Stimulation of oil palm (Elaeis guineensis) seed germination by exposure to electromagnetic fields

Scientia Horticulturae 220 (2017) 66–77

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Stimulation of oil palm (Elaeis guineensis) seed germination by exposure to electromagnetic fields Chadapust J. Sudsiri a , Nattawat Jumpa b , Pinpong Kongchana b , Raymond J. Ritchie c,∗ a b c

Faculty of Sciences and Industrial Technology, Prince of Songkla University-Surat-thani Campus, Muang District, Surat-thani Province, 84100, Thailand Sciences Laboratory and Equipment Centre, Prince of Songkla University-Surat-thani Campus, Muang District, Surat-thani Province, 84100, Thailand Tropical Plant Biology, Faculty of Technology and Environment, Prince of Songkla University-Phuket Campus, Kathu, Phuket, 83120, Thailand

a r t i c l e

i n f o

Article history: Received 24 June 2016 Received in revised form 7 March 2017 Accepted 23 March 2017 Keywords: Electromagnetic effects on plant growth Magnetically treated water Plant growth enhancement Plant seedling Seed germination

a b s t r a c t Difficult-to-germinate seeds are a common plant conservation, plantation and biotechnological problem. Oil palm (Elaeis guineensis) seed germination takes 6 months to 1 year with ≈40% failure rate. We investigated the affect of various electromagnetic DC field strengths on oil palm germination. Kernels imbibed water more effectively when exposed to a magnetic field. The kernels were divided into three groups. The first group (50) was soaked (imbibed) in distilled water (2 h) with no exposure to applied magnetic fields (blank control). The second group was exposed to magnetic fields with intensities of 2.5 mT, 5.0 mT, 7.0 mT, 9.0 mT and 11.0 mT for 1, 2, 3, 4 and 5 h (Dry Treated Kernels, DTK). The same electromagnetic protocol was conducted on a third group but with kernels which were immersed in water (Magnetically-Treated-Water-Kernels, MTWK). 96% germination was achieved by day-30 for the MTWK treatment using 9.0 mT for 4 h: slightly lower results were found for DTK kernels. None of the controls germinated within 30 days. Young oil palm seedlings kept in a shade house (≈110 ␮mol quanta m−2 s−1 PPFD) watered every day with MTW grew 3 times faster (3.1 mm day−1 ) than controls watered using unmagnetised water. © 2017 Elsevier B.V. All rights reserved.

1. Introduction There is a need for new, safer technologies for raising agricultural production for food and for raw materials for industry. Environmental studies on the factors effecting plant growth such as light, moisture, temperature, anthropogenic changes of the soil, water, and the atmosphere, the use of different chemical additives for raising plants productivity have led to the search for alternative ways to raise productivity with less environmental impact. Safe methods for increasing yield include the reasonable use of chemicals, preference for environmentally degradable chemicals over persistent agrochemicals and substitution of some of them by appropriate physical treatments. Physical methods for increasing plant production are based on the use of physical factors for plant treatment, with the major goal of increasing the yield and accelerating plant growth and development.

∗ Corresponding author. E-mail addresses: [email protected] (C.J. Sudsiri), [email protected] (N. Jumpa), [email protected] (P. Kongchana), [email protected], [email protected] (R.J. Ritchie). http://dx.doi.org/10.1016/j.scienta.2017.03.036 0304-4238/© 2017 Elsevier B.V. All rights reserved.

The effects of magnetic fields on plant growth have been the subject of many research studies including our recent study on Oil Palm (Elaeis guineensis Jacq.) (Sudsiri et al., 2016). Many authors have reported the effects of static magnetic fields using permanent magnets on the metabolism and growth of different plant species (Muraji et al., 1998; Hirota et al., 1999; Carbonell et al., 2000; Penuelas et al., 2004), including our patent (Sudsiri et al., 2014, 2015) and our previous paper on the use of permanent magnets and Magnetically Treated Water (MTW) to stimulate Oil Palm germination (Sudsiri et al., 2016). Numerous authors have found that magnetic fields increased the germination rates of seeds and increased the growth rate of seedlings, activated protein synthesis and increased root growth (Carbonell et al., 2000; Moon and Chung, 2000; Martíne et al., 2009; Flores et al., 2007). Many studies have found better germination percentage and larger growth in plants (Chauhan and Agarwal, 1977; Kavi, 1983; Dayal and Singh, 1986; Alexander and Ganeshan, 1990; Namba et al., 1995; Phirke et al., 1996a,b; Nargis and Thiagarajan, 1996; Negishi et al., 1999; Hilal and Hilal, 2000; Moon and Chung, 2000). A strong positive influence of magnetic fields on the initial growth stages of seedling plants after germination is often reported (Phirke et al., 1996a,b; Aladjadjiyan, 2002; De Souza et al., 2006) but in our previous study on oil palm we found that magnetic treatment improved germina-

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tion dramatically but did not affect the growth rate of the seedlings once they had germinated (Sudsiri et al., 2016). Samy (1989) found earlier flowering and yield increase in cauliflower (Brassica oleracea L.) as a result exposure to a static magnetic field for 8-h. De Souza et al. (2006) found out that treatment with a static magnetic field with induction of 0.08, 0.1 and 0.17 T increased the germination rate of tomato seeds (Solanum lycopersicum L.) by 5–25%. Phirke et al. (1996a,b) reported larger effects on soybean (Glycine max (L.) Merr.) and Indian tree cotton (Gossypium arboreum (L.) and wheat (Triticum aestivum L.) and plotted response surfaces (field strength and exposure time) to find the optimum treatment. Moon and Chung (2000) found larger effects on tomato germination using alternating electromagnetic fields. Alternating magnetic fields (AC-generated) appear to be as effective as fixed magnetic fields (DC-generated or using permanent magnets). Similar results for rice (Oryza sativa L.), sunflower (Helianthus annuus L.) and maize (Zea mays L.) were reported by Carbonell et al. (2000) and Flores et al. (2007). An important factor in the magnetic field effect on living organisms is water which is necessary for all living processes and water is very sensitive to magnetic field influence. Water that is subjected to treatment by a magnetic field is called Magnetically Treated Water (MTW) and offers an environmentally clean method for promoting growth of irrigated crops (Bugatin et al., 1999; Radeva and Mamarova, 1988; Rokhinson et al., 1994; Tian et al., 1989). Many experimental results on MTW show that it is more easily absorbed by the seed tissue and in this way it can stimulate internal metabolic processes which are conducive to germination (Belov et al., 1988; Henkenius and Retseck, 1992) even though a general theory of its mechanism of action has not yet been worked out (Belyavskaya, 2004; Galland and Pazur, 2005). Alexander and Doijode (1995) noted that aged low viability onion (Allium cepa L.) and rice seeds exposed to a weak electromagnetic field for 12 h via MTW increased their germination rate and shoot and root length of seedlings. Celestino et al. (2000) reported enhanced germination and growth of Cork oak (Quercus suber L.) seedlings when exposed to high-strength magnetic fields. Harichand et al. (2002) reported that exposure to a magnetic field (10 mT; 40 h) increased plant height, seed weight per spike and yield of wheat. Aladjadjiyan (2002) observed that the magnetic field stimulated shoot development of maize and led to an increase in germination rate and survival, fresh weight and shoot length. Growth of germinated broad bean (Vicia faba L.) seedlings, had an increased mitotic index and 3 H-thymidine uptake (an indicator of DNA synthesis)was also enhanced by the application of high frequency magnetic fields (100 mT) generated by AC currents (Rajendra et al., 2005). In lima bean (Phaseolus lunatus L.) and pea (Pisum sativum L.) cultivars the magnetic stimulation of seeds improved the sprouting and emergence of seed and resulted in higher final pod number and seed yield (Podlesny et al., 2005). Harichand et al. (2002) reported that exposure to a high energy magnetic field (10 mT; 40 h) increased plant height, seed weight per spike and yield of wheat. Nawroz and Hero (2010) investigated the seeds of different varieties of chickpea (Cicer arientinum L.) which were exposed in batches to static magnetic fields (1500 G of magnetic force) for 30, 50 and 70 min. Their results showed that magnetic field application enhanced seed performance in terms of laboratory germination rate, seedling length and seedling fresh and dry weight compared to the unexposed controls. However, the response varied with duration of exposure. Among the various duration exposures, 50 and 70 min exposures gave best results. They concluded from their results that magnetized seeds irrigated with magnetized water have enhanced seed performance in terms of plant height, number of branches, number of leaves, number of leaflets, root and shoot fresh weight, root and shoot dry weight, the total photosynthetic pigments (chlorophyll a, b, and carotenoids) and yield in some varieties of chickpea

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plants. For routine seed treatment in a nursery the use of permanent magnets is probably preferable (Sudsiri et al., 2016) but for experimental purposes an electromagnetic field is superior because it offers the opportunity to use a full range of field strengths to optimise responses and gain insights into the mode of action as well as offering a more uniform magnetic field. Oil palm is widely cropped in Thailand as an industrial plantation crop. It is also used in a wide variety of ways by the local population. Unfortunately, the germination of untreated oil palm seeds takes more than 8 months and germination is unreliable and the percentage rarely exceeds 60%. Heat treatment and scarification methods are used routinely in Oil Palm nurseries to improve germination and survival of Oil Palm (Myint et al., 2010). Germination difficulties are major complicating factors for oil palm agriculture and greatly increase the cost of young oil palm trees. Long germination times increases the risk of fungal infections killing the seeds or kernels. In this present work we applied a graded series of electromagnetic fields and MTW to stimulate the germination process of oil palm kernels in order to achieve a shorter germination time and a more reliable germination rate. In our previous paper we demonstrated that MTW, generated using permanent magnets and fixed magnetic field, was effective in stimulating germination of Oil Palm kernels (Sudsiri et al., 2016). In the present paper, we have used an electromagnetic apparatus using a DC current to stimulate germination of oil palm kernels which has allowed us to investigate the effects of a range of applied magnetic fields to optimise the germination rate. A directly applied magnetic field to seed kernels has been shown to be more effective than soaking in MTW and achieves a more rapid germination response and a higher growth rate of the seedlings. 2. Materials and methods 2.1. Plant materials Ripening oil palm (Elaeis guineensis Jacq., var. Suratthani 2) fruits harvested from a single crop were used as the experimental material. Oil palm seeds of nearly the same size and coloration were selected (Suppl. Fig. 1a). Their mesocarp was peeled to obtained oil palm seeds (Suppl. Fig. 1b). To obtain oil palm kernels, the seeds were left for 48 h at temperature 25 ◦ C prior to breaking the seed coats to obtain the kernels (Suppl. Fig. 1c), following standard Oil Palm nursery procedure in Thailand (Myint et al., 2010). Where magnetic activation methods are used to germinate Oil Palm seeds it is very unlikely that removal of seed coats to obtain kernels is actually necessary (Sudsiri et al., 2016). Kernels were treated with a standard fungicide (Metalaxyl, PATO Chemicals, Samut Prakarn, Thailand, 3 kg m−3 ) as previously described Sudsiri et al. (2016). 2.2. Selection of experimental material Oil palm kernels of similar weight and appearance (mean 0.661 g, range 0.55–0.76 g) were selected and divided into three groups. The dry weights of the kernels in the three groups were recorded. The first group of 50 dry kernels was designated the control. The second group was prepared to be directly exposed to the electromagnetic field without water. This group was designated Dry-Treated-Kernels (DTK). The kernels in the third group were immersed in distilled water during the magnetic treatment. This group was labelled as Magnetically-Treated-Water-Kernels (MTWK). The experiment was designed to have five varied magnetic field intensities at 2.5 mT, 5.0 mT, 7.0 mT, 9.0 mT, and 11.0 mT. For each magnetic field intensity, 5 periods of exposure times (1, 2, 3, 4 and 5 h) were conducted to find the optimum combination of field strength and exposure time (Phirke et al., 1996a,b). The ker-

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Table 1 Experimental Magnetic Treatment Protocol Arrays. Field Strength (mT)

Exposure Time 1h

Dry Treated Seed Kernels (DTK) D DTK1 2.5 5.0 D DTK6 7.0 D DTK11 9.0 D DTK16 11.0 D DTK21

2h

3h

4h

5h

D DTK2 D DTK7 D DTK22 D DTK17 D DTK22

D DTK3 D DTK8 D DTK23 D DTK18 D DTK23

D DTK4 D DTK9 D DTK24 D DTK19 D DTK24

D DTK5 D DTK10 D DTK15 D DTK20 D DTK25

D MTWK4 D MTWK9 D MTWK24 D MTWK19 D MTWK24

D MTWK5 D MTWK10 D MTWK15 D MTWK20 D MTWK25

Magnetically Treated Water Seed Kernels (MTWK) D MTWK1 D MTWK2 D MTWK3 2.5 5.0 D MTWK6 D MTWK7 D MTWK8 7.0 D MTWK11 D MTWK22 D MTWK23 9.0 D MTWK16 D MTWK17 D MTWK18 11.0 D MTWK21 D MTWK22 D MTWK23

nels in the second and the third group each were divided into 25 subgroups of 50 kernels each (see Table 1 protocol). To clarify the effects of different experimental variables, the following experimental protocol was designed. The magnetic treatment was provided as a known dose (D), varying the exposure time (t) and the magnetic field induction strength (M) with different magnetic induction values. The treatments in DTK group were labelled DDTK1 -DDTK25 whereas those exposed to a magnetic field while immersed in water were designated MTWK were marked as DMTWK1 -DMTWK25 (Table 1).

accomplished by applying DC voltage and calculated according to the Ohm’s law where the wire resistance was known. The calculated magnetic field strength was compared to actual experimental measurements using the Tesla meter. Kernels were exposed to electromagnetic fields in the reactor under two conditions, as dry kernels (Dry-Treated-Kernels, DTK) (Suppl. Fig. 2a) and as kernels immersed in water during exposure to the applied electromagnetic field (Magnetically-Treated-WaterKernels, MTWK) (Suppl. Fig. 2b). During the exposure, the temperature in the container was measured using a thermostat (see Suppl. Fig. 2b). To avoid an uneven electromagnetic field distribution exposure of the kernels, a manual regular stir of the kernels is needed periodically. At the end of the irradiance treatment, the kernels were then taken out and weighed as fresh weight. Similar procedures were conducted to the groups of kernels treated in an electromagnetic field immersed in water (MTWTK, DMTWK1 DMTWK25 ). Normal water was used to fill the seed container and would have been magnetized when the current was turned on. The kernels for the control group were immersed in normal water (NW) for 48 h and then their imbibed fresh weight was recorded. The two classes of kernels treated with the electromagnetic field were instead immersed in MTW for 48 h. Mean percent water content in the kernel was calculated as previously described (Sudsiri et al., 2016): %Wc

=

 Imbibed weight − Dry weight  Imbibed weight

× 100

(2)

where, %WC is the percentage water content. 2.3. Magnetic field generation and seed treatment 2.4. Modelling of water uptake Electromagnetic fields were generated using a laboratory workshop designed magnetic field generator able to vary magnetic fields. The electrical power was provided by a 220 V, 50 Hz DC power supply (30 V/10A) with variable voltages and currents. The magnetic field reactor for the seed treatments consisted of a steel tube cylindrical former with an outer diameter of 7.5 cm, inner diameter of 7.0 and 12.0 cm in length (Suppl. Fig. 2a). The tube was designed for treating 100 kernels at a time. It was wound with 1000 turns of 0.5 mm diameter insulated copper wire. The resistance of the winding was 38 ohm. The instrument was also designed such that it would withstand temperature rise even when operated for a long period of time. A thermocouple was used to monitor the temperature (Suppl. Fig. 2b). The apparatus generated an electric field comparable to the apparatus described by Moon and Chung (2000) and field strengths typically generated by experimental setups using permanent magnets (Phirke et al., 1996a,b; Flores et al., 2007; and see Sudsiri et al., 2016). A digital Tesla-meter (PHYWE No. 13610-93, Germany) operating on the principle of the Hall Effect was used to measure the strength of the induced magnetic field in the steel tube. The probe of the Teslameter was made of Indium Arsenide crystal and encapsulated to a non-magnetic sheet of 5 mm × 4 mm × 1 mm and could measure 0–2T full-scale. Oil palm kernels were exposed to a DC-generated constant magnetic field in the cylindrically shaped sample holder of 282 × 10−3 m3 capacity, made from a non-magnetic thin transparent plastic sheet (a plastic bottled water bottle is convenient). The ៝ circulating in the steel tube can be themagnetic flux density (B) oretically expressed in the following equation (Huang and Wang, 2007): N N B៝ =  i + o i l l

Water uptake vs. time was modelled using a logistic model described by Eq. (3) (Torres and Frutos, 1990; Garcia and Arza, 2001). Logistic growth parameters a (dimensions%W) and b (dimensions t−1 ) are constant parameters obtained from the fit to the logistic model. % Wt = a × (1 − exp(−b × t))

(3)

2.5. Preparation of magnetically treated water (MTW) The permanent magnet equipment for the preparation of irrigation water for the experiments was built according to Morejon et al. (2007) and is fully described and a diagram of the apparatus is included in our previous paper (Sudsiri et al., 2016). A cylindrical aluminium container with normal water was exposed to four isotropic permanent strontium magnets, arranged in a bipolar configuration (facing the magnetic poles). The intensity of the magnetic field in the container was 2.5–11.0 mT measured with a Tesla-meter (PHYWE No. 13610-93, Germany). The water used in the experiments was normal water (water + ions, pH of about 8.2 and electrical conductivity of ≈600 ␮S m−1 ). For the magnetic treatment, the container was filled with water and left for a routine period of 48 h (although 24 h is sufficient). Water in its magnetized state (Magnetically-Treated Water, MTW) had a conductivity of ≈1400 ␮S m−1 and remained magnetized for up to 130 h (Sudsiri et al., 2016). To check on whether the MTW was still in a magnetized state before watering kernels and seedlings its conductivity checked with a conductivity meter (Mettler Toledo No MC 126–2 M, Switzerland).

(1)

where, ␮ and ␮o are the magnetic permeability of the steel tube and air respectively, N is the number of turns of wire, l is the length of the tube and i is the current flowing through the wire. To obtain a desired magnetic field as in Eq. (1), the required current was

2.6. Kernel germination The kernels from all sub-groups were allowed to germinate on an absorbent sponge support (Fig. 1) in a germinating box at a room temperature of about 30 ◦ C in darkness in an incubator. The kernels

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where, Nt of the logistic curve is the total number of germinated kernels in the time t (%), Nmax is the number of germinated kernels obtained from the fit (%), b and k are exponential coefficients (Eq. (6)). To characterize the germination process, characteristic parameters were calculated: t1/2 is the time elapsed since the beginning of the experiment to the point of inflection of the logistic curve (t1/2 = b/k), N1/2 represents the number of kernels germinated at the logistic point of inflection of the curve, v max is the maximal rate of germination at the point of inflection of the logistic curve (vmax = Nmax k/4). 2.7. Young oil palm seedlings

Fig. 1. Labelling of germinating sponge tablet with numbers and with kernels randomly located to carry out the germination incubation. The numbers 1–5 referto the magnetic field exposure time (1–5 h, respectively). The controls are marked as number 0.

shown had met the germination criteria of ISTA (2009). The humidity on the watered sponge support in the germinating box was controlled to be not more than 80% because saturating humidities encourage fungal growth. Holes were cut in the sponge to accommodate the kernels. Each kernel was number marked according to exposure time (Fig. 1). The control kernels were watered with normal water (NW). To determine the effects of MTW and NW upon the two groups of magnetically treated kernels (MTWK and DTK) the kernels from each of these treatments (DMTWK1 -DMTWK25 and DDTK1 -DDTK25 ) were divided into 2 sub-groups each containing 25 kernels with different watering regimes. One pair of treatments was watered with NW, the other with MTW. Thus, the first sub-group was designated DMTWK1-NW -DMTWK25-NW and DDTK1-NW -DDTK25-NW and the second group of treatments DMTWK1-MTW -DMTWK25-MTW and DDTK1-MTW -DDTK25-MTW , respectively (see the protocol set out in Table 1). Germination test scoring was performed according to the rules issued by the International Seeds Testing Association (ISTA, 2009). Therefore, kernels were considered as germinated when the emerged seedling radicle was at least 2 mm long (as illustrated in Suppl. Figs. 3 and 4) (ISTA, 2009). The lengths of the radicles were measured daily and recorded as the seedlings grew over a period of 30 days after germination. The germination percentages, Nk (%) were determined according to Eq. (4) (Torres and Frutos, 1990). The germination rate, Skmax (% day−1 ), was calculated following Vashisth and Nagarajan (2010) as in Eq. (5). The germination experiments were run in 3 replicates. To fit the experimental points, a logistic relation (Eq. (6)) was applied. Nk =

nk × 100% nc

(4)

where, nk specifies the total number of germinated kernels of a given sample, nc the total number of kernels sown. Sk =

nmax t

(5)

where, n max represents the number of germinated kernel recorded during the counting for the time interval t between two countings. The time course of cumulative germination over time can be described by a logistic equation (Torres and Frutos, 1990; Sudsiri et al., 2016): Nt = Nmax [1 + exp(b − kt)]

−1

(6)

After the 30th day of the germination experiment, the sprouts of each group (both MTWK and DTK) were divided into 4 sub-groups and put out to grow in pots under natural environmental conditions in a shade house plant nursery (≈28 ± 3 ◦ C, humidity of about 65%). The light available was ≈110 ␮mol (quanta) m−2 s−1 PPFD (MQ200, Apogee Instruments, Logan, UT, USA). The first group from MTWK and DTK treatments was watered with magnetically treated water (MW) and the groups were designated MTWK-MW and DTK-MW, respectively. The other two groups from MTWK and DTK groups were watered with unmagnetised normal water (NW) and designated MTWK-NW and DTK-NW, respectively. All treatments were left to grow for 20 days. Heights of shoot of each treatment were recorded. The seedling growth, G over the germination time, t were fitted using a simple exponential growth model (Eq. (7)). G = a × exp(b × t)

(7)

where, a is the initial seedling length and should be quoted in SI units (mm) and b is a growth constant (day−1 ). 2.8. Statistical analyses Statistical analysis of the experimental data was performed with SPSS 11.5 for Windows software. The results were subjected to an analysis of variance (ANOVA) and Least Significant Difference (LSD) or Tukey test interval (TTI) to detect differences between mean parameters at the p < 0.05 level. Snedecor and Cochran (1989) was used as the standard statistical reference text. 3. Results 3.1. Water absorption The result of percent water imbibed in oil seeds (% Wc ) calculated according to Eq. (2) were plotted over absorption time (t) and are shown in Fig. 2(a and b). The experimental points were fitted using the logistic model described by Eq. (3). Logistic growth parameters a (dimensions %W) and b (dimensions t−1 ) are constant parameters obtained from the least square fit. Values of parameters a and b for all treatments are tabulated in Table 2. The parameters were plotted for each magnetic field treatment, including the controls given no magnetic field exposure. Table 1 shows the protocol for the germination experiments. From Fig. 2a and b, it is evident that water uptake rate in oil palm kernel increases as a result of exposure to a magnetic field, whether it is applied on Dry-Treated-Kernels (DTK) or on kernels immersed in water (Magnetically-Treated-Water-Kernels, MTWK). The fitted lines for Eq. (8) are shown on Fig. 2 (a and b) and the parameters a and b obtained from the fit are shown in Table 2. For both experimental treatments, the higher the electromagnetic dose the quicker the kernels hydrated and the higher the degree of hydration at the asymptote. Plot of the parameters a and b of the MTWK

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Fig. 2. The % increase in water content in oil palm kernels under the influence of magnetic fields varied from 2.5 mT to 11.0 mT for irradiance times of 1 h to 5 h for the group DMTWK1 -DMTWK30 (2a) and DDTK1 -DDTK30 (2b). Normal water (NW) was used to imbibe the kernels. Means and Error bars (standard deviation) are based on 3 replicates.

Table 2 Germination Parameters %Wmax and b obtained by fitting plots with Eq. (8) for the dry kernels exposed to an electromagnetic field not immersed in water (DTK-group) and those exposed to an electromagnetic field while immersed in water (MTWKgroup). The kernels were watered with normal water (NW). Kernels not exposed to any electromagnetic field or magnetically treated water had no germination at all during the course of the experiment (35d). They absorbed water (NW) but did not germinate. Magnetic field (mT)

0 2.5 5.0 7.0 9.0 11.0

DTK-group

MTWK-group

%Wmax

b (h−1 )

%Wmax

b (h−1 )

9.87 12.88 13.5 13.5 13.93 14.67

0.96 0.99 1.14 1.14 1.27 1.40

9.87 16.78 19.44 20.91 23.83 26.37

0.96 0.96 0.96 1.14 1.17 1.08

Fig. 3. Plots of average parameters a (Fig. 3a) and b (Fig. 3b) over a range of magnetic fields with fitted linear regressions for the exponential constant (b) vs. applied magnetic field.

with the applied magnetic field (Fig. 3b). The relations between the parameter a as function of magnetic field a(B) and parameter b(B) as function of magnetic field are shown in Eqs. (8) and (9) (Garcia and Arza, 2001).



⎡

1

(1+2 B2 ) 2+

RTL b(B) = co ⎣1 + Vo b(B) =

(8)

B

RTco 1 +



B 1 + 2 B2

RTL εL co be + Vo V0

⎤ 2+1

⎦ + εL

V0

(9)

(10)

where,

⎡

be = ⎣1 + and DTK groups vs. the applied magnetic field (Table 2) are shown in Fig. 3 (a and b). The parameter a (the asymptotic final% uptake of water) of the MTWK group (Fig. 5a) exponentially increases with the magnetic field, whereas parameter a of the DTK group (Fig. 3a) was constant at applied magnetic fields higher than 2.5 mT. For the DTK group the asymptotic% water uptake maximum was only about 15%, whereas the 11.0 mT treated MTWK group reached over 26%. For both groups, the exponential constant b linearly increased

P



a(B) =

⎤ 

B 1 + 2 B2

2+1

⎦

(11)

where, R is the universal gas constant (L mol K−1 ), T is the absolute temperature (K), L is the hydraulic conductance (m s−1 ), Vo is the initial water volume in kernels (L), B is the magnetic induction in Teslas (T), P is the variation of turgor pressure (Pa), ␮ is an “effective mobility (m2 V−1 s)” which includes all the effects of the magnetic field on permeability and conductivity of the cellu-

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lar membranes, co is the ionic concentration in the cytoplasm (M) when B = 0, and ␧ is the elastic modulus of the cellular membrane 1 (Pa). The exponent 2+ signifies that only the positive square root is relevant in Eqs. (8), (9) and (11). Plotting parameter b over magnetic field (Fig. 3b), and fitting the points with Eq. (5), the slope and the intercept of the regression line were obtained as follows: Index of correlation: r2 = 0.978 Slope: RTLco /Vo = 0.017 (kg m−2 s−2 ) (DTK group) and 0.011 (MTWK group) Intercept: ␧L/Vo = 0.93 (kg m−3 s−1 ) (DTK group) and 0.92 (MTWK group) Unfortunately, it is impossible to establish an analogous correlation analysis for the asymptotic relative water uptake (parameter a) shown in Fig. 3a and the applied magnetic field of treated kernels (B) using Eq. (9). The reason is that P is not constant for different values of B. Therefore, it is clear that the rate of water uptake not only increased, but the total mass of absorbed water and the turgor pressure within the kernels would also have increased. From the definition of a as the quotient of P, we should expect that it changes significantly with increments in B. Our experiments showed that a dramatically increased with the applied magnetic field (B) for the MTWK group but was almost constant for the DTK group. Nevertheless, it is important to note that electromagnetic treatment of dry kernels did have very significant effects on water uptake compared to the controls. Control kernels took up very little water (Fig. 2 (a and b), <10%) as found in our previous paper (Sudsiri et al., 2016).

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3.3. Growth of young oil palm seedlings Fig. 6 shows the maximum height of shoots for each treatment. The two parameters obtained by fitting the experimental points (Fig. 6) to Eq. (7) are shown in Table 3. The parameter b (exponential growth constant) had a value of about 0.06 day−1 for all treatments. However, different values of parameter a were obtained for varied field intensities. The maximum value of 22.3 mm (2.23 cm) was found in the treatment MTWK-MW. This behaviour indicates that different magnetic field strength treatments treatment does not significantly change the growth rate of young oil palm seedlings, but does influence seed germination. Thus the main effect of magnetic field treatment is upon germination rate not growth rate. Suppl. Fig. 8 is a set of photographs of oil palm seedlings at different nursing times. Initial oil palm sprout at the first day of the nursing phase of the experiment is shown in Suppl. Fig. 8(b). First leaf of the young seedlings were observed on the 10th day of the experiment as in Suppl. Fig. 8(c), and on the 20th day, the second leaves of the seedlings were seen as in Suppl. Fig. 6(d). During the course of the entire 30 day experiment no development in the kernels was observed for the control group in Suppl. Fig. 8(a). These results show visually that oil palm seed germination is stimulated by magnetic field treatment.

4. Discussion 4.1. Water absorption

3.2. Early growth The experiment showed that DC-generated constant magnetic fields significantly improved oil palm germination related characters such as germination percentage and speed of germination (Suppl. Figs. 5–7), and for experimental purposes are more versatile than using permanent magnets (Sudsiri et al., 2016). The results indicated that the two magnetic treatments for both experimental groups (DTK and MTWK) strongly improve germination percentage and germination speed whereas no development of any of the germination-related characters was found in the control group. Germination percentages of all treatments for both experimental groups are illustrated in Fig. 4 (a1–a5) and 4 (b1–b5) with nonlinear least squares fitted curves to the logistic absorption model (Eq. (6)). The maximum rate of germination of the kernels (max ) (germinations per day) can be calculated from the curve fits or estimated graphically (Suppl. Fig. 7). Photographs of germinating kernels at 7 and 30 days are shown in Suppl. Figs. 3 and 4. The controls showed zero germination even after 30 days (Suppl. Fig. 8a). For MTWK kernels, a 4 h electromagnetic treatment in a 200 mT field shows the most augmentation. The earliest germination of both groups (MTWK and DTK) were observed on the 5th day after experimental treatment with a germination percentage of 12% (P < 0.05) for TW group and 10% (P < 0.05) for the DTK group. The other treatments however exhibited signs of early growth on the 10th day after treatment. On the 30th day of the germination experiment, maximum germination percentages of all magnetic treatments was consistently observed in kernels given a 4 h exposing time. The highest value was obtained in 200 mT with 96.1% germination (P < 0.05) and 86.6% germination (P < 0.05) for MTWK and DTK groups, respectively. Comparison of maximum percent germination under different magnetic fields vs. time for the DTk group is illustrated in Fig. 5. Maximum numbers of both groups were found at magnetic field of 9 mT for 4 h with values of 3 kernels/day and 2 kernels/day for TW and DTK group, respectively.

Dependence between the time courses of water absorption by kernels (%) at different magnetic field irradiances are shown in Fig. 2. The results showed that the water uptake mechanism in oil palm kernel is altered by application of a magnetic field. Experimental evidence shows that when normal water is exposed to magnetic field, some of its readily measureable properties are changed, such as conductivity, surface tension, solubility of salt, refractive index and pH (Smikhina, 1981; Yakovlev and Kolobenkov, 1976). Unfortunately, the lack of a coherent theory to explain how water changes its properties when exposed to a magnetic field hampers the development of hypotheses about the possible mechanism of action of magnetic fields in biological systems (Belyavskaya, 2004; Galland and Pazur, 2005). Experimental experience has led to the development of a hypothesis about the mechanism which attempts to explain the effects of electromagnetic fields on the biological systems. The explanation has been based on the possible impact of magnetic fields on the permeability of ion and aquaporin channels in biological membranes (Maurel et al., 2008), in which, at least, three factors are involved: the water conductance (L) of the seed coat; dry matter; plasma membrane and tonoplast membranes; turgor pressure; P of inner water and the osmotic pressure, ␲ of solutes. Later, Reina and Pascual (2001) proposed that membrane properties such as membrane permeability, P, the ionic current density (J), and ionic concentration (c) within membranes are a function of the magnetic field environment as in the case effective mobility in aqueous media. They suggested that the magnetic field enhances water uptake into the cell and the magnetic field interacts with ionic currents in the embryo cell membrane which consequently induces changes in the ionic concentrations and the increase osmotic pressure insides of the membrane, and therefore force more water flows into the cell. Reina et al. (2001) showed that the increase in the rate of water uptake in the seed leads to a higher total mass of absorbed water. Increasing ␲ and water uptake would stimulate the germination rate of the cell however its behaviour starts only if a strong influence is exerted on water inside the kernels (Cosgrove, 1993). He suggested that ionic solutes passively leak from the cell into cell

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Table 3 Seedling growth parameters obtained by fitting growth data to an exponential growth relation. The parameters a and b initial sprout height (mm) and mean growth constant (d−1 ), respectively. Treatment

Magnetic field (mT) 2.5

5.0

7.0

9.0

11.0

Parameters

a

b

a

b

a

b

a

b

a

b

DTK-NW DTK-MW MTWK-NW MTWK-MW

9.6 11.2 11.7 13.3

0.061 0.063 0.064 0.066

11.2 14.2 15.9 19.4

0.055 0.063 0.062 0.058

11.5 12.0 13.4 15.4

0.066 0.070 0.069 0.07

15.4 16.7 17.6 22.3

0.063 0.065 0.069 0.061

16.4 18.7 19.3 20.4

0.063 0.060 0.062 0.063

wall region and, since the volume of cell wall is smaller than the volume of the cell volume, ␲ is increased in that increase volume of the cell wall until reaching equilibrium. This mechanism is thought to

be the principal one in the embryo cells that controls water uptake, due to the high density of ionic channels in the plasma membrane of the cells (Tyerman, 1992; Maurel et al., 2008).

Fig. 4. Percent germination of oil palm kernels magnetically pre-treated using magnetic field strengths of 2.5–11.0 mT for 1–5 h of MTWK group (4a1–6a5) and DTK group (4b1-4b5). The curves are fitted to a logistic model (Eq. (6)) using non-linear least squares methods. Suppl. Figs. 5 and 6 show detailed response surfaces for the effects of magnetic field strength and exposure time on germination percentages over time.

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Fig. 4. (Continued)

The agreement of the experimental results with the theoretical model (Reina and Pascual, 2001) gives strong evidence that the magnetic field interacts with water uptake in embryo cells. The observed variation of more than 40% in parameter b of Eqs. (8) and (9) (Table 2) with a change in magnetic field from 0 to 11 mT establishes that the link of magnetic field with osmoregulation is very strong. The logistic growth parameter (b) for both DTK and TW groups was linearly related to magnetic field (Fig. 5b) but different values of the slope of b were obtained. A value of 0.017 and 0.011 was found for the DTK and MTWK groups, respectively. The difference suggests kernels in the DTK group (no water added during magnetic treatment) absorb water into the wall space faster than that of MTWK group. In the dry state the plasma membrane would contain a very little water but a high ionic concentrations and so

the water potential gradient would be larger (Vertucci and Ross, 1990). In the case of the MTWK group, the kernels were immersed in distilled water during electromagnetic treatment during which water would have flowed into the cells across the cells membranes and ionic concentrations would have decreased. The parameter a of Eq. (8) is the asymptotic uptake of water by the imbibed kernels and so is the point at which the osmotic water potential between the inner and outer sides of the cell membrane is in equilibrium. The logistic growth parameter (a) of the MTWK group increased linearly with the strength of the applied magnetic field. To describe this behaviour, only ionic concentration and P can be taken into consideration (see Eqs. (9)–(11)). During magnetic treatment, the magnetic field may alter P to enhance water flux across the cell membrane and hence reduce the ionic concentration gradient. For

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Fig. 5. Comparison of maximum percent germination obtained from the DTK group.

the DTK group, however, there was no alteration of P during the treatment; water flowed through the membrane only under the influence of osmotic pressure caused by ionic concentration in cell membrane. The logistic growth parameter (a) is constant for all magnetic fields. The increment in water uptake rate is the reason that magnetic treated kernels of TW group germinate faster than DTK group, while in control samples there is little increment in water uptake. Dahal et al. (1997) and Chen and Bradford (1999) proposed that the effect of applied magnetic fields on germination occurs only if there is a strong influence on water inside the kernels. In addition, magnetic field may induce other effects such as a weakening in the cell wall that makes the radical emergence easier. Germination of some types of seeds commonly fails at the very last stages of the germination process where the radicle fails to emerge. 4.2. Early growth Data presented in Fig. 4 (a1–a5 and b1–b5) show that the percent germination of oil palm kernels rises linearly with their exposure time (dose) in the magnetic field until 4 h of magnetic treatment and drops off in kernels given 5 h treatment. The maximum percent germinations for both groups were found in kernels exposed to a magnetic field of 9.0 mT. The result is correlated to the work of (Reina et al., 2001) who observed an increase for the initial growth stages and an early sprouting of Lettuce seeds exposed to a 0–10 mT stationary magnetic field. The comparison of the effect of the magnetic field treatment on soaked kernels (MTWK) during the treatment and non-soaked kernels (DTK) showed that the presence of water (TW) led to the increase of the percentage germination. The maximum percent germination for the DTK group at the 30th day of the experiment using different exposures to a magnetic field (magnetic dose) is shown in Fig. 5. The percentage germination vs. magnetic dose for the TW group was about 10% higher than for the DTK group. This agrees with the germination speed data shown in Suppl. Fig. 7 (a and b). 4.3. Growth of young seedlings Young oil palm seedlings for all magnetic treatments grew exponentially after germination (Fig. 6a–e). The experimental points were fitted to Eq. (12). The calculated parameters ag and bg are shown in Table 3. The germination rate, ag of about 0.06 day−1 was obtained for all treatments (4 groups), effectively the same result as found in our previous study (Sudsiri et al., 2016). The result is agree very well with the work of Matwijczuk et al. (2012) which investigated the effect of magnetic field on sunflower seed who also found

that the germination rate of magnetically treated sunflower seeds was ≈0.06 d−1 . In the present study, and our previous study (Sudsiri et al., 2016) we found that germinated oil palm seedlings once they had germinated grew with the same rate constant whether or not they were watered by magnetically treated water (MTW) or even normal water (NW). The finding that applied magnetic fields stimulated germination in dry kernels shows that an applied magnetic field plays an important role only on the very first stages of seed germination. Not even imbibition of external water is necessary. However, the electromagnetic effect is greater on kernels immersed in water during irradiation (MTWK) and the highest germination rate was found in kernels irradiated in water and then imbibed with magnetically treated water (MTWK-MTW) group. Therefore, the response depends not only on the magnetic induction but also on the physiological state of experimental organism as proposed by Smith et al. (1992). This can be seen from different growth rates found in different growth condition (such as immersed seeds or kernels in water) in response to magnetic treatment. Higashitani et al. (1996) found that a static magnetic effect caused an increase in the ordered structure of water formed around hydrophobic molecules and colloids. Later, Kitazawa et al. (2001) reported that such fields can also increase the evaporation rate of water and the dissolution rate of oxygen. Madsen (2004) found an increase in proton spin relaxation which may speed up some reactions dependent on proton transfer and dissolving properties of water increase when treated with magnetic field. In addition, when the young oil palm trees are planted in pots of soil watering using magnetically treated (MTW) water leads the young oil palm trees to grow faster than if watered using normal water. Plants and trees need mineral salts and microelements from the soil to function and photosynthesize properly but much of the essential elements present in soils are in bound forms to organic matter and clay particles. When watering plants with normal water, only small amounts of bound plant nutrients are actually available to plants. MTW probably has its observed positive effects as a result of mobilization of otherwise not readily available plant nutrients. This results in an increased crop production and in an increased quality of agricultural products. Our results are in agreement with those reported by Yinan et al. (2005) who observed an increase on rate of germination of cucumber seeds (Cucumis sativus L.) and basil seeds (Ocimun basilicum L.) (Soltani et al., 2006a,b) exposed to a magnetic field. Application of an external magnetic field as a pre-germination treatment improved the germination and seedling vigour of low viability rice and onion seeds (Alexander and Doijode, 1995), chickpeas (Nawroz and Hero, 2010) and sunflowers (Vashisth and Nagarajan, 2010). Garcia and Arza (2001) observed that lettuce seeds (Lactuca sativa L.) previously treated in a stationary magnetic field of 1–10 mT germinated earlier than the untreated, and proposed that it could be due to an increase in water uptake rate. In addition, Podlesny et al. (2004) published the positive effect of magnetic treatment on the germination and emergence of two broad bean cultivars. 4.4. Mechanism of action The above observations can be explained by assuming that some organelles of plant cells (i.e. mitochondria) possess paramagnetic properties as found in chloroplasts by Commoner et al. (1956). This is consistent with findings that mitochondria are sensitive to magnetic fields (Belyavskaya, 2004). This should not be surprising because both mitochondria and chloroplasts have electron transport chains imbedded in their membranes generating an electrochemical potential gradient for protons across the membranes. Commoner et al. (1956) suggested that metabolically active tissues of plant cells contain free radicals and they play an important role in electron transfer and in the kinetics of the chemical reactions.

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Fig. 6. Growth of young oil palm trees which were exposed to magnetic fields at varied field intensities of 2.5 mT–11.0 mT (a–e, arranged left to right). For each field intensity, maximum height of shoot obtained from the TW-MW group are compared in (f).

These free radicals possess non-paired electrons with magnetic moments that can be oriented in the outer magnetic field. As a result of the interaction between the outer magnetic field and the magnetic moment of unpaired electrons microwave energy is absorbed (Commoner et al., 1954). This energy is later transformed into chemical form and accelerates the activation of germination processes in the seeds. In our study, the effect of the magnetic field treatment was stronger for preliminary soaked kernels. This observation could be due to the fact, that the water molecule also possesses paramagnetic properties and absorbs the energy of the magnetic field. This energy is also transformed into chemical form

and is an additional amount to that absorbed by the free radicals, existing in plant tissues of non-soaked kernels. The above reasoning seems to explain the effect of magnetic fields on seed germination where water plays an important role, but currently there is no complete and uniform theory to explain the effects of magnetic fields on the properties of water. Aladjadjiyan and Ylieva (2003) proposed a hypothetical mechanism for this phenomenon, which attempted to explain the biological effects of electromagnetic fields. It is based on their possible impact on the permeability of ion channels in the membrane, which may affect the transport of ions into the cells and lead to biological

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changes in the organism. Similar proposals have been made for a role of ferretin in activating membrane channels (Galland and Pazur, 2005). Variable magnetic fields may affect the biological functions of organisms through changes of hormone concentrations, changes of enzyme functions or of transport of ions through the cell membrane and through changes in DNA synthesis or transmission (Strasak et al., 2002). In addition, if we are dealing with electrical anisotropy of the structure of plant tissues and cells, the magnetic field may affect the working of ion pumps for the transport of Ca2+ ions (Piacentini et al., 2001). Garcia and Arza (2001) offered a hypothesis along similar lines. In a study on the effects on water absorption by lettuce seeds in a stationary magnetic field of 1–10 mT, they found an applied magnetic field increased water uptake into the cells. It seems that changes in intracellular levels of Ca2+ and ionic current density across the cell membrane causes alteration in the osmotic pressure gradient and changes in capacity of cellular tissues to absorb water. From changes in the Ca2+ contents in cells of plants such as pea flax, lentil, onion and radish seedlings exposed to weak magnetic field, Walleczeh and Budinger (1992) concluded that magnetic fields cause effects related to interference with cytoplasm ion current or ion distribution. From such observations, applied magnetic fields seem to target the signalling system of plant cells involving Ca2+ ions. This is fully consistent with the assumptions of the parametrical resonance model (Belyavskaya, 2001; Belyavskaya, 2004). Interference with the role of Ca2+ in cell signalling mechanisms is strongly suspected to be a key effect of magnetic fields on organism (Walleczeh and Budinger, 1992; Belyavskaya, 2004). The other strong candidate is the Fe-containing compound ferretin which until recently was not thought to be present in vascular plants but has now been identified in plants (Galland and Pazur, 2005).

6. Young oil palm trees grow exponentially with time with the growth rate of about 0.06/d which agrees well with our previous work (Sudsiri et al., 2016). 7. Young oil palm trees irrigated by magnetically treated water (MTW) grew faster than those watered using normal water. This is an improvement over simply using MTW as the magnetic treatment in our previous study (Sudsiri et al., 2016). 8. The work provides a good method to enhance oil palm germination which usually takes about 6 months to 1 year to reach the spouting stage, but with magnetic treatment, it takes only about 1 week. A mechanized electromagnetic treatment device for treating large numbers of oil palm seeds or kernels appears to be feasible. 9. Magnetic induction of seeds is likely to be important in bioresource conservation because the seeds of many endangered plants have low viability or the natural environmental cues for germination are not known and old seed stocks in Seed Banks need to be revived. Magnetic induction is non-destructive whereas scarification and other means of stimulating germination may kill seeds if not performed properly. Specifically, magnetic induction has been successfully used on low viability seeds of aged low viability onion (Allium cepa L.) and rice (Oryza sativa L.) seeds (Alexander and Doijode, 1995), Pinus tropicalis (Morejon et al., 2007) and our studies of Oil palm in this and another study (Sudsiri et al., 2016). A non-destructive method for germinating difficult-to-germinate seeds is of great importance to horticulture.

5. Conclusions

Appendix A. Supplementary data

1. Oil palm kernels treated with an electromagnetically applied magnetic field germinate in about 1 week after the experimental treatment. No germination of control treated kernels was observed over the course of the experiment (≈30 d). This is a great improvement on the usual 8 month germination time for Oil palm kernels. 2. An adjustable electromagnetic field apparatus offers the advantage over a static permanent magnet setup (Sudsiri et al., 2016) that the applied magnetic field can be optimally adjusted for different types of kernels and is more suitable for experimental studies on the mechanism of action of magnetic fields on seeds and kernels. With an adjustable magnetic field response surfaces such as those shown in Suppl. Figs. 3 and 4 can be easily measured. 3. Maximum percent germination obtained was about 90% if the kernels were immersed in water during the magnetic field treatment. Germination rates were better with the electrically generated magnetic field than previously found for permanent magnets. This is most likely due to the lack of “dead spaces” in the electromagnetic field apparatus (Fig. 2a,b) compared to a field generated by permanent magnets (Sudsiri et al., 2016). 4. Comparison of dry magnetically treated kernels (DTK) with kernels magnetically treated in water (TW) show that imbibition of water is not necessary for the magnetic triggering of germination of the oil palm kernels although the germination success rate is lower. Intrinsic water in dry kernels is all that is needed for a magnetic treatment to stimulate germination. 5. The best magnetic parameters to stimulate oil palm germination are at an applied magnetic field of strength of 9.0 mT for 4 h, immersed in water.

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.scienta.2017. 03.036.

Acknowledgement The senior author wishes to thank Prince of Songkla University (Surat-thani Campus) for partially funding the project.

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