Oxygen Measurements in Seed Testing · Respiration is known to be associated with seed vigor and viability, as is shown through tetrazolium tests. UC DAVIS. Respiration As A Seed

Oxygen Measurements in Seed TestingKent J. Bradford, Pedro Bello, Jing-Chen Fu and Margarita Barros

Department of Plant Sciences, Seed Biotechnology Center

University of California, Davis, USA

[email protected]

Respiration Rates and Seed Quality

VIABLE NONVIABLE

Respiration is known to be associated with seed vigor and viability, as is shown through tetrazolium tests.

UCDAVIS

Respiration As A Seed Quality Parameter

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• Respiration is essential for germination.

• Respiration is related to seed quality.

• Individual seeds vary in their respiration rates and capacity.

• Single-seed assays are labor-intensive, but bulk samples give only an average value for the seed population.

• It is difficult to relate respiration directly to components of seed quality in bulk samples.

• A convenient method to assay respiration of many individual seeds would be useful.

Astec Q2 for Measuring Seed Respiration

www.astecglobal.netUCDAVIS

Single-seed Respiration Measurements

Individual seeds are sealed into the wells of a microtiter plate or in screwcap vials. The sealing membrane or screwcap has dye dots on the inner side whose fluorescence is proportional to the oxygen concentration in the well.

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As the seeds imbibe and respire, the oxygen content of the air space of the well decreases. This increases the fluorescence of the dye in the dot inside the well when illuminated with light.

Single-Seed Respiration Measurements

A light source/fluorescence sensor unit moves over the plates at user-specified intervals and records the oxygen content of each well.

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The instrument records the oxygen level in each well over time as it is depleted due to seed respiration.

Air Volume Can Be Varied and Germination Observed

Using agar, the air volume in the wells can be varied to match the size and respiration rates of the seeds.

Agar also allows radicle emergence to be observed through the bottom of the plate at desired intervals.

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Q2 Temperature Controller

We developed a custom temperature controller that can maintain constant and different temperatures in groups of four plates.

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ASTEC Analysis Software for Q2 Data

Raw data

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Categorized and modeled data

Respiration Patterns and ASTEC Values

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Time (h)

SMR = Starting Metabolism Rate

SMRSMR

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Respiration Patterns and ASTEC Values

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Time (h)

SMR = Starting Metabolism RateIMT = Increased Metabolism Time

SMRSMR

IMT

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Respiration Patterns and ASTEC Values

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SMR = Starting Metabolism RateIMT = Increased Metabolism TimeOMR = Oxygen Metabolism Rate

SMRSMR

IMT

OMR

OMR

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Respiration Patterns and ASTEC Values

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SMR = Starting Metabolism RateIMT = Increased Metabolism TimeOMR = Oxygen Metabolism RateRGT = Relative Germination TimeSMRSMR

IMT

OMR

RGTRGT

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OMR

Respiration Patterns and ASTEC Values

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SMR = Starting Metabolism RateIMT = Increased Metabolism TimeOMR = Oxygen Metabolism RateRGT = Relative Germination TimeCOP = Critical Oxygen Pressure

SMRSMR

IMT

OMR

RGTRGT

COPCOP

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OMR

Respiration Patterns and ASTEC Values

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SMR = Starting Metabolism RateIMT = Increased Metabolism TimeOMR = Oxygen Metabolism RateRGT = Relative Germination TimeCOP = Critical Oxygen PressureQT50 = Time to 50% Oxygen

SMRSMR

IMT

OMR

RGTRGTQT50 QT50

COPCOP

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OMR

Respiration Patterns and ASTEC Values

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SMR = Starting Metabolism RateIMT = Increased Metabolism TimeOMR = Oxygen Metabolism RateRGT = Relative Germination TimeCOP = Critical Oxygen PressureQT50 = Time to 50% Oxygen AUC50 = Area Under Curve to QT50

SMRSMR

IMT

OMR

RGTRGTQT50 QT50

AUC50COP

COP

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OMR

Q2 Experience with Diverse Species

AlfalfaCabbageChinese cabbageCauliflowerCantaloupeCoreopsisCornflowerLettuce

MarigoldOnionPansyRadishSugar beetTomatoTurnipVinca Viola

17 Species Tested

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Vigor Correlation with ASTEC Values -- Brassica

IMT and OMR correlated significantly with a common vigour index (early count in a standard germination test) across 17 cabbage, 6 Chinese cabbage, 5 turnip and 5 cauliflower seed lots.

y = -0.3194x + 43.629R² = 0.2133**

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First count (%)

A IMT

y = 0.0708x - 3.374R² = 0.1858*

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TurnipChinese cabbage CauliflowerCabbage

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Vigor Correlation with ASTEC Values -- Brassica

RGT and HOM also correlated significantly with a common vigour index (early count in a standard germination test) across 17 cabbage, 6 Chinese cabbage, 5 turnip and 5 cauliflower seed lots.

SMR and COP did not have significant correlations with vigor index.

y = -1.1324x + 151.66R² = 0.2315**

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E RGT

y = -0.6892x + 76.92R² = 0.2967**

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TurnipChinese cabbage CauliflowerCabbage

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Priming Treatments -- Tomato

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Screening priming treatments by comparing times to 50% germination (T50) with QT50 respiration values.

The times to germination of individual seeds and the T50 of the population were highly correlated with QT50 respiration rates.

The IMT was also highly correlated with QT50 and with T50.

Temperature and Respiration -- Tomato

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Temperature has predictable effects on germination rates.

Similar effects are observed on respiration rates measured by the Q2.

Water Potential and Respiration -- Tomato

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Reduced water potential also has consistent effects on germination rates.

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-0.2 MPa

-0.4 MPaSimilar effects are observed on respiration rates measured by the Q2.

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Priming and Aging Treatments -- Lettuce

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Priming advanced and aging (75% RH and 40ºC) delayed both germination and respiration relative to the control (untreated) seeds.

Note the wide variation in the shapes of the oxygen consumption curves, and the trend for more seeds to have linear patterns as they aged.

Color scale at bottom of each panel indicates the time of radicle emergence for the seeds whose respiration curves are the same color.

Priming and Aging Treatments -- Lettuce

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Some ASTEC values were highly correlated with T50 for germination in response to priming or aging, particularly SMR and RGT.

The QT50 and AUC50 values were highly correlated with T50 for germination, and are not dependent upon the shapes of the respiration curves.

Alternative Data Analysis Method for Q2 Data

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We developed an alternative analysis method based upon the time required for each seed to reduce the oxygen level to a specified percentage, e.g., 50% of the initial value. When plotted versus time, this results in a curve resembling a germination time course.

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Germination versus QT Time Courses

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Priming and aging effects on germination time courses were similar to their effects on respiration time courses.

This representation takes advantage of the single-seed measurements and can reveal differences in behavior among subpopulations of the seed lot.

This analysis method also allows the application of threshold-based population models to the respiration data, such as for temperature, water potential or aging.*

*Alvarado and Bradford (2002) Plant Cell Environ. 25: 1061-1069. Bradford et al. (1993) J. Exp. Bot. 44: 1225-1234.

Application of Q2 to CD Tests -- Radish

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Controlled deterioration viability tests at lower storage MC take a long time to conduct if final viability is scored. CD at 33% RH

and 50 CGermination rates are more sensitive to aging.

Respiration rates are sensitive to aging, and show large differences before significant viability has been lost.

Respiration rates can be assessed after shorter aging periods to estimate potential longevity.

Conclusions

UCDAVIS

• The Q2 instrument can measure respiration time courses of individual seeds.

• There is wide variation among respiration rates of individual seeds, but overall patterns are correlated with seed vigour and germination rates.

• Various components of oxygen depletion time courses can be quantified to assess respiratory patterns.

• Respiratory time courses of seed populations, analogous to germination time courses, can be generated.

• Q2 assays can semi-automate collection of time course data and enable greater use of germination rates in seed quality evaluation.

WRSPRG

Financial Support

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Astec Global: technical and in-kind support

American Seed Trade Association Vegetable & Flower Seed Permanent Research Fund

Western Regional Seed Physiology Research Group

Q2 Users’ Group and multiple companies who provided seeds.

Q2 Users’ Group

Pedro Bello