Effects of NaCl Stress on Seed Germination of Four Canavium album Raeuseh Cultivars

Zhengqing GAO, Yuqiang LOU, Jiaxun LIU, Ziqin LU

1. Horticulture Research Institute, Yunnan Academy of Agricultural Sciences, Kunming 650205, China;

2. Institute of Tropical and Subtropical Economic Crops, Yunnan Academy of Agricultural Sciences, Baoshan 678025, China;

3. Yunnan Academy of Agricultural Sciences, Kunming 650205, China

Currently, due to effects of global climate warming,population growth, aggravated industrial pollution, irrigation agriculture development and fertilizer abuse, soil salinization is aggravated day by day,threatening the sustainable development of agricultural production[1].In addition to prevention and treatment of soil salinization, enhancing salt and drought resistances in plants is also an important way to solve this problem[2].In the hot-dry valleys of Lujiangba and Jinsha River in Baoshan,Yunnan,appropriate economic fruits are planted to develop rationally, govern and improve the ecological environment[3].This can not only improve peoples’well-being, but also prevent the problem of desertification in arid regions[4].Olive has become a major fruit in that region. In April, 2009, Yunnan Province issued "Advice of the People’s Government of Yunnan Province on Accelerating the Development of Woody Oil Industry",which pointed out that the planting area of olive had reached 1 333.3 km2in Baoshan[5],and it would reach 66 700 hm2across the province till 2020[6]. Canavium album Raeuseh (Burevaceae: Canavium) is one of unique subtropical evergreen trees in Southern China. It is well known as "Paradise Fruit". Canavium album Raeuseh is nutritious, and its flesh is rich in proteins,carbohydrates,fats, vitamin C, calcium, phosphorus,iron and other minerals.The vitamin C content in Canavium album Raeuseh is 10 times higher than that in apple,and 5 times higher than those in pear and peach. The calcium content in Canavium album Raeuseh is also higher,and the calcium is easier to be absorbed by human body. Therefore,Canavium album Raeuseh has high economic value and broad market prospects. It is well known thatCanavium album Raeuseh has strong drought tolerance, but there are rare researches on salt tolerance of Canavium album Raeuseh.

In this study, under different intensities of salt stresses, the water intake processes, germination rates,amylase activities and embryo and radicle growth situations of different olive cultivars were studied, and the sensitivities of different olive seeds to salt stress were analyzed, providing some reference for the selection of suitable olive cultivar.

Materials and Methods

Materials

The Canavium album Raeuseh seeds were provided by the Institute of Tropical and Subtropical Economic Crops, Yunnan Academy of Agricultural Sciences. The test olive cultivars included Huiyuan, Chayao No.1, Sanfang and Frantoio[7].Total 11 concentrations(0(CK),0.2%,0.4%,0.6%,0.8%,1.0%,1.2%,1.4%,1.6%,1.8%,2.0%)of NaCl solutions were arranged.

Methods

Treatment and culture methods of seeds The olive fruits were collected 10-20 d after the beginning of winter.They were retted for few days till the flesh and peel were decayed naturally.The residues were soaked in water for 2-3 d at 7-10 ℃, and then the seeds were collected. The olive seeds were washed and dried and subjected to stratification treatment. In February of next year, the seeds were soaked in 0.2% KMnO4solution for 10 min. Subsequently,they were rinsed with sterile water and the moisture on seed surface was dried with sterile towel.In the clean and dried petri dishes,two layers of filter paper were laid. A certain amount (100 ml) of NaCl solution at each of the concentrations was added to a petri dish,in which,30 olive seeds were subsequently placed.The four olive cultivars were tested simultaneously. There were nine replicates(three for the determination of germination rate and seedling growth; three for the measurement of water absorption by seed germination; three for the determination of enzyme activity) for each treatment[8]. The petri dishes were placed in an artificial light incubator (RXZ) at 25 ℃. The filter paper was replaced regularly every day to remain the NaCl concentration unchanged. Since the shell of olive fruit core is thick, the experiment lasted for 14 d.

Determination of germination rate(Gr),germination index(Gi)and vigor index (Vi) The germinated seeds were counted every day. The germination index and vigor index were calculated according to the following formula:

Wherein, Gt represents the number of germinated seeds within t days;Dt represents the corresponding number of day(s);S represent the height of young seedling.

Determination of water absorption by seed germination On hour 5,10,15, 20, 25, 30, 35, 40 and 45 after the beginning of salt stress, the seeds were taken out and weighed respectively after drying the moisture on their surface with paper. The water absorption was calculated according to the following formula:

Wherein, g1and g2represent the weights of seeds before and after the absorption of water,respectively.

Determination of amylase activity Total six olive seeds were taken out from each of the treatments on day 2,4, 6, 8, 10, 12 and 14, respectively.They were peeled and subjected to amylase activity determination. The results were expressed as the averages of three replicates.The determination was completed by the Yunnan Academy of Agricultural Sciences.

Determination of seed moisture content On day 14 after the sowing,total 10 seedlings were sampled from each of the treatments for measurement of lengths, diameters, fresh weights and dry weights of shoots and roots. The moisture content was calculated according to the formula:

Wherein, W1represents the fresh weight of the seedling; W2represents the dry weight of the seedling.

Results and Analysis

Effects of different intensities of NaCl stresses on water absorption by seed germination of four different olive cultivars

As shown in Fig.1, the Frantoio seeds could rapidly absorb water at various salt concentrations. Within the first 25 h, the water absorptions were lower than that in the CK. The decrement of water absorption at higher salt concentration was larger than that at lower salt concentration, but there were no significant differences in overall.After 25 h,the imbibition rate in the CK was increased sharply; the imbibition rate at NaCl concentration of 0.2% was also trended to be increased, but it was significantly lower than that of CK. When the NaCl concentration exceeded 0.8%, the imbibition rates in various treatment groups were trended to be remained constant.The results above validate the conclusion that low-intensity salt stress has certain promoting effects on seed water absorption and germination, but high-intensity salt stress inhibits seed water absorption and germination[9].

Effects of different intensities of NaCl stresses on seed germination rates of four different olive cultivars

Fig.2 showed that with the increased NaCl concentration, the seed germination rates of the four olive cultivars were all trended to be decreased. The seed germination rates in treatment groups were all lower than that in CK at various times,suggesting that salt has certain inhibitory effect on olive seed germination. However, Zhu et al. found that low-intensity NaCl stress did not promote seed germination of all olive cultivars, and different olive cultivars showed different sensitivities to salt stress[10]. In this study,the four olive cultivars also showed different tolerances to salt stress. When the NaCl concentration was lower than 0.8%, the seed germination rates of the four olive cultivars were all higher than 90%, but they were still lower than those in CKs by 10%-15%. The critical salinities inhibiting radicle growth of the four olive cultivars were as follows: Huiyuan 1.0% ; Chayao No.1, 1.2%; Sanfang 1.4%; Frantoio,1.6%. So, the sensitivities of the four olive cultivars ranked as Huiyuan ＞Chayao No.1 ＞Sanfang ＞Frantoio.When the NaCl concentration exceeded the critical salinity, olive seeds almost did not germinate (Fig.2-Fig.5).This was because that Na+, as a decisive factor, destroyed the living envi-ronment of olive seeds[11].

Effects of different intensities of NaCl stresses on seed germination rate and vigor index of Frantoio

Among the four olive cultivars,the data about Frantoio was used to study the laws in seed germination under different-intensity NaCl stresses. The seed germination rate referred to the ratio between germinated seeds number and test seeds number within a certain interval. Moreover, the growth of seedlings was observed. As shown in Table 1, the germination index and vigor index were all decreased greatly with the increase in NaCl concentration.When the NaCl concentration exceeded 0.4%, the decrease amplitude reached 60%, indicating that germination index and vigor index were more sensitive to NaCl stress than germination rate.

Effects of different intensities of NaCl stresses on seed amylase activity of Frantoio

As shown in Fig.6, amylase in Frantoio seeds had certain activity,and within 6 min, the activity reached 1.8 units. With the increased NaCl concentration, the amylase activity was trended to be increased. In the CK, the amylase activity was increased most sharply on the 4th d.Under low-intensity salt stress,the amylase activity in Frantoio seeds was also trended to be increased. With the increase NaCl concentration, the amylase activity was reduced gradually.When the NaCl concentration was up to 1.6%, the amylase activity in Frantoio seeds was basically remained unchanged,which was consistent with the critical salinity inhibiting radicle growth of Frantoio.It suggests that reduced amylase activity is an important reason leading to blocked conversion of stored substances in olive seeds and delayed seed germination and stunted seedling growth under salt stress[12-13].

Effects of different intensities of NaCl stresses on seedling growth of Frantoio

With the increase in NaCl concentration,the Frantoio seeds could basically germinate, but the germination was inhibited in varying degrees com-pared with that in CK (Table 2). The root diameters and dry weights in various treatment groups were all lower than those in CK. When the NaCl concentration exceeded 0.4%,the root diameter and dry weight were decreased sharply. However,when the NaCl concentration was 0.2%, the root length in the treatment groups was higher than that in CK,suggesting that low-concentration salt might promote the development of plant roots[14].When the NaCl concentration was higher than 0.4%, the root length and diameter and dry weight were decreased simultaneously.Table 2 showed that the moisture content in fresh seedlings was trended to be decreased with the increased NaCl concentration,but the decrease amplitude was relatively small,indicating that salt stress has inhibitory effect on water absorption by plants[15]. Different concentrations of NaCl solutions showed different inhibitory effects.The higher the NaCl concentration was,the stronger the inhibitory effect was. On the contrary, the lower the NaCl concentration was, the weaker the inhibitory effect was.

Table 1 Effect of NaCl stress on seed germination of Frantoio

Table 2 Effect of NaCl stress on seedling growth of Frantoio

Conclusions and Discussion

In this study,in the CK,the imbibition rate of olive seeds reached 85%20 h after the beginning of salt stress,and then the olive seeds began to germinate; in various treatment groups,the imbibition rates of olive seeds all reached 85% within 45 h. It suggests that the plant cell membrane system is the main part of salt damage, and the effects of salt stress on membrane structure and functions are generally represented by selective permeability loss, leading to extravasation of large amounts of cellular content[16].With the prolonged imbibition time and increased NaCl concentration, the extravasation amount of cellular contents was increased sharply. The increased NaCl concentration affected imbibition speed and weakened imbibition capacity. But with the prolonged imbibition time, the water absorption could still be adequate for seed germination.

When the NaCl concentration was 0.2%, the root length of Frantoio was significantly higher than that of CK,which is consistent with many previously reported results. But low-concentration NaCl was adverse to seed germination. Wang reported that lowintensity NaCl stress improved seed germination rates of zucchini and winter squash to some degree[17]. Shen et al.also obtained similar results on other crops[18].

Amylase,composed of α-amylase and β-amylase,is the necessary material for seed germination. Its functions are exhibited during seed germination and early seedling growth. The αamylase can degrade starch in seeds into small molecules for seed germination and seedling growth, and its activity reflects seed vigor[19]. In this study, with the increased intensity of NaCl stress, the amylase activity in Frantoio seeds was reduced, and the seed germination was delayed,which might be due to weakened imbibition capacity and osmotic regulation[20].Thus the degradation of starch by α-amylase into small molecules is affected in seeds.

The growth of Frantoio seedlings under salt stress was also observed.In overall, when the NaCl concentration was less than 0.4%, the effect of NaCl stress on seedling growth was relatively weak, and the effect on shoots was stronger than that on roots. However, when the NaCl concentration exceeded 0.4%,the lengths,diameters and dry weights of shoots and roots were all significantly reduced compared with those in CK. It suggests the presence of critical salinitiesfor sensitivities of some plants[21].When the NaCl concentration is higher than the critical value,the inhibitory effects of salt stress on plant growth are more obvious. But this is not applicable to all of the plants. Zhu found that low-concentration NaCl does not promote the seed germination of all watermelon cultivars, and some watermelon cultivars are insensitive to salt stress[22].

The analysis above shows NaCl stress has multiple effects on plants.But for seed germination and growth of both salt tolerant plants and salt sensitive plants, there are all corresponding salt stress sensitivities and maximum growth critical salinities. According to crop type and environment and climate, soil conditions and air composition that are closely associated with plant growth,plant growth salinity table is expected to be prepared in the future studies, thereby providing certain reference for agricultural researches and production.

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