Seedlings of dry forest adapted species resume growth after desiccation

Desiccation tolerance (DT) in germinated seeds is directly linked to the success of seedling survival of seasonally dry tropical forests (SDTF) species. The objective of this study was to evaluate whether the seeds of Anadenanthera colubrina and Cenostigma pyramidale present post-germinative DT and until what stage of seedling development the tolerance persists. Seeds were separated from fruit remains, branches, and seeds of other species and stored for 6 months in cloth bags in a cold and dry chamber (±10 °C/45% RH) until the beginning of the experiment. Fresh A. colubrina and C. pyramidale seeds showed 85% and 90% germination and WC was 8% and 8.1%, prior to DT trials. Seeds were sowed on germination paper moistened with distilled water 2.5 times the dry paper weight and incubated at 25 °C for A. colubrina and 30 °C for C. pyramidale and photoperiod of 12/12 h (white light with photon flux density of 30 W/m2) light/dark, according to specific instructions for best conditions for seed germination (BRASIL, 2013). Since these are very fast germinating species, after 24 hours, radicle protrusion was observed and measured using a digital caliper (0.001 mm accuracy). The seedlings were separated into four IRL categories: 1.00–2.99 mm, 3.00–4.99 mm, 5.00–6.99 mm, and 7.00–10.99 mm. Seedlings (5 replicates of 10 seeds) from each IRL category were desiccated for 24 and 72 hours. Desiccation was performed by transferring seedlings to aluminium screens placed in germination boxes (11 × 3.5 cm) with 100 g of silica gel blue (4–8 mm) and incubated at 25 °C for A. colubrina and 30 °C for C. pyramidale, photoperiod of 12/12 h light/dark (BRASIL, 2013). After 24 and 72 hours desiccation, seedlings were rehydrated and allowed to grow by transferring to germination paper moistened with distilled water and incubated at same temperature and photoperiod conditions as previously described, for 7 and 14 days. WC of seed lots was determined gravimetrically by oven-drying two samples of 25 quiescent seeds (approximately 3 g) of each species at 105 ± 3 °C for 24 h (BRASIL, 2009), since this is a destructive method. The results were expressed as a mean percentage (fresh weight basis) and this result was considered the initial WC of each individual dry seed. Each quiescent seed was individually weighed prior to germination. Seedlings of each category were individually weighed after radicle protrusion, after 24 or 72h of desiccation, and after 7 or 14 days of subsequent rehydration. To evaluate changes in WC, we used the following equation adapted from Hong and Ellis (1996). DT was considered when seedlings resume growth after desiccation and rehydration to original water content. At the end of the experiment, at 14 days after rehydration, final root and shoot length of desiccated and rehydrated seedlings were individually measured using a digital caliper (0.001 mm accuracy).萌发种子的脱水耐性（Desiccation Tolerance, DT）与季节性干旱热带森林（Seasonally Dry Tropical Forests, SDTF）物种的幼苗存活成功率直接相关。本研究旨在探究Anadenanthera colubrina与Cenostigma pyramidale的种子是否具有萌发后脱水耐性，以及该耐性在幼苗发育的哪个阶段仍可维持。实验前，将种子从果肉残体、枝条及其他物种的种子中分离，装入布袋置于低温干燥箱（±10 ℃/45%相对湿度（Relative Humidity, RH））中贮藏6个月。在脱水耐性试验前，新鲜的Anadenanthera colubrina与Cenostigma pyramidale种子的萌发率分别为85%与90%，种子含水量（Water Content, WC）分别为8%与8.1%。将种子播种于以蒸馏水润湿的发芽纸上，加水量为干纸重量的2.5倍，随后分别在25 ℃（Anadenanthera colubrina）与30 ℃（Cenostigma pyramidale）、12/12 h光暗周期（白光光量子通量密度为30 W/m²）的条件下培养，该培养条件参照BRASIL（2013）发布的最佳种子萌发规范。由于这两个物种萌发速度极快，播种24 h后即可观察到胚根突破，并使用精度为0.001 mm的数显游标卡尺测量胚根长度。将幼苗按初生根长度（Initial Radicle Length, IRL）分为4个等级：1.00–2.99 mm、3.00–4.99 mm、5.00–6.99 mm以及7.00–10.99 mm。每个初生根长度等级的幼苗（每组设5个重复，每个重复含10粒种子）分别进行24 h与72 h的脱水处理。脱水操作如下：将幼苗转移至置于发芽盒（11 × 3.5 cm）内的铝制筛网上，盒内加入100 g粒径4–8 mm的蓝色硅胶，随后分别在25 ℃（Anadenanthera colubrina）与30 ℃（Cenostigma pyramidale）、12/12 h光暗周期的条件下培养（BRASIL, 2013）。脱水24 h与72 h后，将幼苗转移至用蒸馏水润湿的发芽纸上进行复水，并在与此前相同的温度与光暗周期条件下培养7 d与14 d，以观察幼苗恢复生长情况。种子含水量采用重量法测定：参照BRASIL（2009）的规范，称取每个物种的2份各25粒静止种子（每份约3 g），置于105 ± 3 ℃的烘箱中烘干24 h，通过称重法计算种子含水量（以鲜重为基准），该值即为单粒干燥种子的初始含水量。实验前需对每粒静止种子单独称重；胚根突破后、脱水24 h或72 h后以及复水7 d或14 d后，也需对每株幼苗单独称重。为测定含水量变化，本研究采用改编自Hong与Ellis（1996）的计算公式。当幼苗经脱水与复水后可恢复至初始含水量并重新生长时，即判定其具有脱水耐性。实验结束时（复水后14 d），使用精度为0.001 mm的数显游标卡尺分别测量经脱水与复水处理的幼苗的最终根长与地上部分长度。