Germination ecophysiology of Annona crassiflora seeds

Abstract

Background and aims: Little is known about environmental factors that break morphophysiological dormancy in seeds of the Annonaceae and the mechanisms involved. The aim of this study was to characterize the morphological and physiological components of dormancy of Annona crassiflora, a tree species native to the Cerrado of Brazil, in an ecophysiological context.

Methods: Morphological and biochemical characteristics of both embryo and endosperm were monitored during dormancy break and germination at field conditions. Seeds were buried in the field and exhumed monthly for 2 years. Germination, embryo length and endosperm digestion, with endo-beta-mannanase activity as a marker, were measured in exhumed seeds, and scanning electron microscopy was used to detect cell division. The effect of constant low and high temperatures and exogenous gibberellins on dormancy break and germination was also tested under laboratory conditions.

Key results: After burial in April, A. crassiflora seeds lost their physiological dormancy in the winter months with lowest monthly average minimum temperatures (May-August) prior to the first rainfall of the wet season. The loss of physiological dormancy enabled initiation of embryo growth within the seed during the first 2 months of the rainy season (September-October), resulting in a germination peak in November. Embryo growth occurred mainly through cell expansion but some dividing cells were also observed. Endosperm digestion started at the micropylar side around the embryo and diffused to the rest of the endosperm. Exogenous gibberellins induced both embryo growth and endo-beta-mannanase activity in dormant seeds.

Conclusions: The physiological dormancy component is broken by low temperature and/or temperature fluctuations preceding the rainy season. Subsequent embryo growth and digestion of the endosperm are both likely to be controlled by gibberellins synthesized during the breaking of physiological dormancy. Radicle protrusion thus occurred at the beginning of the rainy season, thereby maximizing the opportunity for seedlings to emerge and establish.

Fig. 1.

(A) Intact Annona crassiflora seed at time of dispersal. (B) Seed with ruptured seed coat. (C) Seed longitudinally cut showing the micropylar endosperm (MEn), the lateral endosperm (LEn) and the embryo (Em). (D) Embryo at the time of seed dispersal. Note the presence of axis and cotyledons. (E) Scanning electron micrograph of the micropylar endosperm (MEn) and embryo (Em) of imbibed seeds. Observe that the micropylar endosperm contains eight or nine cell layers in front of the embryo. The dotted line indicates the border between embryo and endosperm. (F) Lateral endosperm. Arrows indicate ruminations in the endosperm. Scale bars: A–C = 5 mm; D = 200 µm; E = 10 µm; F = 100 µm.

Fig. 2.

(A) Germination at field conditions during the years 2003 and 2004. Data points are averages of eight replications of 500 seeds each; error bars indicate standard deviation. (B) Average monthly temperature, and average monthly maximum and minimum temperatures and average monthly precipitation (bars) at the experimental site during the germination experiment.

Fig. 3.

(A) Length of axis and cotyledons during germination at field conditions. (B) Images of embryo growth during germination at field conditions in 2003, following dispersal in April. Note that the embryo grows inside the seed prior to radicle protrusion in December. First seed = seed at time of dispersal; second seed = August; third seed = September; fourth seed = October; fifth seed = November.

Fig. 4.

Scanning electron micrograph of cells in the embryonic axis in September 2003, when the embryo started to grow, but prior to radicle protrusion. Arrows indicate dividing cells. Scale bar = 3 µm.

Fig. 5.

Endo-β-mannanase activity in micropylar and lateral endosperm during germination at field conditions from April 2003 (seed dispersal) to November 2003 (after radicle protrusion). Error bars are too small to be shown. Coefficients of variation were 10 % and 6 % for micropylar and lateral endosperm, respectively.

Fig. 6.

Tissue printing of longitudinally cut seeds before and after radicle protrusion, showing endo-β-mannanase activity as clearings in micropylar and lateral endosperm. (A) Absence of endo-β-mannanase activity. (B) Activity in the micropylar region only. (C) Activity in the micropylar and lateral endosperm before radicle protrusion. (D) Activity in the micropylar and lateral endosperm after radicle protrusion. Drawn representations of embryo growth and endo-β-mannanase activity are included for clarity.

Fig. 7.

(A) Germination in various concentration of GA₄₊₇ at 30 °C. Data points are the average of four replicates of 25 seeds. Standard deviations were smaller than the symbols. (B) Endo-β-mannanase activity in micropylar and lateral endosperm from seeds incubated in 500 µm GA₄₊₇. Inset: tissue prints of seeds allowed to imbibe in water (1) and in 500 µm GA₄₊₇ for 15 d (2), displaying endo-β-mannanase activity in micropylar (top side) and lateral endosperm. Standard deviations were smaller than the symbols. (C) Total embryo length in seeds imbibed in 500 µm GA₄₊₇. Bars indicate s.d.