Seed Viability, TZ testing

 

Protocol

Author

Gemma Hoyle

Overview

Determining percentage viability of a seed lot using the Tetrazolium Chloride (TZ) staining technique. The Tetrazolium Chloride (TZ) test aims to determine what percentage of any seed lot is viable (alive), and what proportion is non-viable (dying or dead). This test is useful in determining what proportion of a seed lot can be expected to germinate under favourable germination conditions, and can also assist in the diagnosis of seed dormancy.

Background

Originating in Germany in the 1940s, the TZ test is one of today’s most widely known methods of seed viability testing. It is commonly applied to cereals and less commonly to other agricultural, vegetable and flower seeds.

The TZ test relies on the premise that only living cells have the respiratory enzymes (dehydrogenases) capable of converting a colourless, soluble compound in the TZ solution (2,3,5 triphenyl tetrazolium chloride), into an insoluble red product (2,3,5 triphenyl formazan).

Seeds are soaked in a colourless solution of TZ, which enters both living and dead cells, but only the living cells catalyze the formation of the red, formazan precipitate. Thus living tissue stains red.

Interpretation of staining then follows. Stained and unstained areas are first used to differentiate between living and dead tissues. The size and position of the stained areas and extent of staining are then interpreted as an indication of live versus dead seeds.

A TZ test is often recommended for use at the end of a germination test where more than 5% of seeds remain un-germinated, in order to answer the question ‘are remaining seeds dead or possibly dormant ‘

Although the TZ test is extremely well known and in principle thoroughly attractive, in practice it is a complicated technique and the interpretation phase can be difficult and highly subjective. In addition, it is not applicable to fruits/seeds which are too small to dissect, or others (such as some species of oak, Quercus spp.) in which inhibitors can retard or even prevent the formation of the red, formazan precipitate in living seeds.

Due to the extreme diversity of species for which no official guidelines exist, a simplified approach has been developed. The methods outlined below aim to categorise seeds in a sample as ‘viable’, ‘dying’ or ‘dead’, and will give reliable results in the majority of cases.

Materials/Equipment

To make 1% TZ solution:

  • 2,3,5 triphenyl tetrazolium chloride (powder)
  • Conical flask
  • Distilled water
  • Balance
  • Magnetic stirrer
  • Brown medicine bottle or clear bottle
  • Al-foil
  • 4oC fridge for storage

For TZ test:

  • 1% TZ solution
  • 3 x 10 seeds plus a minimum of 5 extra
  • 3 Petri dishes of agar – 1% water
  • 3 x small glass Petri dishes
  • Al-foil
  • 30oC incubator/oven
  • Scalpel
  • Forceps
  • Dissecting microscope
  • TZ score sheet
  • Paper and pencil
  • Calculator

Units, terms, definitions

Tetrazolium Chloride – TZ

Agar

Pericarp

Relative humidity

Procedure

  1. Make up 1% TZ solution
    Bare in mind that TZ solution is light sensitive – once powder begins to dissolve in solution, keep protected from light.

    • Measure out 1 g of TZ powder
    • Add 1 g of TZ powder to a 100 mL conical flask (use squirty bottle of distilled water to transfer into flask)
    • Continue adding distilled water to conical flask up to 100 mL.
    • Add magnetic stirrer to conical flask
    • Wrap conical flask in Al-foil, place on stirrer and stir gently until powder has dissolved.
    • Pour solution into brown medicine bottle or clear bottle wrapped in Al-foil, label and store at 4oC for up to 3 months.
  2. Sample seeds
    The TZ test assumes that the collection under test has been cleaned (processed) and any obviously empty, infested or necrotic seeds have been discarded. A TZ test is carried out on fully developed, completely intact, seemingly mature seeds.

    • Seeds for testing must be randomly selected.
    • At least 3 replicates of 10 seeds is optimal
    • Allow up to 5 extra seeds per collection, depending on availability, for seed examination and mistakes.
  3. Familiarise yourself with the seed anatomy
    Dissection and evaluation stages of the TZ test will differ depending on seed morphology. Therefore it is important to become familiar with the anatomy of the test seeds prior to carrying out the TZ test. This stage is particularly important when dealing with unfamiliar seeds.

    • It may be easier to carry out this step when seeds are imbibed. If so, place additional seeds in re-hydration conditions (step 4) for this purpose.
    • Under a microscope, use a scalpel to dissect at least 3 seeds.
    • Make annotated drawings of a longitudinal and cross section on the TZ test sheet.
    • In particular, note seed coat thickness, the location and size of the embryo, and the presence or not of endosperm. Note colours.
    • Establish whether or not outer covering structures need to be removed i.e. fruit, glume, caryopsis etc.
  4. Re hydrate seeds in preparation for immersion in TZ solution.
    This stage softens seed tissues to assist subsequent dissection and/or staining. It also prevents imbibition shock when seeds are submerged in TZ solution.

    • Place seeds on agar, in a Petri dish (replicates in separate dishes), or if agar is unavailable, filter paper wetted with distilled water.
    • Store in Petri dishes in ambient conditions (20-25oC) such as a bench top or in an appropriate incubator.
    • Small to medium sized seeds will only require 24 hours to become fully imbibed, larger seeds may take up to 48 hours.
    • Seeds that are impenetrable to water will need to be scarified (chipped) prior to re-hydration and may require up to 48 hours to fully imbibe.
    • A more gentle approach is to place seeds in a sealed container over water (in an atmosphere of 100% RH) for 24 h before placing on agar. This is especially suitable for tiny seeds and those that are suspected of being of low viability.
  5. Expose vital tissues (embryo).
    This is probably the most important step and requires an understanding of the structure and anatomy of the seed (see step 3. above). The objective is to dissect each seed in a way that will facilitate the penetration of TZ solution, particularly into the seed embryo.

    • Carefully cut (chip or pierce) the seed coat / pericarp away from the embryo without damaging it.
    • Alternatively, remove part of the seed you are confident does not contain the embryo.
    • It is not necessary to completely isolate the embryo from the seed.
    • Tiny seeds may only require a pinprick piercing of outer tissues.
    • Species with tough, impermeable exteriors may require considerable ingenuity.
    • Particular care is needed for seeds without endosperm.
  6. Soak seeds in TZ solution and incubate
    Once dissected, seeds are ready to be immersed in TZ solution and stored at 30oC for 24 hrs.

    • Place seeds in glass Petri dishes and pour over just enough TZ solution to cover the seeds. It is important that the seeds are fully immersed yet freely moving in the TZ solution.
    • Wrap each Petri dish in Al-foil in order to prevent photo-conversion of the TZ solution.
    • Label each dish and place at 30oC for 24 h.
  7. Interpret TZ staining
    To interpret staining, each seed must be cut open and internal tissues examined. The aim is to distinguish between seed embryos that are viable, seeds that are dying and seeds that are dead/non-viable.

    • Use forceps to transfer seeds to wetted filter paper (usually at a dissecting microscope), and discard TZ solution down the drain with excess water.
    • Use forceps and scalpel to inspect internal seed tissue under a dissecting microscope.
    • Take care to avoid handling damage, which can influence the staining pattern.
    • For each seed, record TZ staining colouration and staining pattern on TZ test sheet.
    • Try to evaluate each seed in relation to others in the replicate by adjusting the magnification of the microscope so that all seeds in the sample can be viewed simultaneously. This may help to remove some of the inevitable subjectivity.
    • Most species exhibit individual idiosyncrasies, and most seed lots, unique features.
    • Differentiate staining patterns and colours into categories, considering whether the fruit/seed is more likely to be viable, dying or dead.
    • Allocate each seed to one of the following categories:
      1. Alive / viable: Whole fruit/seed including embryo intact, firm, fresh, healthy and embryo fully stained the characteristic rich, formazan red.
      2. Alive / viable: Some unstained tissues, but embryo intact, firm, fresh, healthy and fully stained with the characteristic rich, formazan red, or an acceptable plum or pink colour.
      3. Dying:       Embryo patchy or weakly stained pink, other tissues appear patchy.
      4. Dead / non-viable: Entire seed and embryo flaccid, unhealthy, unstained or uncharacteristically coloured.
    • For a range of species, the ISTA guidelines indicate the relationship between precise patterns of staining and seed viability. These may be useful guides for other related species.
    • Calculate percentage of seed lot -viable’ (omit any empty seeds or seeds lacking embryos from the calculation).
    • For some seed lots it may be appropriate t calculate a potential range of viability.
  8. Using the TZ result to assess seed dormancy
    If the viability of the seed lot appears to be significantly higher than the proportion of seeds that will germinate under favourable conditions (adequate temperatures, moisture and light), the seed lot may contain a proportion of dormant seeds.

    • Carry out a proportions test using Pearson’s chi-squared statistic with Yates’ continuity correction to compare the proportion of individuals that germinated in the germination test with the proportion indicated as viable in the TZ test.
    • If the results are not significantly different (P > 0.05) the germination result is accepted as a true indication of viability. If the results are significantly different, consider investigating dormancy alleviation treatments and/or more specific germination conditions in order to increase percentage germination.
      Note:The statistical test chosen is up to the user, for helpful information see [http://http://www.seedtest.org/en/content-11014–271.html|ISTA Statistical Links for Seed Analysts]

Notes and trouble shooting tips

The whole TZ test is carried out over 3-4 days:

Day 1: Sample seeds. Rehydrate seeds in preparation for dissection and soaking in TZ solution.
Preparation time: 15 mins.
If preparing TZ solution, add 15 mins.
May be possible to examine dry seeds.

Day 2: Become familiar with seed morphology. Dissect seeds before immersing in TZ solution.
Preparation time: 1-2 hrs.

Day 3: Dissect seeds, examine and interpret staining patterns. Calculate percentage viability.
Time: 30 mins – 1 hour.

Three or four species / collections can be tested at once, depending on how easy seeds are to dissect and examine, and the ease with which staining can be interpreted.

International Seed Testing Association – ISTA(external link)

Seed Conservation: Turning Science Into Practice. Edited by Roger Smith, John Dickie, Simon Linington, Hugh Pritchard and Robin Probert(external link) Note: many references below come from this publication

Literature references

Gosling, PG (2004). Chapter 24 – Viability testing. In: R.D. Smith, J.B. Dickie, S.H. Linington, H.W. Pritchard and R.J. Probert (eds.) Seed Conservation: Turning Science Into Practice. Royal Botanic Gardens, Kew, UK: 445-481.

Terry, J, Probert, RJ, Linington, SH (2004). Chapter 17 – Processing and maintenance of Millennium Seed Bank collections. In: R.D. Smith, J.B. Dickie, S.H. Linington, H.W. Pritchard and R.J. Probert (eds.) Seed conservation: Turning science into practice. Royal Botanic Gardens, Kew, UK: 307-325.

Dickie, JB, Stuppy, WH (2004). Chapter 15 – Seed and fruit structure: significance in seed conservation operations. In: R.D. Smith, J.B. Dickie, S.H. Linington, H.W. Pritchard and R.J. Probert (eds.) Seed conservation: Turning science into practice. Royal Botanic Gardens, Kew, UK: 253-279.

Further reading:

General information on TZ testing:

Anon (1999). Biochemical Test for Viability. Chapter 6 of the International Rules for Seed Testing 1999. Seed Science and Technology 27 (supplement): 33-35.

Anon (1999). Biochemical Test for Viability (The Topographical Tetrazolium Test). Annex to Chapter 6 of the International Rules for Seed Testing 1999. Seed Science and Technology 27 (supplement): 201-244.

Moore, RP (1985). Handbook on tetrazolium testing. Publ. ISTA, Zürich, Switzerland.

Grabe, DF (1970). Tetrazolium testing handbook – for agricultural seeds. Contribution No. 29 to the Handbook on Seed Testing. Publ. AOSA.

Tree seeds:

Enescu, V (1991). The tetrazolium test of viability. In A.G. Gordon, P.G. Gosling and B.S.P. Wang (eds.). Tree and shrub seed handbook. ISTA, Zürich, Switzerland.

Tropical tree seeds:

Bhodthipuks, J, Pukittayacamee, P, Saelim, S, Wang, BSP and Yu SL (1996). Rapid viability testing of tropical tree seed. Training course Proceedings No. 4., ASEAN Forest Tree Seed Centre Project. Kuak-Lek, Saraburi, Thailand.

Health, Safety & Hazardous Waste Disposal Considerations

  • Laboratory coats, protective disposable gloves and safety goggles must be worn when preparing the TZ solution.
  • Avoid inhalation of TZ powder.
  • Wash contaminated skin with copious water.
  • Rinse out any eye contamination with sterile eyewash.
  • If irritation persists, seek medical advice.
  • Lab coat must be worn when carrying out TZ test.
  • Take great care when dissecting seeds with a scalpel.
  • The COSHH assessment indicates that the chemicals used in this technique are of low risk when correctly used: http://www.jtbaker.com/msds/englishhtml/t6981.htm(external link)


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