College of Biology Sciences and Engineering,Shaanxi University of Technology,Hanzhong 723000,PRC

Abstract In order to discuss the dormancy characteristics of seeds about Acer miaotaiense Tsoong,an endangered species,the permeability of husk and the effects of inhibitors on seeds germination were tested.The results showed that water absorption rate and velocity both increased significantly (P＜0.01) when the husk spun off seeds,which meant that seed coat was one of the factors affecting dormancy because the lignification restricted absorption of water from the environment.Some inhibitors existing in the pericarp,seed coat and embryo could inhibit the germination of cabbage seeds.There were obvious differences on the germination rate,radicle length and seedling height (P＜0.01),and the inhibition degree was in the order of seed coat＞pericarp＞embryo.

Key words Acer miaotaiense Tsoong;Seeds;Dormancy;Permeability;Extracts

1.Introduction

Acer miaotaienseTsoong is an endemic deciduous tree of the genusAcerof Aceraceae originated in Miaotaizi,Liuba County,Shaanxi Province,China.Scattered in the mountain slopes or jungles of Qinling Mountains in Shaanxi and Gansu Provinces of China at an altitude of 1 000～ 2 000 m,Acer miaotaienseTsoong is featured with towering height,beautiful leaves and unique fruits,which can be planted as an important gardening and greening species[1].Acer miaotaienseTsoong is currently a rare and endangered plant with a narrow distribution range in China.Since its listing as one of the first tertiary protected plants in China,many studies have been conducted on the morphological characteristics[2],reproductive characteristics[3],population distribution[4],genetic diversity[5]and other aspects ofAcer miaotaienseTsoong.Nevertheless,only very few of them investigated the dormancy characteristics ofAcer miaotaienseTsoong seeds.This study used the seeds ofAcer miaotaienseTsoong as the experimental material and explored the possible dormancy mechanism of the seeds based on two aspects:seed coat permeability and inhibitory effects of extracted methanol from different parts on seed germination,aiming to provide reference for the expansion of artificial reproduction ways ofAcer miaotaienseTsoong.

2.Materials and Methods

2.1.Materials

Ripe samara seeds of 20-year-oldAcer miaotaienseTsoong planted in Hanzhong area,Shaanxi Province were selected as the experimental materials of this test.The samara samples were peeled off and cleaned.The seeds that were neatly shaped and full-grained were dried out for use.Whether the extracted methanol from different parts of the seeds contained inhibitors on seeds germination was determined by testing their inhibitory effects on cabbage germination[6-7].This test used the cabbage with 95% of seed purity and 98% of neatness.

2.2.Methods

2.2.1.Determination of seed coat permeability

Select 30 grains of seeds with full husk and 30 with husk spun off.Weigh them on a 0.1 mg scale respectively.Put them in a beaker and add deionized water for soaking.Then place the beaker under constant 25℃ for imbibition.Weigh the soaked seeds every 6 h in the first 24 h and every 12 h after 24 h,until the weight kept constant.Each treatment was repeated three times.Calculate the water absorption rate and velocity,and average the results.

Water absorption rate//%=[weight after absorption (w2)-weight before absorption (w1)]/weight before absorption (w1)×100%;

Water absorption velocity//mg/h=[weight after absorption (w2)-weight before absorption (w1)]/interval (h2-h1).

2.2.2.Determination of inhibitors

Refer to the approach by LIU Y Yet al.[6]and only make a few adjustments based on the characteristics of the seeds ofAcer miaotaienseTsoong.Take 2 g of each pericarp,seed coat and embryo,dry and smash,and put them into a 50 mL conical flask,respectively.Add 10 mL 80% methanol into each flask,mix and place in the refrigerator for sealed extraction under 4℃ condition.Constantly shake the flask to ensure an adequate extraction.Filter the liquid after 24 h.Repeat the extraction three times and mix the extracts evenly.Then reduce the constant volume to 20 mL after pressure reduced and the concentration became 0.1 g/mL.Dilute the above original liquids to 0.02,0.04,0.06,0.08 and 0.10 g/mL,respectively.Take 5 mL of each of the above concentration gradient solution,and add in a 9 mm diameter culture dish prelined with filter paper.Place them under 25 ℃ with 1 500～2 000 Lux illumination intensity for cultivation.Put 50 grains of cabbage seeds randomly into each culture dish.Use deionized water as control,and count the germination rate after 48 h.Each treatment was repeated three times and significance tests were conducted.Measure the seedling height and radicle length after 72 h.Select five representative seedlings for measurement and perform difference significance analyses.

2.2.3.Data processing

Use Excel for data drawing and SPSS 23.0 statistical software for One-way ANOVA.Apply Duncan’s new multiple range test method for multiple comparisons in case of equal variances,and use Games-Howell for comparison in case of unequal variances.P＜0.05 means significant difference,P＜0.01 means extremely significant difference.

3.Results and Analysis

3.1.Effects of husk on permeability of seeds

3.1.1.Effects of husk on water absorption rate of seeds As shown in Fig.1,the water absorption rate of seeds with husk grew rapidly in the first 12 h,which reached 54.84%.Then it slowed down and was almost saturated at 48 h and maintained at about 71% (F=82.31,P＜0.01).In comparison,the water absorption rate of seeds with husk spun off showed significant difference at different soaking periods(F=13.28，P＜0.01).In the beginning,the absorption rate developed very fast,which reached 99.45% at 6 h of soaking,and then maintained a high rate during 6～36 h.After that,it was saturated and the final rate was as high as 160.57%.On the whole,the water absorption of seeds with husk spun off was shortened significantly to reach saturation (36 h),and the water absorption rate was significantly higher than that of seeds with husk,which was 2.3 times that of the seeds with husk when the absorption reached saturation.Therefore,the mechanical obstruction by the lignified husk ofAcer miaotaienseTsoong had a large impact on the water absorption of seeds during the first 36 h of soaking,restricting the water absorption of seeds to a large extent,which was one of the major factors that influence the dormancy ofAcer miaotaienseTsoong seeds.

3.1.2.Effects of husk on water absorption velocity of seeds

The water absorption velocity ofAcer miaotaienseTsoong seeds with and without husk varied significantly (Fig.2).For seeds with husk,the water absorption velocity increased very fast in the first 12 h,then it slowed down in 12～48 h and was close to 0 at 48 h when the absorption reached saturation(F=250.62,P＜0.01).As for seeds without husk,the water absorption velocity in different absorption periods was extremely different (F=261.70,P＜0.01).In the first 12 h,the velocity increased very fast,which was more than twice of the seeds with husk.After that,the absorption velocity gradually decreased and was stable around 48 h.This indicated that the removal of lignified husk could overcome the mechanical obstruction of the hard husk and greatly improve the water absorption velocity ofAcer miaotaienseTsoong seeds in the first 12 h of soaking.

Fig.2 Effects of different treatments on water absorption velocity

3.2. Effects of extracted methanol from different parts on seed germination

As the dormantAcer miaotaienseTsoong seeds in natural state can’t make direct response to the inhibitory effect of extracted methanol from different parts,this study referred to other scholar’s method by using the germination of cabbage seeds as the determination basis[6-7].Such method has high germination rate and the germination effect is easy to measure.

3.2.1.Effects of extracted methanol from different parts on germination rate of cabbage seeds

As shown in Fig.3,extracted methanol from different parts ofAcer miaotaienseTsoong presented obvious inhibitory effect on the germination of cabbage seeds.The germination rate of cabbage gradually reduced as the concentration of pericarp,seed coat and embryo extracts increased,and different treatments varied significantly in results (P＜0.01).Of the three extracts,the seed coat extract had the highest inhibitory effect on the cabbage germination,followed by pericarp extract and then embryo extract.Especially for the concentration of 0.06 g/mL,the difference of the three extracts in inhibitory effects was the most obvious(F=195.53，P＜0.01).Therefore,it can be preliminarily inferred that the pericarp,seed coat and embryo ofAcer miaotaienseTsoong contained some inhibitors that could inhibit the germination of seeds,and lignified seed coat had the highest quantity of inhibitors.

Table 1 Inhibitory effects of extracted methanol from different parts on cabbage germination

Fig.3 Effects of extracted methanol from different parts on germination rate of cabbage seeds

3.2.2. Effects of extracted methanol from different parts on seedling growth

The variance analysis showed that the extracted methanol from the pericarp,seed coat and embryo ofAcer miaotaienseTsoong had extremely significant effects on the height of cabbage seedlings (Fpericarp=101.99,Fseedcoat=108.84,Fembryo=81.46,P＜0.01).At 0.08 g/mL concentration,the extract inhibited the growth of cabbage seedlings completely;at 0.02～0.06 g/mL concentration,all the three extracts had inhibitory effects,and the inhibition degree was seed coat＞pericarp＞embryo (Table 1).In addition,the extracts from different parts ofAcer miaotaienseTsoong also affected the radicle length of cabbage(Fpericarp=54.15,Fseedcoat=75.70,Fembryo=30.48,P＜0.01).Among them,the pericarp extract had the strongest inhibitory effect,followed by seed coat extract and embryo extract which had similar inhibitory effect.

4.Discussions

Seed dormancy is not only a self-protection of plants to resist the bad environment in their longterm growing process but also an adaptive strategy to the harsh environment in their evolution process[8].The reason for the natural dormancy of seeds is complicated.Researches have shown that seed coat,due to its hard and dense structure or covered with wax,leather or sticky materials or thick cuticle,makes the seed airtight and impermeable,causing mechanical obstruction to the growth of embryo and hindering the germination of the seeds[9-10].ZHAO Jet al.studied the seeds ofStyrax tonkinensisand found that the mechanical obstruction of seed husk constituted one of the major causes to seed dormancy[11].CHENG G Yet al.found thatTaxus cuspidatahad hard and cuticularized seed coat,which affected the waterair exchange and hindered the imbibition and germination of the seeds[12].XU X L studied the water absorption ofAcer yanjuechiand concluded that the pericarp and seed coat had restricted the water absorption of seeds and reduced the water absorption velocity[7].This study investigated the effects of different treatments on water absorption rate and velocity ofAcer miaotaienseTsoong seeds and emphasized the physical obstruction of seed husk on the water absorption of the seeds.The water absorption rate and velocity can be significantly improved after removal of husk.Hence,it can be seen that the hard lignified seed coat could hinder the volume and velocity ofAcer miaotaienseTsoong seeds exchanging with surrounding water content.As a result,the seeds are unable to absorb water rapidly and adequately,and the physiological and biochemical processes related to seed germination such as respiratory action and energy metabolism of seeds were temporarily blocked,inhibiting the germination ofAcer miaotaienseTsoong seeds.Therefore,the mechanical obstruction is regarded as a major cause to the dormancy ofAcer miaotaienseTsoong seeds.

Seed dormancy and germination is a very complicated process.A large number of studies have shown that germination inhibitors usually exist in different parts of dormant seeds.LIU C Get al.[13]found that fresh Taxus chinensis var.Mairei seeds contained germination inhibitors and the extracted methanol had extremely significant inhibitory effect on the germination of cabbage seeds.LIU Y Yet al.[6]studied the seeds ofCeltis koraiensisand discovered germination inhibitors in both the pericarp and embryo of the seeds.HU J Jet al.[14]also found germination inhibitors inTaxus cuspidataS et Z.seeds.The embryo had the strongest inhibitory effect,followed by endosperm,inner seed coat,middle seed coat and outer seed coat.The results of this study indicated that the pericarp,seed coat and embryo ofAcer miaotaienseTsoong may all contain germination inhibitors,and their inhibitory effects were significantly enhanced with the increase of concentration.Among them the seed coat had the largest number of inhibitors,followed by the pericarp.The inhibitors existed in different parts of the seed were the major cause to the seed dormancy ofAcer miaotaienseTsoong.The contents,physiological activities and differential characteristics of these inhibitors need further exploration.

Future studies would also discuss whetherAcer miaotaienseTsoong seeds are morphophysiology dormancy (MPD) and whether the embryo structure and developmental state could restrict the seed germination.These studies would further investigate other reasons that caused the dormancy ofAcer miaotaienseTsoong seeds based on the anatomical structure of embryo and the degree of embryo development,thus providing reference for expanding the artificial reproduction ways and enhancing the protection,breeding and utilization of rare and endangered tree species.