Ethylene Sampling Protocols

The main challenge in determining fruit maturity classes is the actual correlation between I_AD value and ethylene production as it requires sophisticated instrumentation, such as  a gas chromatograph to measure ethylene. In general, this equipment is only available in fruit research laboratories. However, the correlation with ethylene production and the identification of the maturity classes is fundamental to the use of the DA meter and if not done will reduce the accuracy and effectiveness of the DA-Meter.

To enable owners of DA meters access to ethylene analyses, Agriculture Victoria scientists developed a procedure for the collection of ethylene produced by the fruit that can then be sent to a central laboratory for measurement with GC. This procedure will enable the accurate measurement of ethylene production by individual fruit from anywhere in the production and postharvest handling chain. When combined with the DA measurement it will allow the segregation of fruit with similar ethylene production into maturity classes. Accurate identification of fruit maturity will allow for the determination of appropriate harvest, storage and marketing practices. The procedure uses pre-evacuated vials for the easy and rapid collection of ethylene samples from fruit in the field. By following the procedure, growers and other handling chain participants can collect the ethylene and ship it to a DEDJTR laboratory for analysis. Central processing of the ethylene samples makes the process less costly and allows for systematic identification of maturity classes for each cultivar. Consolidation of results will provide a valuable database for industry.

For each cultivar tested ethylene measurement should be conducted at DA values between 0.2 and 1.7 for stone fruit and between 0.2 and 1.9 for pome fruit. Best results are achieved if ethylene is measured at DA value increments of 0.1. At least 5 individual fruit are required for each ethylene production measurement. This procedure will allow the creation of cultivar specific graphs correlating DA and ethylene production from which it will be possible to accurately identify maturity classes (Figure 1).

Figure 1: Nectarine Autumn Bright ethylene production and segregation in maturity classes of similar ethylene behaviour correlated to DA values. Mature = ripe (climacteric fruit), Commercial = usual maturity at commercial harvest (On-set of climacteric) and Immature = unripe (pre-climacteric fruit).

Fruit pre-measured and labelled for DA and grouped into 0.1 DA value increments.

Respiration chambers: Sealable air tight jars/containers: between 500 mL and 1 L capacity depending on availability; see Appendix A

Evacuated vials: supplied by DEDJTR Horticultural Production Science team

25 cc syringe: with 25G needle

Timer/clock

Scale to record fruit weight

Recording sheets (download recording sheet)

Picture 1: Equipment needed to perform the procedure.

Method:  See video - How to sample ethylene from stonefruit

DA monitoring for harvest prediction date should start at least 4-5 weeks prior to harvest and be performed at least weekly. It is important to find the DA values corresponding to pre-climacteric, onset and post-climacteric fruit development.. This will ensure the identification of fruit at high DA values, corresponding to fruit not producing ethylene (Pre-climacteric). Fruit selection should continue as the DA values decrease with at least 5 fruit for each 0.1 DA increment (i.e. 0.80 and 0.89 would be placed in the 0.8 group) until fruit between DA values of 1.7 to 0.2 are measured for ethylene production. This should result in a minimum of 75 fruit sampled for ethylene. It is important that fruit are free from any blemish, wounds, disease,  pest damage or bruise as it will affect ethylene production rate. The 5 fruit for each 0.1 DA increment do not have to be collected on the same day but can be spread across sampling times. It is most important that a minimum of 5 fruit are collected for each 0.1 DA increment.

It is possible to sample more than one variety at the same time But the samples must be correctly labelled for each variety and DA value.  Sampling more than one variety at a time does not increase the overall time commitment  much but it increases the number of chambers to be used at each single time or it requires multiple samplings with a fixed number of chambers to be able to collect from 5 fruit in each 0.1 DA group.

Fruit selection can be performed in two ways:

1. Harvest minimum of 100 fruit at random into cardboard trays with single fruit liners to separate fruit and minimise bruising; fruit should be collected at shoulder height and from multiple trees, usually no more that 8-10 fruit/tree

Measure and write the DA value on each fruit with a permanent marker

Segregate the fruit in groups within the same 0.1 of DA increments with a minimum of 5 fruit for each increment from 1.7 to 0.2

1. Measure fruit with the DA, directly on the tree, Write the DA value on the fruit with a permanent marker. Select a minimum of 100 random fruit at shoulder height and from multiple trees, usually no more that 8-10 fruit/tree

Harvest the fruit that will be needed to segregate the fruit in groups within the same 0.1 of DA increments with a minimum of 5 fruit in each group (from 1.7 to 0.2) and place them in cardboard trays with single fruit liners to separate fruit and minimise bruising

Print (download) or have sample record sheet on computer/tablet open and ready to edit;

Label the fruit numerically (eg 1-20);

Record the DA, the weight of each fruit and the volume of the chamber in which the fruit goes into (Table 1);

Place each fruit into its allocated chamber;

Seal all the chambers at once and record the time, in hours and minutes.

A chamber with no fruit in it should also be sealed at this time to determine if there are any background ethylene contamination that will affect the results.

The chambers should be left for 3 to 4 hours in the shade or inside to allow the accumulation of any ethylene production. It is important that chambers are never exposed to direct sunlight.

While this time elapses label the evacuated vials that match the fruit in the chambers.

Include Grower/Orchard identification, Sample type (cultivar), fruit number, chamber label and collection date.

After the time has elapsed sample the chamber using a 25cc syringe fitted with a 25G needle.

Insert the needle through the silicon seal into the chamber as far as it can go.

Slowly pump the syringe 3-5 times to create air movement inside the chamber stirring the contents.

Draw 20cc into the syringe and remove the syringe from the chamber. Insert the needle into the correspondingly labelled vial. The syringe should release most of its contents automatically as the vial is evacuated.

When the syringe plunger stops moving gently push down the plunger forcing the remaining contents into the vial. While holding the plunger down, remove the needle from the vial.

Store the vials at room temperature in the dark.

Send the vials with a copy of the relevant sample recording sheet to Agriculture Victoria for ethylene analysis.

Size preference is 1L capacity, glass or gas tight plastic; depending on availability chamber volume could be between 500 mL to 1L. Most important is that the chamber opening is large enough to insert and remove large fruit easily.

Exact volume must be determined (see below); each container must contain at least one hole filled with regular glass silicone sealant.

Picture 2: Example of silicone to be used.

Note: Sealant will require a minimum of 24-48 hours curing time. Do not use any other type of silicon sealant as they contain compounds that will be detected by the instrumentation (GC) used to determine ethylene presence and concentration, and therefore will interfere with the analysis of the samples.

Label each chamber and the corresponding lid, numerically or other, for ease of recording and re-using the volume information against the individual chamber.

This is done by weighing the total amount of water required to fill both base and lid of the chamber. This should only need to be done once for each chamber. While the totals will be similar for most of the chosen chambers sometimes there are differences large enough to alter the results so it is important to do this for every chamber used.

Equipment: a balance or set of scales, accurate to 0.1g is preferred, and worksheet – Jar Volume Determination (download).

①record the chamber and lid labels.

②separate lid from base of chamber;

③place base and lid on the balance and record the weight;

④remove the lid, pour water into the base until is completely full (to the brim), try to avoid any spillage onto the scales as this will add to weight; replace the lid upside down on top of the base (still containing water) and fill the lid with water;

⑤record the total weight (base and lid);

repeat from ① for all remaining chambers;

Record this value for the specific chamber and keep this information for reporting with the sample information each time the chambers are used.

Table 1: Example of recording sheet (Download spreadsheet)

Grower:  

Variety: O’Henry

Date: 16/2/2016

At the time of writing, the cost of the ethylene analysis per variety is estimated at $1,250 including supply of syringe, needles and pre-evacuated vials but not the chambers.

The DA-Meter can be sourced through Summerfruit Australia Limited.

The procedure was developed as part of the SF15001 “Comparing Stonefruit ripening, quality and volatile composition” project co-financed by stonefruit industry levy and Agriculture Victoria.