laminas have a shiny side

and a dull side (Figure 19), so that the shiny side should be placed down and dull side up,

in order to avoid reflections at the time of taking the photograph.

Figure 19. Shiny side (left) and dull side (right) of the lamina of polystyrene and acrylic

Join the two laminas with the hinges. Place one side of the hinges on the acrylic lamina in

the desired position, and with a marker, paint points through which the screws should enter

(Figure 20).

13

Figure 20. Hinge location and marking plates

Subsequently, with the drill, open the holes through which will pass the screws at two sides

of the lamina. Make sure you have all the tools and security necessary to use the drill

(Figure 21).

Figure 21. Opening holes with the drill in the plates

Locate the hinge on the holes made previously and insert the screws that come with the

hinge, securing each one with nuts (Figure 22).

14

Figure 22. Locations of screws a hinge and plate

The result on the acrylic lamina is a hinge side installed, and secured with screws and nuts

(Figure 23).

Figure 23. Assurance hinges on acrylic sheet

Then, place the other side of the hinges on the red polystyrene lamina painting the points

where the screws must enter. Then, with the drill open the holes and join both laminas

(Figure 24).

15

Figure 24. Union of acrylic and polystyrene laminas.

Finally, create the scale that will be used in the pictures in cm, on the red polystyrene

lamina. For this, initially, with a ruler, draw a scale of approximately 6 cm in two corners

of the lamina in pencil. Then use a non-erasable marker to accentuate the scale (Figure 25).

Figure 25. Dialing scale

16

In the case where there is no availability of a computer with the software to analyze the

images, a grid in the acrylic lamina can be generated with a known distance between each

line, for example 2cm in length. Thus, when placing the leaf under the grid, one can easily

count the number of squares occupied by the leaf being measured, and therefore provide an

indication of the leaf area that you have (Figure 26).

.

Figure 26. Definition of grid

The end result of the process is the union of the two laminas with a hinge at each corner,

the polystyrene lamina with a scale and if necessary, the acrylic sheet with a grid. This

instrument facilitates taking photographs to determine the leaf area because it is easy to

transport, does not create reflections when taking the picture and can be opened and closed

as many times as necessary without risk of damage (Figure 27).

Figure 27. Instrument to measure leaf area

17

ANNEX 3: IMAGE PROCESSING WITH IMAGE J SOFTWARE

ImageJ is a program that makes use of digital image processing in Java, it is public domain and

easy to use. You can run ImageJ through JAVA online or on any computer via the Java virtual

machine; please note that ImageJ is downloadable for different computer operating systems. The

program enables the user to view, edit, analyze, process and save images of 8, 16 and 32 bits;

furthermore it reads many image formats. ImageJ allows the user to make simple and

multithreaded operations on the images and run the program for any purpose, studying how it

works and changing it according to the intended need, it also allows the user to redistribute

copies and add improvements to the program which can then be available to the public.

A. INSTALLATION AND IMPLEMENTATION OF IMAGE J

This application is free; download and run the file for your operating system from the following

link: http://imagej.nih.gov/ij/ selecting the download option. When the installation window is

opened, select the location of the folder where you want to save the program files, and the

additional icons that you want to install (Figure 28).

18

Figure 28. ImageJ installation

B. IMAGE PROCESSING

Once it is installed, you can access the program from the icon that was installed on the

desktop or through Start, selecting All Programs, and then Image J. When the program is

opened, the following window will appear which is the taskbar main program and consists of

several menus and various buttons (Figure 29).

Figure 29. Working window for ImageJ

To open an image, select the File menu, then select the Open option and in the dialog box

choose the image that you want to open (Figure 30). The image should have a scale that uses a

known distance to define the scale of photography; this is defined by the distance in pixels in

accordance with the distance of the real scale.

19

Figure 30. Menu to open the image and emerging window

Now that you have opened the image, we are ready to start the analysis process. Select with

the cursor the icon Straight in the taskbar, it has a diagonal line with a red arrow. Then select

a known distance according to the scale of the image (Figure 31).

Figure 31. Straight icon from the principal menu of ImageJ

Subsequently, in the Analyze menu select the option Set scale. A table will now open showing

the distance in pixels from the previously plotted line. Thus, having knowledge of the real

length of this distance, the scale of the photograph is defined through the evaluation of the

size of a pixel in cm. In the Known Distance box enter the real length of the selected line and

in the box Unit of length enter the units for this distance. Select the Global option at the

bottom of the table; afterwards, if you analyze other images you will just have to modify the

distance in pixels that represents a known unit of measurement (Figure 32).

20

Figure 32. Scale definition according with the image

Afterwards, you have to convert the color image to grayscale by selecting in the Image menu,

the options of Type and 8 bits (Figure 33). Then, go to the Process menu and select the Binary

option and finally Make binary option (Figure 34). Additionally, you have to select the

variables that you want to analyze in the image by selecting the Analyze menu, Set

Measurements option and verifying that only the area box is checked because it is the

variable that we want to calculate, then select Ok (Figure 35).

Figure 33. Image conversion to 8 bits

21

Figure 34. Image conversion to binary

And now select in the Analyze menu, the Analyze Particles option and in the Size box set the

unit of measure of the scale of the original image. The Pixel units option should not be

checked, the Circularity option must be filled with the number one (1) and the Show option

should display the word Nothing. Next, mark the following options; results display, Clear

results, Include holes and Add to manager (Figure 36).

Figure 35. Selection of the variable to measure, area

22

Figure 36. Menu Analyze particles

Observing the image, we find a number of polygons, with our main interest being the one

with the largest area. Additionally, the Results window shows the area of the polygons

identified in the image. With the ROI Manager window you can select the number of the

polygon of interest (first 4 digits); when you select the correct polygon, its outline will take a

different color in comparison with others (Figure 37).

Figure 37. Results visualization for the images without shade and reflection

23

C. IMAGE PROCESSING WITH SHADOWS

When you take the picture of the leaf, depending on the light’s intensity and location, it is

possible that the image has shadows or reflections that interfere with the analysis. If this

happens you must follow another methodology to analyze the image. In this case, after defining

the scale according to the last explanation, you have to select in the Image menu the adjust

option and then the Threshold option (Figure 38).

Figure 38. Threshold menu

In this section the properties of hue, saturation and brightness are set, then the threshold to fit

only the shape or contour of the leaf to be analyzed should be selected (Figure 39). Next, select

in the Analyze menu, the Analyze Particles option, the size box should be filled with letters 0.1 to

Infinity, Pixel units option should not be checked, the Circularity option must be filled with the

numbers 0-1 and the Show option should be displaying the word Nothing. Next, mark the

options; results display, Clear results, Include holes and Add to manager (Figure 40).

Figure 39. Menu to define hide, saturation and brightness properties

Reflejo

24

Again, observing the image, we find a number of polygons, and as before, our main interest is

in the one with the largest area. Additionally, the Results window shows the area of the

polygons identified in the image. With the ROI Manager window you can select the number

of polygons that are of interest (first 4 digits); when you select the correct polygon, its outline

will take a different color in comparison with others (Figure 41).

Figure 40. Menu Analyze particles

Figure 41. Results visualization for images with shades and reflection

25

Finally, in the Results window, export the results as an Excel table by selecting in the File

menu the Save As option. When you open the saved Excel file, you can access the data of leaf

area which will be used as part of the non-destructive methodology for the cassava crop.

26