Reliable Method for Assessing Seed Germination, Dormancy, and Mortality under Field Conditions

Summary

Here we present a protocol for assessing seed survivorship, germination and dormancy under field conditions using buried, labeled seed strips and tetrazolium chloride (TZ) viability testing.

Abstract

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester fabric and glue can be folded and glued to construct a strip of compartments that house seeds and identifying information, while allowing contact with soil leachate, water, microorganisms, and ambient temperature. Strips may be constructed with a wide range of compartment numbers and sizes and allow the researcher to house a variety of genotypes within a single species, different species, or seeds that have experienced different treatments. As opposed to individual seed packets, strips are more easily retrieved as a unit. While replicate packets can be included within a strip, different strips can act as blocks or can be retrieved at different times for observation of seed behavior over time. We used a high temperature glue gun to delineate compartments and sealed the strips once the seed and tags identifying block and removal times were inserted. The seed strips were then buried in the field at the desired depth, with the location marked for later removal. Burrowing animal predators were effectively excluded by use of a covering of metal mesh hardware cloth on the soil surface. After the selected time interval for burial, strips were dug up and seeds were assessed for germination, dormancy and mortality. While clearly dead seeds can often be distinguished from ungerminated living ones by eye, dormant seeds were conclusively identified using a standard Tetrazolium chloride colorimetric test for seed viability.

Introduction

The overall goal of this method is to reliably assess seed survival over time under field conditions.

Soil seed banks are a reserve of dispersed, viable yet ungerminated seeds distributed either on the soil surface, within surface litter, or within the soil profile, which may persist transiently or for many years^1,2. When seed burial methods similar to those presented here were applied to a 17-year study using several dozen species, viable seeds were found in many of the species tested³. Seed dormancy is a block to seed germination until the appropriate combination of conditions for seedling survival arise⁴.....

Protocol

1. Gather Seed from Multiple Species or Controlled Crosses of a Single Species

Note: This example used seed from 15 cross types within the species Helianthus annuus (sunflower) using wild, hybrid, and crop types as the maternal (seed producing) parent.

1. At the end of the growing season, collect mature seed heads in labeled bags. Clean seed from chaff and place seed in envelopes labeled with parental cross type in standard format (i.e., maternal x paternal).
2. Bulk seed together in large envelopes using equal amounts of seed from an equal number of identical parental cross types. For example, 100 seeds from 10 cross types wit....

Results

Cross types with varied maternal parentage and crop allele percentage (Table 1) differed across removal dates in percent germinated, ungerminated, and dead seed (Fig. 2 and 3). Using TZ testing of ungerminated seeds, we found some truly dormant seeds at the second removal (early spring) (Table 2), while all seeds ungerminated by the third removal (spring) were found to be truly dormant.

Discussion

Here we present methods for using seed burial strips to observe seed germination, dormancy and mortality of diverse seed stocks at preselected time periods in the field. The advantages of using strips rather than individual packets lie in (1) the speed of strip and compartment construction over creation of individual packets; and (2) the ease and speed of removing multiple compartments in one motion without the danger of omitting a packet or removing one unintentionally. As two of the removal dates in the example present.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Small coin envelopes	Any
Large coin envelopes	Any
fine meshed polyester mosquito netting	Any
high-temperature glue gun	Any
high-temperature glue stick refills	Any
Industrial permenant markers	Any
plastic garden labels	Any
scissors	Any
Shovel	Any
Metal mesh hardward cloth	Any
Surveyor's flags, multiple colors	Any
Wet newspaper	Any
cooler	Any
blotter paper	Any
petri dishes	Any
Temp. controlled growth chamber	Any
razor blades	Any
Petri dishes	Any
Tetrazolium chloride	Any
water	Any
heat incubator	Any