Determining the Dormancy Type of the Endangered Linum mulleri by Testing 7000 Seeds

Abstract

Linum mulleri is an endemic taxon of southwestern Sardinia (Italy), categorised as Endangered (EN) on the IUCN Red List and included in Annexes II and IV of the Habitats Directive (92/43/EEC) as priority species for conservation. This study investigated the germination ecophysiology of L. mulleri and the possible presence of dormancy by using 7000 seeds, providing useful information for conservation strategies. The germination response of fresh seeds was evaluated under different temperatures, photoperiods, pre-treatments [cold stratification (C); warm stratification (W); W+C; C+W+C; dry after-ripening (DAR)], and different gibberellic acid (GA₃) concentrations. L. mulleri germinated under controlled conditions, particularly at 15 and 20 °C, while germination percentages (GP) never exceeded 5% at 5 and 30 °C. C and C+W+C induced secondary dormancy, delaying germination, whereas W, DAR, and GA₃ stimulate it. Light and dark incubation showed no significant differences in regards to GP. W, DAR, and 250 mg/L GA₃ effectively overcame physiological dormancy (PD), expanding the germination temperature range to below 10 and above 25 °C. These responses suggested type 3 non-deep PD, as germination temperatures extended from a moderate range to both low and high temperatures. Analyzing 7000 seeds provided crucial information regarding dormancy and germination strategies, supporting both ex situ and in situ conservation efforts.

Keywords: Linum; dormancy; endemic plant; pre-treatment; seed germination.

Figure 1

(A) Germplasm collection of Linum mulleri; (B) seed lot after cleaning, with removal of impurities and empty seeds; (C) sowing seeds in 1% agar substrate; (D) Petri dishes sealed with Parafilm and ready for incubation in growth chambers; (E) example of germinated seeds at 20 °C after DAR; (F) seedlings of Linum mulleri in the soil substrate; (G) detail of Linum mulleri seedling.

Figure 2

Germination percentage during pre-treatments. I, II, and III correspond to the three pretreatment cycles applied to Linum mulleri seeds, with I = 90 days for W (warm stratification) and C (cold stratification), II = 180 days for W+C (warm stratification followed by cold stratification), and III = 270 days for C+W+C (cold stratification followed by warm stratification and another cold stratification period). Data represent the mean of four replicates (±SD). The GLMs were applied during the germination, and values with the same letter are not statistically different by post hoc pairwise t-test comparisons.

Figure 3

Germination percentage (GP) of Linum mulleri seeds under different treatments and pre-treatments [CTR (control), C (cold stratification), W (warm stratification), W+C (warm stratification followed by cold stratification), C+W+C (cold stratification followed by warm stratification and another cold stratification period), DAR (dry after-ripening), and dark] and incubated at constant (5, 10, 15, 20, 25, 30 °C) and alternating (25/10 °C) temperatures. Data represent the mean of four replicates (±SD). GLMs were carried out on the germination, and values with the same letter are not statistically different by post hoc pairwise t-test comparisons.

Figure 4

Effect of different concentrations of GA₃ (0, 250, 500, 1000 mg/L) on seed germination of Linum mulleri incubated at constant (5, 10, 15, 20, 25, and 30 °C) and alternating (25/10 °C) temperatures. The data represent the mean of four replicates (±SD). Values with the same letter are not statistically different by post hoc pairwise t-test comparisons.

Figure 5

Gaussian curves representing the effects of CTR (control), W (warm stratification), DAR (dry after-ripening), and 250 mg/L of GA₃ on the germination dynamics of Linum mulleri seeds. Points correspond to actual data, and solid lines indicate the fitted lines from Gaussian regressions. Vertical dotted lines indicate the minimum temperature at which a 50% germination rate can be achieved under the different treatments. The data represent the mean of four replicates.