Work Package 3 High-Throughput Phenotyping Protocols (HTPP ...

Training Report

Work Package 3 High-Throughput Phenotyping

Protocols (HTPP) Report Template: Training

Fabrice DAVRIEUX, CIRAD, Saint Denis de La Réunion, France

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This report has been written in the framework of RTBfoods project.

To be cited as:

Fabrice DAVRIEUX. 2018. Work Package 3. High-Throughput Phenotyping Protocols (HTPP). Report Template: Training. Saint Denis de La Réunion, (France). RTBfoods Project Report, 11 p.

Image cover page © Dufour D. for RTBfoods.

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Work Package 3

High-throughput phenotyping protocols (HTPP)

REPORT TEMPLATE: TRAINING

Title (Topic): Near infrared Spectroscopy: Theory and Application Date & Location:

• 23 May – 28 May 2018 • NACRRI, Research center Institute, Namulongue, Uganda

Trainer(s) The main trainer was Davrieux Fabrice from CIRAD with details as highlighted in the Table 1 below. However, during the training, the partcipants also interacted with other project based resource persons such as the project leader Domique Dufour and the leader of work package 5 Hana Chair. The presence of the breeders in the training was also an added advantage as they provided important information on what was expected and how the tool would be useful to the different people involved in the training. Table 1: Details of the chief trainer (Mr. Davrieux Fabrice)

NAME Surname Gender (F/M)

Education (ex: MS in Biochemistry)

Institute Role in

RTBfoods project

Country Email Contact

Davrieux Fabrice M MS Chemistry MS Statistics CIRAD WP3 Leader France [email protected]

Objectives of the Training: The main objective of the training was to equip the technical group working with the NIRS tool with an understanding of operations of the NIRS so as to provide timely information for breeders during the breeding cycle. Specifically, the training was aimed at providing an extended overview of the principles and theory of NIR spectroscopy with an emphasis on the potential of NIRS as an HTTP tool and the different applications it can be put to. The training was also aimed at providing an introduction to multivariate analysis as a tool for data manipulation in the NIRS. In addition, the training was meant to provide the participants with relevant information on the development of calibration models for the different crops where theoretical aspects were provide on how spectral data can be used in the development and utilization of predictive models. Finally, the last objective was the hands on engagement aimed at understanding the procedures involved in spectral acquisition and measurement protocols.

1. Overview of the principle and theory of NIR spectroscopy 2. Initiation to multivariate analysis 3. Calibration development: From spectra to predictive model 4. Spectral acquisition and measurement protocols

Participants The participants in this training were drawn from broad areas of research including breeding, food science, biochemistry, and other crop based disciplines. The participants were also drawn from different institutes that are participating in RTB projects including national agricultural Research Organization (NARO) represented by the National Crops Resources Research Institute (NaCRRI) and the National Agricultural Research Laboratories (NARL). The participants from these two institutes represented the core part of the trainers as they are directly

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involved in work package 3. In addition, other participants were drawn from other participating partners including Makerere University, international potato center (CIP) and the International Institute of Tropical Agriculture (IITA). Important to note is that most of the participants did not have any working knowledge of the NIRS either theoretically or practically making the training highly impactful. The details of the participants and their contacts are as described in the table 2 below. Table 2: Details of the participants and their contact information

NAME Gender Education Institute Function Country Email Contact

1 Robert Kawuki M PhD NaCRRI Uganda [email protected] 2 Tawanda Muzhingi M PhD CIP

2 Nuwamanya Ephraim M PhD Plant Physiology NaCRRI NIRS

management Uganda [email protected]

3 Brigitte Uwimana F PhD IITA Uganda [email protected] 4 Esuma Williams M PhD NaCRRI Uganda [email protected] 5 Apio Hellen F MSc NaCRRI [email protected]

3 Wembabazi Enoch M MSc Biochemistry NaCRRI Data

collection Uganda [email protected]

4 Senyonjo Andrew M CIP - NaCRRI NIRS management Uganda [email protected]

5 Fatumah Babirye F BSc Biotechnology NaCRRI Uganda [email protected]

6 Nanyonjo Ann Ritah F MSc Crop Science NaCRRI Uganda [email protected]

7 Katungisa Arnold M BSc NaCRRI Uganda [email protected]

8 Mukasa Yusuf M BSc Biochemistry NaCRRI Uganda [email protected]

9 Kisakye Sarah F BSc NARL Uganda [email protected]

10 Atwijukire Evans M MSc Biochemistry NARL Uganda [email protected]

11 Asiimwe Moses M CIP Uganda 13 Monica kemigisha F BSc MUK Uganda 14 Nalukwago Betty F Dip NaCRRI Uganda 15 Reuben Ssali M CIP Uganda [email protected] 16 Robert Misanga M CIP [email protected] 17 Amwano Rose F Dip NaCRRI Uganda [email protected] 18 Jacinta Akol F BSc NaCRRI Uganda [email protected] 19 Aol Jane F Dip NaCRRI Uganda

Background & Experience Level in Training Content All the selected participants were not experienced in Near Infra-Red Spectroscopy. This was the basis of the selection and serves as the justification for taking part in this training. The selected participants however had basic level training in chemistry, spectroscopy, statistics and multivariate analysis. From the ten participants that undertook the hands on training in the laboratory, it was observed that only have of these had basic experience in chemistry having undertaken chemistry courses at undergraduate level. However, only four out of the ten participants had basic level training in spectroscopy while three out of the ten had basic level training in multivariate analysis. It was also observed that about 80% of the participants had basic level training in statistics much as one of the participants did not have any experience in the four important fields that the training was being conducted on. The summary background and experience levels for each of the participants are as provided in the abridged table below.

Participant/expertise Level of experience Participant 1 No experience Basics Experienced Expert

Chemistry x Spectroscopy x

Statistics x Multivariate Analysis x

mailto:[email protected]











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Participant 2 No experience Basics Experienced Expert Chemistry x

Spectroscopy x Statistics x

Multivariate Analysis x Participant 3 No experience Basics Experienced Expert




















Multivariate Analysis x Note: Please note that this evaluation was carried out at the beginning of the training and does not reflect participant evaluation of the training. List of materials / training documents distributed: (to be provided as Attachments)

Two main papers where distributed to guide on the methodological approaches in the use of NIRS. The papers describe different quantification techniques using NIRS in two different crops (legume-peas; cereal-Wheat). The

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papers also provide for specific analyses targeting specific plant parts and were thus critical for information for methodological development in spectral acquisition. The snapshots of the material are as attached while the papers are provided as part of the attachments on training materials. In addition two other PDF files on PDF file on Near Infrared theory and PDF file on Calibration methodology were provided and are also attached.

Training methods applied: (Methods include: lecture, field visit, panel discussion, multimedia presentation, group practice, etc.)

• Lecture • PowerPoint presentation • Chemometric Software (Winis 4, and Unscrambler 15) • Group practice

The lectures were conducted by the main trainer in the conservation training hall and were attended by all the participants. During the lectures, interactive learning sessions were carried out which involved sharing of experiences among the participants. The practical sessions involved group practice where the participants were involved in understanding sample preparation and spectral acquisition. This involved the process of acquiring samples from the field, processing of samples, and spectral acquisition techniques. The training also hinted on data management techniques from the spectra obtained.

List of Documents to be attached

Document YES/NO 1. Calendar / Training Agenda

YES

2. Training Material / Documents distributed

YES

3. Attendance Sheet(s) (including Consent Forms) signed by participants

YES*.

4. Individual & Anonymized training evaluation forms from all participants NO

5. Pictures

YES**

6. Current summary progress YES * By the time of the training, the consent form signing aspect was not in place and is thus not reflected ** Some of the pictures of the training are attached as an appendix below. Summary of the training

1. Objectives The main objective of the training was to give an overview of the theory and the applications of near infrared spectroscopy (NIRS) to the scientific and technical staff from NACRII, NARL, NARO, IITA and CIP involved or potentially involved in the RTBfoods project.

1.1 Specific objectives - To perform an audit of the human resources and of the technical capacities. - To learn about the basic of the interaction light and matter and vibrational spectroscopy. - To familiarize with the mathematics associated to NIRS. - To familiarize with the principle of calibration. - To understand the different steps from spectra to calibration. - To set up the instruments and adapt the protocols to the different products (Cassava, Banana).

To achieve the above objectives, training was held at the National Crops Resources Research Institute (NaCRRI) in Namulonge. The one week training was held from the 23rd may 2018 up to the 28th May 2018. The training was attended by a crossection of researchers and scientists including breeders, food scientists, biochemists, and

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technicians working under the breeding and food science research. The participants were drawn from various institutes including NaCRRI as the host institute, National Agricultural Research Laboratories (NARL), CIP and IITA. The training was organized in form of interactive theory sessions where the trainer provided back ground theoretical information to the participants followed by practical sessions in the laboratory. The practical sessions involved acquisition of spectra from cassava, bananas and sweet potatoes. It also involved the comparison of the NIRS installed at the NaCRRI nutritional and bio-analytical laboratories and the CIP based nears installed at the same campus. The main outcome of the training was the ability of the technicians to undertake NIRS spectral acquisition for the different crops which has already been especially for cassava and bananas. In addition, aspects of trouble shooting and sample preparation were also highlighted and have been critical in the understanding of operation of the tool.

2. Calendar

Date Morning/Afternoon Place Activity

May 23 Morning Namulonge

Opening training/Theory of NIRS Afternoon Training

May 24 Morning

Namulonge Chemometrics/Calibration

Afternoon NIRS measurements. Laboratory tests Banana and Cassava

May 25

Morning

Namulonge

Interpretation of banana and cassava spectra/training

Afternoon SCIO and DS2500 spectra

recovering/existing databases recovering

May 28 Morning Namulonge Instruments and software configuration

Afternoon Kawanda (NARL) Meeting with Mr. Kephas

3. Preamble The training took place in Research center Institute laboratory of NACRRI, Namulongue, Uganda. The number of participants (and their qualification) was not fixed before the training in order to allow as many people as possible to become familiar with this technology. In this way, the management teams of the various institutes will be able to select, at the end of the training, the personnel in charge of developing within the RTBfoods project the implementation of the technology. The NACRRI institute is equipped with two types of spectrometers:

- One lab top instrument, model DS2500 (Foss Inc., Silver Spring, MD, USA), working in reflectance mode within the wavelength range 400 nm – 2500 nm

- Two micro spectrometers, model SCIO (Consumer Physics Inc., Ha-Nagar St 6A, Hod HaSharon, Israël), working in reflectance mode within the wavelength range 700 nm -1100 nm.

NACRII staff used these two instrument for analyzing ground-dried cassava. These spectrometers were operational but without any planning of tests and operations and without defined and fixed measurement protocol. Furthermore, the technical and the scientific staff were not trained to routine analysis, database management and calibration development for these two type of spectrometers. The FOSS instrument was equipped with the routine analysis software (Nova and Mozaic®) while SCIO spectrometer was equipped with an internal routine software link to a smartphone and a cloud database. In both cases, no calibration development software were available. There is in the same location, NACRII Research center Institute, a spectrometer operated by CIP. This spectrometer dedicated to Potato and Sweet potato is an XDS FOSS (Foss Inc., Silver Spring, MD, USA) working in reflectance mode within the wavelength range 400 nm – 2500 nm.

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4. Main results

- The theory of vibrational spectroscopy was presented and explained. A focus was done on diffuse reflectance and its application in near infrared.

- A special course presenting the path from spectra to calibration was done. - A course on chemometrics applied to NIRS was realized, with a focus on Principal Components Analysis,

Mahalanobis distances and Partial Least Square Regression. - The courses were closed by an overview of different applications of NIRS on agro-food products. - The following of the training was dedicated to practical exercises and spectra treatment. The first step

was to start the instrument DS2500, to create an operating protocol and to do specific tests for both fresh cassava and fresh banana pulp. For banana, a first experience was set up on Foss DS2500 spectrometer from NACRRI. For the test, four different varieties (7197-2 diploïd, Narita26, Naritabeer8, EntuKura highland) were sampled. For each variety a number of finger were retained, respectively 7, 2, 3, 3. Variety 7197-2 has small fingers and a pulp difficult to grind, the others have big fingers and a pulp easy to grind. The selected fingers per variety were peeled and mix, then ground in a homogenous puree. For each samples (4), the following protocol was applied for scanning the sample: • five spectra were recorded from aliquots of about 10 g of homogenized puree, • the two first spectra were without no change (same sub sample) • the third and fourth spectra were new subsamples (~10g) with no replacement of previous

subsamples into the original sample. • the fifth spectra was done on a subsample after remixing of all the subsamples. Doing this way, 20 spectra were collected. The second experience for banana was realized on XDS Foss Instrument from CIP. The two varieties (7197-2 and Narita26) were analyzed as intact finger with skin and as intact finger peeled. Two fingers were selected per variety. For each variety and each presentation (with and without skin), the spectra of the two fingers were obtained at two different positions on the longitudinal face of the fruit. The operation was done on both side of the fruit. The first position was scanned twice. Doing this way, five spectra were collected by finger. Doing this way, 20 spectra were collected. The same experiment was realized for cassava on the FOSS DS2500 spectrometer from NACRRI. In this case, two varieties were selected: UGC1700131 and UGC1700638. One root was sampled per variety, the whole root was peeled and ground in a homogenous puree. The scanning protocol was the same as the first experience done on banana, 10 spectra were recorded (five per variety). The main results from these tests are:

• Spectra of fresh ground banana and fresh ground cassava are highly repeatable. • Spectra of subsample of fresh ground banana and fresh ground cassava are representative of

the whole sample (set of fingers or one root). • Spectra of fresh ground banana and cassava contain relevant information about the sample,

which allows separating the different varieties. • Spectra of intact banana (with and without skin) are repeatable; the spectral information is

slightly different within fruit as a function of the position and the side. There is a difference between two fingers of the same variety. These differences, intra finger (position) and inter fingers (same variety) were lower than the spectral difference observed between the two varieties.

• Finally, the fresh cassava spectra were close (in term of Mahalanobis distance) to the spectral database developed by CIAT on fresh cassava using a FOSS nirsystem 6500. This result is of importance if ones wants to use the CIAT database and calibrations for the characterization of fresh cassava

The already existing databases were exported from the instrument SCIO_seedling trials (136 samples scanned between 6 to 7 times) and from SCIO_Namulonge UYT Harvest, 17 samples scanned between 29 to 82 times. Finally, the dried Cassava database was exported from the DS2500 (96 samples scanned twice).

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5. Conclusion This training was a great opportunity to touch a large public of technicians and scientists, and to give them a clear information about NIRS. The theory of NIRS and the principle of calibrations development, including mathematics, were discussed and presented. The combination of theoretical courses and practical work allowed the staff to understand the constraints and limitations of this technique while grasping the advantages. The different experiences realized on banana and cassava allowed to define the instruments operating procedure and to set up potential protocols for banana and fresh cassava. Furthermore, the training session permitted to have an audit of NACRRI, CIP and NARL laboratory capacities, and especially regarding NIRS technology.

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Appendix

The installed NIRS equipment at NaCRRI. The equipment is a FOSS

NIRS DS2500 equipment.

The computer interface and one of the samples prepared for analysis. Sample preparation was one of the

aspects of the training and comparisons were made between surface scanning and scanning the puree for spectral acquisition. The cassava used in the training was

sourced from the C0 trials at Namulonge

The trainer (Fabrice, looking into the camera) and project staff (Fabrice, Hana) and Ephraim

Nuwamanya in the laboratory during the training

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Appendix: Attendance list

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Institution: Cirad – UMR QualiSud

Address: C/O Cathy Méjean, TA-B95/15 - 73 rue Jean-François Breton - 34398 MONTPELLIER Cedex 5 - France

Contact Tel: +33 4 67 61 44 31

Contact Email: [email protected]

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