Effectiveness of ultrafine bubbles and gamma irradiation treatments to improve seed viability and vigor of Albizia chinensis(Osbeck) Merr.

Dede J.Sudrajat·Anindyta Putri·Y.Aris Purwanto·Iskandar Z.Siregar

Abstract Ultrafine bubbles and low doses of gamma irradiation had a positive effect on seed germination.This research tested the effectiveness of ultrafine bubbles (UFB)water and gamma irradiation for improving the viability and vigor of Albizia chinensis (Osbeck) Merr.seeds that had been stored for four years.A completely randomized two-factor experiment，i.e.，UFB water and gamma irradiation treatments，were used to determine the best seed treatment.The UFB water treatment had a significant effect on all seed germination parameters;the gamma irradiation treatment did not have a significant effect on improving the viability and vigor of the seeds，with a very low initial germination (11.7%).Soaking the seeds in UFB water for 30 min gave the best germination (51.3%).These improvements also occurred in other germination parameters such as germination rate，germination value，radicle length and vigor index.The UFB water treatment has the potential to improve the germination of seeds with low physiological qualities.

Keywords Albizia chinensis ·Gamma rays·Germination·Ultrafine bubbles

Introduction

Albizia chinensis(Osbeck) Merr.，locally known as red sengon，is a tropical fast-growing tree species widely cultivated in plantation and community forests.This species is well adapted to soil that is less fertile with pH ＞ 7 or that contains high salt on alluvial soils and sandy soils from mine tailings(Orwa et al.2009).The wood is not attacked by subterranean termites because it contains extractive compounds and is often used as crates，boats，housing，and bridge materials.In addition，A.chinensisis often planted for shade，for protecting sloping lands and for improving soil because the root system is nitrogenous (Suita et al.2018).

Cultivation ofA.chinensisrequires good quality seeds.AlthoughA.chinensisseed is orthodox seed according to Orwa et al.(2009)，at room temperature storage conditions viability is only maintained for 12 months.Seed storability is influenced by seed structure，biochemical composition，initial physiological quality，seed dormancy，and handling(Khatun et al.2009;Syamsuwida et al.2020)，seed ecology (Yasaka et al.2008)，genetics (Rustam et al.2017)，and condition and storage period (Suszka et al.2014).A lengthy storage period can cause quality deterioration，physically，physiologically，and biochemically，which results in decrease in seed viability and vigor.

Improving low seed viability and vigor can be done by invigorating treatment through seed priming (Duta 2018)and gamma irradiation (Araújo et al.2016;Zanzibar and Sudrajat 2016;Zanzibar et al.2021).A promising priming technology is using untrafine bubbles (UFB)，a renewable technology utilizing the dissolved oxygen in water (Liu et al.2014).The concentration of oxygen in plant tissues plays an important role in regulating biochemical reactions(Wagner 2008)，increasing seed germination (Liu et al.2014)，and promoting seedling growth (Iijima et al.2020).UFB are miniscule bubbles with diameters between micro(10-4-10-6μm) and nano (smaller than 10-6μm) (Uchida et al.2011).Seed immersion treatment with nanosized bubbles in water (UFB water) has increased the germination ofTriticum aestivumL.(Liu et al.2 013)，Hordeum vulgareL.(Liu et al.2017)，Oryza sativaL.(Iswara et al.2018)，andGlycine max(L.) Merr.(Purwanto et al.2019).Several studies on forest tree seeds also reported the positive effect of UFB water on increasing germination capacity ofNeolamarckia cadamba(Roxb.) Bosser (Fata et al.2020) andGmelina arboreaRoxb.(Siregar et al.2020).

Application of low doses of gamma irradiation can also stimulate early seed germination by mean of enhancing enzyme activities and increasing cell division (Ikram et al.2010;Piri et al.2011;Iglesias-Andreu et al.2012;Araújo et al.2016).The use of irradiation to improve seed vigor has been widely used in agricultural crops (Piri et al.2011;Iglesias-Andreu et al.2012)，and several studies have been conducted on tropical tree species such asMagnolia champaca(L.) Baill.ex Pierre (Zanzibar and Sudrajat 2016)，Neolamarckia cadamba(Roxb.) Bosser (Suhartanto et al.2018)andToona sureni(Blume) Merr.(Zanzibar et al.2021).

UFB water and gamma irradiation applications for improving the viability and vigor of tropical forest tree seeds are still limited (Iglesias-Andreu et al.2012;Siregar et al.2020;Syamsuwida et al.2020).Therefore，the objective of this study was to determine the effectiveness of UFB water and gamma irradiation for improving the viability and vigor ofA.chinensisseeds stored for four years.

Materials and methods

Materials

A.chinensisseeds were collected from a community forest seed stand in Cisitu Subdistrict，Sumedang District，West Java，Indonesia (6°53?23″ S，108°01?35″ E).The seeds were stored at the Tree Seed Testing Laboratory，F(xiàn)orest Tree Seed Technology Research and Development Center，Bogor.Before storage，the moisture content of the seeds was lowered to 8% to increase storability.The seeds were packed in airtight plastic bags and stored in dry storage at temperature 4-8°C and relative humidity of 40-50%.TheA.chinensisseeds used in this study were stored for four years.

Preparation of UFB water

Production of water containing UFB was carried out at the Biosystem Environmental Engineering Laboratory，IPB University using a UFB generator (Ultrafine GaLF type FZ1N-10).UFB water was made by running the generator for 30 min and then for 55 min using distilled water.The difference between UFB water running 30 and 55 min is in the number of bubbles produced.Liu et al.(2014) noted that the length of running time in the generator does not affect bubble diameter but will affect the density of bubbles (number of bubbles per ml of water).

Gamma irradiation treatments

Gamma irradiation treatments were carried out at the Center for the Application of Isotope and Radiation Technology，National Nuclear Energy Agency，Jakarta，Indonesia.The seeds were given irradiation treatment using Gamma Cell 220 with a radiation source of60Co (Cobalt-60) and a rate of 6645.7 Gy h-1.Radiated seeds were repackaged and identified according to treatment.

Experimental design

A factorial completely randomized design with two factors was used to test invigoration treatments of UFB water and gamma irradiation on seed germination.The first factor was the dose of gamma irradiation，i.e.，zero，10，20，30，40，and 50 Gy;the second factor was the immersion of seeds in UFB water:seeds without immersion;immersion in UFB water processed for 30 and 55 min in the generator (Siregar et al.2020).Each treatment was repeated four times with 100 seeds per replication.

Seed germination testing

To test seed viability and vigor，a top of the straw paper media was used (Sudrajat et al.2017).All seeds were pretreated with hot water immersion for five minutes.For controls and gamma ray irradiation treated seeds，soaking continued for 24 h in cold water.For UFB water treated seeds，soaking was also continued for 24 h.The seeds were sown in petri plates on paper media (top paper test) moistened with distilled water and placed in a seed germinator type IPB 73-2A/B.The germinator was set at 24-30°C，relative humidity of 90-95%，and lighting of 8 h bright and 12 h dark(Sudrajat et al.2017).

Germination observations were carried out every day for 14 days by counting seeds that germinated normally，i.e.，the sprouts length (radicle and hypocotyl) was twice the length of the seed (Sudrajat et al.2017).The parameters of seed germination observed and calculated in this study were capacity，rate，mean germination time，germination value，hypocotyl length，radicle length，and vigor index (Czabator 1962;Ranal et al.2009;Gairola et al.2011;Sudrajat 2016).The lengths of the hypocotyl and radicle were measured at the end of seed germination (14 days after seed sowing).The vigor index (total length of sprouts x germination capacity)was calculated based on Bhattacharya et al.(1991).

Data analysis

Analysis of variance with a factorial completely randomized design was used to determine the effects of UFB water and gamma irradiation treatments.The data were tested for normality and if not normal，the data was transformed witharcsineto increase normality (Zar 1996).If the results of the analysis of variance show that the treatment had a significant effect on an observed parameter，then Duncan’s multiple range test at the 5% level was performed to determine the differences among treatments.

Results and discussion

UFB water treatments produced significant effects on all germination parameters except on radicle length.A significant effect was also shown in the interaction of UFB water and gamma irradiation on germination capacity，rate，and value，and radicle length and vigor index (Table 1).However，irradiation alone had no significant effects on all germination parameters ofA.chinensisseeds.A similar result was reported by Suhartanto et al.(2018) forNeolamackia cadamba(Roxb.) Bosser seeds，i.e.，that gamma irradiation did not improve the quality of seeds with low initial germination(＜40%).Several previous studies have shown the positive effect of low-dose gamma irradiation on the germination of forest tree seeds such asTerminalia arjuna(Roxb.) Wight&Arn.(Akshatha et al.2013) andMagnolia champaca(L.)Baill.ex Pierre (Zanzibar and Sudrajat 2016).The difference in results between these studies was due to differences in the genetic (species or provenance)，physiological and chemical characteristics of the seeds tested.

Table 1 Analysis of variance of the UFB water and gamma irradiation treatments on the viability and vigor of Albizia chinensis seed

The positive effect of UFB water on germination has also been reported for several crops and trees species such asTriticum aestivumL.(Liu et al.2013)，Hordeum vulgareL.(Liu et al.2016)，Oryza sativaL.(Iswara et al.2018)，Daucus carotaL.，Lactuca sativaL.，Vicia fabaL.，Solanum lycopersicumL.(Ahmed et al.2018)，Glycine max(L.) Merr.(Purwanto et al.2019)，Neolamarckia cadamba(Roxb.) Bosser(Fata et al.2020)，andGmelina arboreaRoxb.seeds (Siregar et al.2020).However，UFB water effects on germination is influencd by seed characteristics，initial germination capacity，biochemical contents，and germination environments(Fata et al.2020;Iijima et al.2020;Siregar et al.2020).

In this study，the best treatment was seed immersion in UFB water for 30 min，with 51.3% germination capacity，an increase of 336% compared to the controls (11.7%) (Fig.1).The large increase indicates the potential of UFB water to improve the physiological quality of deteriorated seeds.An increase in germination in response to nanobubbles water treatment was reported by Liu et al.(2013) for barley (Hordeum vulgareL.)，with a germination capacity 15%-25%greater than the seeds immersed in distilled water with the same dissolved oxygen concentration.InNeolamarckia cadamba(Roxb.) Bosser seeds from several provenances in Indonesia，UFB water treatment increased germination capacity by 40-75% (Fata et al.2020)，while withGmelina arboreaRoxb.seeds，germination capacity increased by 12.5% (Siregar et al.2020).

Fig.1 Germination capacity of A.chinensis seed on the interaction of UFB water and gamma irradiation treatments.Notes:U0=without UFB water treatment，U1=soaking in UFB water (30 min)，U2=soaking in UFB water (55 min);G0=without irradiation treatment，G1=irradiation 10 Gy，G2=irradiation 20 Gy，G3=irradiation 30 Gy，G4=irradiation 40 Gy，and G5=irradiation 50 Gy

The improvement ofA.chinensisseed germination by immersion 30 min in UFB water also improved germination rates，radicle lengths and vigor index (Fig.2).The treatment increased germination rate from 1.7 to 7.9%day-1，germination value from 0.54 to 8.89，radicle length from 2.1 to 3.9 cm，and vigor index from 0.48.to 2.28.Similar results were also reported by Fata et al.(2020)forNeolamarckia cadamba(Roxb.) Bosser seeds.Nano bubbles affect the physiological activities of plants such as increasing seed germination and seedling growth.The negative load from the micro and nanobubbles affects the bioelectricity of the plant which is closely related to the growth in length.The length of the radicle is thought to be due to the presence of abundant bioelectricity so that the growth of the radicle，which is a future root，develops well(Liu et al.2013).Ushikubo et al.(2008) also reported that common wheat seeds (Triticum aestivumL.) treated with micro and nano bubbles water immersion showed faster cytoplasm flow.This mechanism is responsible for improving seed vigor and uniformity of growth.High growth uniformity indicates high storability of seed as well.Thus，the seeds with a fast germination rate and high growth uniformity would likely possess high vigor levels.

Fig.2 Effect of UFB water and gamma ray irradiation treatments on germination rate a germination value b radicle length (c) and vigor index (d)of Albizia chinensis seeds.See Fig.1 for seed treatment information

For mean germination time and hypocotyle length，the UFB water treatment resulted in significant differences from untreated seeds.Soaking the seeds in UFB water for 30 min gave the best results and the mean germination time was relatively faster，the mean germination time on day 7.47 compared to control (day 8.23) (Table 2).Similarly with hypocotyle lengths，seeds soaked in UFB water had longer hypocotyles than untreated seed.Hypocotyl length is a result of cell division in the apical meristem.This meristematic activity is influenced by the availability of water containing nutrients and minerals that the embryos need to germinate (Kozlowski and Pallardy 1997).Hypocotyl is formed due to the presence of gibberellin hormones produced by the seeds themselves have been sufficient to carry out the germination process (Weiss and Ori 2007;Miransari and Smith 2014).

Table 2 Effect of UFB water treatment on mean germination time，germination value，and hypocotyl length of Albizia chinensis seeds

Ultrafine bubbles enter the seeds through the seed coat because of their size (Ahmed et al.2018).UFB water containing miniscule bubbles with diameters between micro(10-4-10-6μm) and nano (smaller than 10-6μm) could produce reactive oxygen species (ROS)，and the amount of ROS was positively correlated with UFB density.These bubble generators produce water containing high concentrations of micro and nano bubbles ranging from 106 to 108 particles mL-1(Liu et al.2016).Moderate levels of exogenous ROS produced by micro and nano bubble water played an important role in seed germination (Liu et al.2016).The hydroxyl radicals produced by UFB trigger the formation of ROS in seeds which become physiological regulators signaling germination.ROS compounds also cause cell walls to loosen so that water，gas，and embryonic structures can penetrate the seed coat.This cell wall loosening occurs because UFB induces the expression of genes involved in the process (Liu et al.2016).Thus，imbibition is an initial stage in the germination process.

UFB water increases gibberellin levels in seeds (Gomes and Garcia 2013;Liu et al.2016).Gibberellin is a hormone needed in the germination process (Cornea-Cipcigan et al.2020) to activate α-amylase，proctoase，ribonuclease，β-glyconase and phosphatase enyzems which are important in the germination process (Khairani et al.2016).The enzymes enter the endosperm by diffusion to become catalysts for the change of food reserves into sugars and amino acids.Soaking seeds in UFB water also increases the solubility of gases (solubility of oxygen) in liquids(Liu et al.2 016).Changing food reserves in the process of respiration also requires oxygen.

Conclusion

UFB water treatment run for 30 min was more effective than gamma irradiation in significantly improving the germination ofA.chinensisseeds with low germinating capacity (11.7%).This treatment increased germination capacity from 11.7 to 50.3%，the germination rate from 1.7 to 7.9% day-1，germination value from 0.54 to 8.89，radicle length from 2.1 to 3.9 cm，and vigor index from 0.48 to 2.28.In addition，UFB water treatment improved the mean germination time and increased hypocotyle length.Therefore，in addition to increasing the germination of low viable and vigorA.chinesisseeds，UFB water treatment also increased the growth ofA.chinensisseedlings.

AcknowledgementsThe authors thank the Center for the Application of Isotope and Radiation Technology，the National Nuclear Energy Agency (BATAN)，and the Biosystem Environmental Engineering Laboratory，IPB University，for facilitating this research.