Plant Materials
In 2019, the NWR cultivated variety ‘Itasca-C12’ and the breeding line ‘K2EF-C16’, representing genotypes with average and early germination, respectively, were used to establish a dormancy curve. Itasca-C12 was broadcast planted at a density of 112 kg ha−1, in a grower’s dike irrigated, field in Gully, MN. K2EF-C16 was broadcast planted at a density of 15.7 kg ha−1 in a 12 x 15 m, in a dike-lined research paddy at the University of Minnesota North Central Research and Outreach Center (NCROC), in Grand Rapids, MN (47.2372° N, 93.5302° W, and 392 m elevation). The genotypes were both planted on May 16th of 2019, open pollinated in their respective paddies, and then hand harvested on September 3rd and 4th of 2019, respectively.
For the dormancy breaking experiments, the NWR variety ‘Barron’ was chosen, as it is a productive variety with good germination, and excess seed was readily available for the experiment. Germination was an important consideration for this study, as the recalcitrance of NWR seed limits the viability of seed stocks in storage. Seeds were tractor-planted planted on May 12th of 2020, at 15.7 kg ha−1, in a 153 x 27 m dike irrigated research plot, with 3m single rows, spaced 0.38m apart, and 1m alleys, for a final plant density of 1 plant per 0.09m2. Plants were open-pollinated and seed was hand harvested on September 10th of 2020. Following harvest and processing, seed was stored submerged in dH20 in heavy sealed plastic bags, in the dark, at 3°C until the beginning of each trial.
Germination and Tetrazolium Testing
To assess seed viability, seeds were placed in petri dishes lined with filter paper and 20 ml of dH20. Plates were then randomized within blocks, in a Conviron-E15 growth chamber (Winnipeg, Canada), for germination testing. The growth chamber was set to 15-hour days at 20°C and 9-hour nights at 14°C. To keep seeds moist through-out the germination testing period, dH20 was added to petri dishes as necessary. After 14 days, the number of germinated seeds, as defined by coleoptile emergence equal to the length of the seed, were quantified. The germinated seeds from the dormancy treatment experiment were then placed in 4 oz jars with 20 ml of dH20 for 1 week, in preparation for seedling evaluations and remained in the growth chamber under the same conditions as for germination testing. dH20 was added to jars as necessary to keep seeds submerged.
Ungerminated seeds were evaluated for metabolic activity to estimate seed viability with a protocol adapted from the tetrazolium testing handbook (Peters 2000). Briefly, seed was bisected longitudinally to expose the embryo, then suspended in a 0.2% solution of 2,3,5 triphenyl tetrazolium chloride (Mp Biomedicals Inc, Santa Ana, CA) for 2 hours. Seed was then triple-rinsed and the number of stained embryos was quantified.
Evaluation of NWR Dormancy
To produce a dormancy curve for NWR, seed was evaluated over a 9-month period (~38 weeks), starting from harvest, on September 5th, 2019, through May 27th, 2020. Every two weeks during this period, 3 replicates of 20 seeds were pulled from submerged cold storage. Seeds were germination tested, under the same conditions as is described above. Any seed that did not germinate, was subsequently tetrazolium stained, as described above, to assess seed viability. From these data, a dormancy curve was produced and an estimate of the weeks post-harvest at which 50% of viable seed germinated was calculated, as described in the data analysis section.
Dormancy Breaking Experiment
To develop improved methodology for overcoming dormancy in NWR, we evaluated several treatments, over the first three months of the dormancy period. The experiment was repeated 3 times, with the first experiment starting on September 21st, 2020, 1 week post-harvest, the second on October 27th, 7 weeks post-harvest, and the third on November 24th, 11 weeks post-harvest. The trials were split into five steps: primary treatment, targeting the seed coat, secondary treatment, using hormones to stimulate the embryo, germination and tetrazolium testing, seedling evaluations, and plant health ratings. Each trial was set up as a randomized complete block design, with 5 blocks of 20 seeds per primary and secondary treatment combination.
To evaluate the possibility of seed coat imposed dormancy, or that related to the tissues surrounding the embryo, three primary treatments were tested, including a 2 hour soak in 300 ml of 2.63% sodium hypochlorite (NaClO) (Oelke and Albrecht 1980), a 45 second wash in 200 ml of 98% sulfuric acid (SA), and a 45 second wash in dH20, as a control. Following the treatment of seeds with NaClO, SA, or water, seeds were rinsed 3 times with 300 ml of dH20. To evaluate the potential for embryo imposed dormancy, or that related to the embryo or endosperm, secondary hormone treatments were immediately applied following primary treatments. Secondary treatments included: 0.1 mM gibberellic acid (GA3) (Research Products International, Mt Prospect, IL), 1 mM kinetin (K) (Chem Impex International Inc, Bensenville, IL), a combination of 0.1 mM GA3 and 1 mM K, and a dH20 control. Seeds were placed in sealed, 4 oz plastic jars, with 20 ml of secondary treatment solution and stored at room temperature (~24°C), in the dark, for 24 hours (Oelke and Albrecht 1980). After 24 hours, each jar of seed was rinsed for 30 seconds under running water.
Evaluation of Seedling and Plant Vigor
To evaluate the seedling and plant vigor of seeds that germinated, seedlings and plants were assessed by several methods. After 1 week of growth in the growth chamber, seedlings were measured from root tip to the top of shoot tissue. Seedlings were then randomized in a greenhouse, set to 12-hour days at 24°C and 12-hour nights at 18°C. Seedlings were grown in fully submerged, 25 cm deep, 3.2 cm radius cone-tainers, amended with 22.4 kg ha−1 of urea (Alpha Chemicals, Stoughton, MA) and 48.8 kg ha−1 of Sprint 330 iron chelate (BASF, Ludwigshafen, Germany) for 30 days. After 30 days, the root and shoot tissue of each plant were measured separately, using a measuring tape, and plants were given a health rating of 1-4 (1: a dead plant, 2: a plant with significantly stunted growth and thin leaves, 3: a plant with relatively normal growth but with minor stunting of stem or leaf tissue, 4: A healthy plant with no stunting) (Figure 1).
Data Analysis
Analyses were done in Rstudio, version 1.2.5001 (Rstudio-team 2019). Data was first run through standard residual diagnostic plots, including residuals versus fitted, normal Q-Q, scale-location and residuals versus leverage, to assess data quality. An analysis of variance (ANOVA) was conducted for germination and tetrazolium testing results. Significant results from the ANOVA were evaluated using the post hoc Tukey LSD test from the ‘agricolae’ package, using a Bonferroni adjustment for p-values (Mendiburu 2019). Due to uneven sample sizes in the seedling and plant rating data, statistical analyses could not be done on these data. The ‘ggplot2’ package in R (Wikham 2016) was used to produce all graphs in this publication.
The equation\(Ratio of true dormancy break= \frac{\# seeds germinated}{(\# seeds germinated + \# seeds tetrazolium stained)}\) was used to determine the ratio of seeds that had germinated, per time point, relative to the number of tested seeds that were actually viable. A modified LD50 calculation, using germination in place of toxicology data, from the Rstudio package ‘MASS’ (Venables and Ripley 2002), was then used to calculate the time during the dormancy period at which 50 % of all viable seed had germinated (D50) per genotype, using the above calculation of true dormany break.