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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

In the present study, the seeds of brinjal (Solanum melongena L.) var. Mattu Gulla were irradiated with single exposure of He–Ne laser at different doses of 5–40 J cm−2 and germinated aseptically. Thirty day old seedlings were harvested and the germination, growth, physiological and biochemical parameters were estimated and compared with un-irradiated control seedlings. A significant enhancement in growth characters were noted with respect to length, fresh and dry weight of shoots and roots. In addition, chlorophyll (a and b), carotenoid content, anthocyanin and amylases (α and β) activities were found to be altered. Significant alterations in percentage of seed germination (< 0.001) and time to 50% germination (< 0.001) were observed in the irradiated seeds compared with the un-irradiated controls. In conclusion, the results of the present study demonstrated that low dose (5–30 J cm−2) of He–Ne laser irradiation enhanced the germination process and altered growth, by positively influencing physiological and biochemical parameters of the brinjal seedlings compared with un-irradiated control under in vitro conditions.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The imperative needs of agricultural products and fresh vegetables from different ecological zones for food production as well as different food industry requires probing innovative and safer methods to boost the agricultural productivity to meet the demand of increasing human population (1). Use of the chemical additives and other methods currently used for improving plant productivity have been changing the properties of soil, water and atmosphere which in turn led to unfavorable conditions for growth and development of plants. The impact of physical methods in increasing morphological, physiological and agronomical characters is well documented since 1950. In recent years, the biostimulatory role of physical methods have been observed in different agronomical characters of major crop plants and is being used in single or combinations of physical factors including physical and chemical mutagens (2). Among these, the influence of mutagens is well documented on the improvement of major crop yield, quality, disease and pest resistance. These produced several germplasms with novel and desired traits (3), and after characterization, are released worldwide for crop plants (4). Wilde et al. (5) and Paleg and Aspinall (6) were first to report the use of Ruby and He–Ne laser in agriculture for rapid seed germination and growth of the plants through activation of phytochrome system (6). Several studies have demonstrated the potential, significant and positive impact of lasers on major crop plants. Recently, the use of lasers in agriculture is reviewed by Hernandez et al. (7), Aladjadjiyan (8) and Vasilevski (9). Potential beneficial effects were observed in number of parameters including seed germination, growth, biochemical compositions, enzyme activities, fruit size, resistance to pathogenic microbes and stress resistance/tolerance (10–22). The biostimulatory effects of laser irradiation also have shown to improve the growth of Isatis indigotica seedlings and protect the seedlings from enhanced UV-B damage (15,23–25), cold stress (26) and enhanced water tolerance (27).

Brinjal (Solanum melongena L.) is an economically important vegetable crop which provides carbohydrates, protein, minerals and vitamins for human health. It is a nontuberous perennial Solanaceous plant cultivated as an annual crop. Solanaceous vegetables such as eggplant, pepper and tomato have higher phenolic content (phytophenols) with high free radical scavenging properties (28). Eggplant also has several health benefits especially in decreasing the effects of cholesterol of selected diet with being the source of fiber (29). The brinjal fruit is used in the diet to manage type 2 diabetes as per the guidelines and recommendations of National Diabetes Education program of National Institute of Health, USA (30). Recently, Kwon et al. (31) have reported the significant inhibitory activity of phenolic enriched extracts of brinjal on α-glucosidase and angiotensin I-converting enzyme. Besides the fruits, roots of brinjal plant also have antiasthmatic properties and leaves are used externally for the treatment of burns, cold sores and abscesses (32).

Different varieties of brinjal are cultivated throughout India, whose fruits are either elongated or round, violet/pink/white/green hues. The brinjal variety found in Mattu village, vernacularly called Mattu Gulla (MG) in Udupi District, Karnataka, India, is round and green in color. Mattu village is known for this unique variety of brinjal (33) which is cultivated once in a year after monsoon for 500 years (34). This brinjal variety is susceptible to enormous biotic (fungal, insect pests and nematode infection especially the root lesion) and abiotic (drought, heat and water logging) stresses that adversely affect the normal growth and development which lead to huge reduction in the crop yield.

The stimulatory role of laser irradiation on germination percentage, growth, development and yield characters are reported for several crop plants in field conditions. However, no previous literatures are available on influence of laser irradiation in seed germination in vitro and its effect on further growth and development. The present study was aimed to examine the effects of He–Ne laser irradiated seeds of brinjal (Solanum melongena L.) var. Mattu Gulla on in vitro germination, growth, physiological and biochemical characters.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Selection of seeds and laser treatment.  The seeds of brinjal [Solanum melongena L] var. Mattu Gulla (MG) were collected from local farmers and also from ripe fruits of brinjal from Mattu Village, Udupi District, Karnataka, India. The healthy and uniform seeds were selected and soaked in distilled water for 3 h, and the extra moisture content of the seeds was removed by filter paper prior to laser exposure. The laser delivery setup used for the present study which is capable of providing uniform laser power over the entire target area was described in detail elsewhere (35,36). Each experimental group consisted of 20 seeds spread over 15 × 15 mm circular area. The treatment groups were applied with the single exposures of preassigned laser doses (i.e. 5, 10, 15, 20, 25, 30, 35 and 40 J cm−2) using 7 mW He–Ne laser (632.8 nm), with a power density of 4.02 mW cm−2. The details of the source and other experimental conditions are listed in Table 1. The laser irradiation was carried out after 15 minutes of the laser warm-up time to avoid any possible error in the laser power stability. The laser power was monitored before and after each exposure using a laser power meter (Gentec, Canada) to ensure proper energy delivery to the target site. The laser beam was carried via an optical fiber of core diameter 200 μm (transmittance ≥90%; Ocean Optics) and coupled to a beam expander (2.5X-15X; CVI Melles Griot) held at a constant distance of 20 mm above the surface of the seeds (noncontact mode) providing a laser spot size of diameter 15 mm using a focusing lens. Before each experiment, it was confirmed that the laser beam was spread out uniformly over the entire target area. An equal number of seeds was used as un-irradiated control.

Table 1.   Laser source and conditions used to irradiate seeds of brinjal.
VariableExperimental conditions
LaserHelium–Neon (632.8 nm)
Wave emissionContinuous (CW)
Power and power density7 mW; 4.02 mW cm−2
Duration of the irradiation21 min 15 s–5 J cm−2
42 min 30 s–10 J cm−2
63 min 45 s–15 J cm−2
85 min–20 J cm−2
106 min 15 s–25 J cm−2
127 min 30 s–30 J cm−2
148 min 45 s–35 J cm−2
170 min–40 J cm−2
No. irradiationSingle
Laser spot size at surface of seeds15 mm
Distance from the sample20 mm
Room temperature22 ± 1°C
Laser polarizationLinear

Surface sterilization and in vitro germination of seeds.  Immediately after the laser irradiation, the seeds were surface sterilized separately for each experimental group including control. For surface sterilization, the seeds were soaked in soap solution (with 2–3 drops of Tween 20) for 5 min, followed by 70% ethanol treatment for 1 min, sterilized using 0.1% (w/v) mercuric chloride for 5 min and finally five rinses with sterile distilled water. The MS basal medium (macro and micro nutrients, iron source and vitamins) was prepared with 0.8% (w/v) agar and adjusted to pH 5.8 and autoclaved for 15 min at 121°C. Surface sterilized seeds were germinated aseptically on MS (37) basal medium in culture bottles (6.5 × 11cm), incubated at 25 ± 2°C for 24 h in dark. After 24 h, the culture bottles were exposed to light under cool-white fluorescent tubes providing irradiance of 40 μmol m−2 s−1 during a 16-h photoperiod for germination.

Determination of germination growth characters.  Percentage of germination. The germination percentage of seeds for all the groups was observed and noted on every alternate day for 10 days. The percentage germination was calculated by number of seeds germinated/total number of seeds inoculated ×100. To calculate the days required for 50% germination of seeds, germination was recorded every day for different treatments, the values were noted and calculated according to Coolbear et al. (38) and modified by Farooq et al. (39).

Length of shoot and root measurements. The 30 day old seedlings were removed carefully from the culture bottles and the roots were washed with tap water to remove the agar. The excess moisture was removed by toweling with blotting paper. The length of the shoot and root (cm) was measured from shoot/root junction to the tip of the shoot and main root respectively from ten individual seedlings of each treatment group.

Fresh and dry weight. Ten seedlings were selected randomly and the fresh weight of the shoots and roots were measured separately using electronic balance and the values were noted. After measuring the fresh weight, the shoots and roots were dried in a hot air oven at 60°C for 48 h and the dry weight was calculated.

Number of leaves and roots. Number of leaves (excluding cotyledons) and the main and lateral roots were counted from 10 seedlings of each experimental group.

Seedling vigor index (SVI). The seedling vigor index was calculated by seedling length (cm) × germination percentage according to Orchard (40).

Determination of physiological and biochemical characters.  One gram of shoot and root of 30 day old seedlings were homogenized separately for the physiological and biochemical analyses.

Chlorophyll and carotenoid content. One hundred milligrams of leaves from each treatment were homogenized individually with 10 mL of 80% acetone and the homogenate was centrifuged at 956 g for 3 min. The supernatant was saved and the pellet was extracted twice with 5 mL of 80% acetone. All the supernatants were pooled and saved, and the total chlorophyll content (chlorophyll a and chlorophyll b) was calculated by taking absorbance at 645 and 663 nm against the blank according to method of Arnon (41). The carotenoid content was calculated using absorbance value at 473 nm according to Goodwin (42).

Anthocyanin content. One gram of leaves was homogenized with 20 mL of ethanol in 2% HCl and the homogenates were centrifuged at 956 g for 5 min. The supernatant was saved and the absorbance was measured at 529 nm and anthocyanin content was was calculated according to Caldwell (43).

Activity of amylases. Five hundred milligrams of leaves were ground in chilled pestle and mortar using sterile distilled water, centrifuged at 2655 g for 5 min at 4°C, and the supernatant was used as enzyme source to estimate the α and β-amylase activity.

α-Amylase. Each sample was incubated in water bath at 70°C for 5 min to inactivate β-amylase. To 1 mL of the extract, 1 mL of 0.1 M citrate buffer (pH 5.0) and 0.5 mL of 2% soluble starch was added and incubated at 30°C for 10 min. 2 mL of dinitrosalicylic acid reagent was added, incubated again in boiling water bath for 5 min. After cooling, the enzyme reaction was made up to 10 mL with sterile distilled water and the absorbance was measured at 540 nm according to Dure (44).

β-Amylase. The enzymatic reaction mixture consisted of 1 mL of enzyme extract, 1 mL of 0.1 M citrate buffer (pH 3.4), 2 mL of 2% starch and this was incubated at 30°C for 10 min. Two milliliters of dinitrosalicylic acid reagent was added to the reaction mixture and incubated again in boiling water bath for 5 min. After cooling, the enzyme reaction was made up to 10 mL with sterile distilled water and the absorbance was measured at 540 nm according to Dure (44). The activity of both the amylase enzymes were expressed as μg of maltose hydrolyzed g−1 FW h−1.

Proteases. One gram of plant was homogenized in prechilled pestle and mortar with 8 mL of 0.1 M sodium phosphate buffer (pH 6.5) in ice and centrifuged at 10 621 g for 25 min. The supernatant was collected and made up to 10 mL with same buffer and used as source of protease enzyme according to Kar and Mishra (45). The reaction mixture for measuring protease activity consisted of 1 mL of enzyme extract, 0.1 mL of 0.1 M magnesium sulphate and 1 mL of BSA (50 μg mL−1) and incubated for 1 h at 37°C. The reaction was terminated by the addition of 1 mL of 50% TCA according to method described by Snell and Snell (46) and modified by Biswas and Choudhuri (47). The residual protein was estimated following the method of Bradford (48) and the activity of the enzyme was expressed as μg of BSA hydrolyzed g−1 FW h−1.

Total protein. Five hundred milligrams of shoot and root were homogenized in prechilled mortar and pestle with 10% ice-cold TCA and incubated overnight at 4°C. The homogenates were centrifuged at 10 621 g for 10 min and the pellet was washed with 100% acetone to remove the pigments from the pellet. The pellet was washed further with 80% ethanol followed by ethanol/chloroform (3:1 v/v), ethanol/diethyl ether (3:1 v/v) and finally with diethyl ether to remove the phenolic compounds according to method described by Damerval et al. (49). The pellet was dissolved in known volume of 0.1 N NaOH and the concentration was estimated following the method of Bradford (48) with reference to BSA as standard.

Total soluble sugars. Fresh shoot and roots (500 mg) were ground thoroughly with 5 mL of 80% methanol using mortar and pestle. The homogenate was centrifuged at 2655 g for 10 min and the supernatant was saved. The pellet was re-extracted once with addition of 5 mL of 80% solvent and the supernatants were combined. The chlorophyll content in supernatant was removed by the addition of equal volume of petroleum ether in a separating funnel and the methanol phase was taken for the determination of total soluble sugar according to Dubois et al. (50). The reaction mixture consisted of 0.5 mL of sample, 1 mL of 5% phenol, 5 mL concentrated H2SO4 and 8.5 mL distilled water. The reaction mixture was incubated at room temperature for 15 min and the brown reduction product was measured at 490 nm using ELISA Plate reader (Infinite M200, Tecan, Austria) and calculated with reference to glucose as standard.

Statistical analysis.  Each treatment consisted of 20 seeds and each experiment was conducted in triplicate. The data were presented as mean ± SD and the statistical significance among un-treated control and laser treatment group was determined using one-way anova with Bonferroni’s test for multiple comparisons using biostatistics software GraphPAD Prism 4 (Graph-Pad Software, Inc.). The level of < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Germination and growth parameters

The data for percentage of germination and days to 50% germination is shown Figs. 1 and 2 respectively. It is evident that each laser dose has influence on in vitro germination and time to 50% germination of seeds. The percentage of germination was found to be increased over control from 5 J cm−2 gradually, reached maximum percentage of germination at 30 J cm−2 and thereafter decreased trend was observed at 35 and 40 J cm−2. Lowest seed germination (49.3 ± 3.5%) was recorded from un-irradiated control whereas single exposure of 30 J cm−2 resulted in 82.6 ± 4.5% of germination. The increased percentage of germination at 30 and 35 J cm−2 was found to be significant (< 0.001) compared with un-irradiated control. Though the decreased percentage of germination was noted in 35 and 40 J cm−2, it was 13.7% higher than un-irradiated controls. The time to 50% germination also showed similar trend with percentage of germination (Fig. 2). The control set of seed needed 17.6 ± 1.52 days for 50% germination, whereas it was reduced by 4.6 days (13.0 ± 1 days) at 5 J cm−2 and gradual reduction in time was noted at 10, 15, 20 and 25 J cm−2 and reached minimum of 6.6 ± 0.57 days for 50% germination at 30 J cm−2. The time to 50% germination was recorded as 15.3 ± 3.05 and 25.6 ± 2.08 days for 35 and 40 J cm−2 respectively. Reduction in time for 50% germination for laser treatment group (20, 25 and 30 J cm−2) was found to be significant (< 0.001) compared with un-irradiated control group. Influence of different laser doses on growth parameters of in vitro raised brinjal seedling is indicated in Table 2. Increased shoot length and root length following single exposure of different laser doses are shown in Fig. 3. The laser treatment at 25 J cm−2 shows the maximum response in terms of length, fresh and dry weights of shoot and root. Single laser exposure at 25 J cm−2 increased the shoot (< 0.001) and root (< 0.05) length, fresh weight of shoot (< 0.001), dry weight of root (< 0.001) and number of leaves compared with un-irradiated control set. Even though number of roots was found to be higher in 25 J cm−2 compared with other laser doses and un-irradiated control, the difference was not found to be statistically significant. In the case of root weight, single exposure of red light at 20 J cm−2 caused increase (< 0.001) compared with un-irradiated group. The seedling vigor index also showed linear increase from 5 J cm−2 and showed maximum at 25 J cm−2 and decreased thereafter. The highest seedling index of 2006.4 ± 6.32 was recorded for 25 J cm−2 (< 0.001), which was found to be statistically significant compared with un-irradiated control (Fig. 4).

image

Figure 1.  Percentage of germination of seeds of brinjal (Solanum melongena L.) var. Mattu Gulla treated with different doses of laser irradiation. Data are expressed as mean ± SD and significant at ***< 0.001 compared with un-irradiated control.

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image

Figure 2.  Days to 50% germination of seeds of brinjal (Solanum melongena L.) var. Mattu Gulla treated with different doses of laser irradiation. Data are expressed as mean ± SD and significant at ***< 0.001 compared with un-irradiated control.

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Table 2.   Influence of laser dose on growth parameters of the in vitro raised seedlings of brinjal.
Laser dose (J cm−2)Growth of seedlingsNo. leavesNo. roots
Length (cm)Fresh weight (g)Dry weight (g)
ShootRootShootRootShootRoot
  1. *Significance levels aP < 0.05, bP < 0.01, cP < 0.001 compared with un-irradiated control.

04.1 ± 0.549.6 ± 3.21.87 ± 0.220.32 ± 0.100.78 ± 0.110.021 ± 0.00642.0 ± 0.04.0 ± 1.3
55.4 ± 0.6110.2 ± 2.11.99 ± 0.270.59 ± 0.110.98 ± 0.280.052 ± 0.00432.0 ± 0.06.2 ± 1.2
106.6 ± 0.7612.1 ± 0.92.23 ± 0.300.73 ± 0.151.11 ± 0.130.064 ± 0.00324.0 ± 1.07.4 ± 1.4
156.6 ± 0.7713.1 ± 1.12.24 ± 0.250.86 ± 0.191.13 ± 0.250.072 ± 0.00654.0 ± 1.08.6 ± 2.8
208.2 ± 1.2814.2 ± 1.22.55 ± 0. 321.13 ± 0.24c1.26 ± 0.810.075 ± 0.00584.0 ± 1.08.2 ± 1.8
2510.9 ± 0.96c15.5 ± 2.2a2.61 ± 0.22c0.89 ± 0.241.12 ± 0.170.086 ± 0.0046c6.0 ± 1.0b9.5 ± 3.2
308.2 ± 1.1814.8 ± 1.42.16 ± 0.260.81 ± 0.231.07 ± 0.380.063 ± 0.00576.0 ± 1.08.5 ± 2.3
356.4 ± 1.0812.8 ± 1.21.93 ± 0.320.77 ± 0.180.99 ± 0.520.062 ± 0.00574.0 ± 1.07.6 ± 1.8
404.2 ± 2.088.2 ± 1.70.87 ± 0.150.17 ± 0.130.45 ± 0.360.018 ± 0.00354.0 ± 1.06.2 ± 1.2
image

Figure 3.  Length of in vitro raised seedlings of brinjal (Solanum melongena L.) var. Mattu Gulla developed from laser-treated seeds.

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Figure 4.  Seedling vigor index of brinjal (Solanum melongena L.) var. Mattu Gulla. Data are expressed as mean ± SD and significant at ***< 0.001 compared with un-irradiated control.

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Physiological and biochemical characters

The influence of laser irradiation on pigment content of brinjal seedlings are presented in Table 3. The pigment contents in the control and laser irradiated groups are comparable. All the pigment contents showed increasing trends up to 25 J cm−2 and reduced thereafter. Highest levels of total chlorophyll content (chlorophyll a and b) as well as carotenoid content was recorded for 25 J cm−2 which was statistically significant (< 0.001) compared with un-irradiated control. However the changes in anthocyanin content were not significant as that was only marginal with increasing dose of laser irradiation (Table 3).

Table 3.   Influence of laser dose on pigment content (mg g−1 FW) of the in vitro raised seedlings of brinjal.
Laser dose (J cm−2)Chlorophyll content (mg g−1 FW)Carotenoids (mg g−1 FW)Anthocyanin (mg g−1 FW)
Total chlorophyllChlorophyll aChlorophyll b
  1. FW, fresh weight.

  2. *Significance levels: aP < 0.001 compared with un-irradiated control.

01.54 ± 0.090.82 ± 0.020.66 ± 0.110.27 ± 0.0231.38 ± 0.31
51.59 ± 0.070.87 ± 0.090.81 ± 0.090.33 ± 0.0241.57 ± 0.21
101.79 ± 0.070.93 ± 0.050.85 ± 0.200.33 ± 0.0221.62 ± 0.15
151.88 ± 0.220.98 ± 0.030.95 ± 0.160.34 ± 0.0181.76 ± 0.09
201.88 ± 0.120.93 ± 0.030.98 ± 0.250.34 ± 0.0161.81 ± 0.45
252.15 ± 0.06a1.17 ± 0.09a1.08 ± 0.130.39 ± 0.015a1.84 ± 0.51
301.89 ± 0.020.97 ± 0.030.93 ± 0.150.33 ± 0.0191.66 ± 0.12
351.54 ± 0.090.81 ± 0.020.83 ± 0.160.31 ± 0.0131.46 ± 0.08
401.19 ± 0.030.75 ± 0.040.61 ± 0.120.23 ± 0.0161.24 ± 0.12

The amylases and proteases are very important enzymes to hydrolyze the stored polysaccharides, proteins required for the growth and development of seedlings. The activity of both α- and β-amylases in brinjal seedlings grown in vitro from seeds irradiated with different laser doses and un-irradiated control are presented in Fig. 5a,b. Significant positive effect was observed in both α- and β-amylase activity in seedlings of brinjal. The activity of amylases increased gradually and the maximum activities of α-amylase were noted in shoots and roots of seedlings derived from 25 J cm−2 laser irradiated seeds, whereas the activity of β-amylase showed maximum activity at 20 J cm−2 which was found to be significant (< 0.001) compared with respective un-irradiated control group. The activities of both α- and β-amylases were found to be increased up to 25 J cm−2 (Fig. 5a,b). The different energy density of laser irradiation also increased the activity of seedling proteases significantly during germination and further growth. Similarly, the proteases of shoots and roots also showed increasing trends from 5 J cm−2 and maximum activity was observed at 25 J cm−2 and it decreased thereafter (Fig. 6). The increased activity of proteases, recorded from roots and shoots exposed to single irradiation of 25 J cm−2 was also statistically significant (< 0.001) compared with the respective control groups.

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Figure 5.  (a) Activity of alpha amylase in shoots (line) and roots (bar) of brinjal (Solanum melongena L.) var. Mattu Gulla seedlings. Data are expressed as mean ± SD and significant at ***< 0.001 compared with un-irradiated control. (b) Activity of beta amylase in shoots (line) and roots (bar) of brinjal (Solanum melongena L.) var. Mattu Gulla seedlings. Data are expressed as mean ± SD and significant at ***< 0.001 compared with un-irradiated control.

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image

Figure 6.  Activity of protease in seedlings of brinjal (Solanum melongena L.) var. Mattu Gulla. Data are expressed as mean ± SD and significant at ***< 0.001 compared with un-irradiated control.

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The data on total protein and soluble sugar are presented in Figs. 7 and 8. The different doses of laser irradiation resulted in significant increase in total protein and sugar content. Protein content of shoot and root also showed increased trends up to 25 J cm−2 with reference to control. The maximum protein content was noted in both shoots and roots at 25 J cm−2 laser irradiated seedlings (Fig. 7) which was significantly (< 0.001) higher compared with un-irradiated control. There was a gradual decrease in total soluble sugar content of shoots and roots raised from seeds irradiated at 15 and 20 J cm−2. However, maximum soluble sugar content was observed at 25 J cm−2 in shoots and roots (Fig. 8), which was significant (< 0.001) compared with control.

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Figure 7.  Total protein content in seedlings of brinjal (Solanum melongena L.) var. Mattu Gulla. Data are expressed as mean ± SD and significant at ***< 0.001 compared with un-irradiated control.

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Figure 8.  Total soluble sugar in shoots (line) and roots (bar) of brinjal seedlings (Solanum melongena L.) var. Mattu Gulla. Data are expressed as mean ± SD and significant at ***< 0.001 compared with un-irradiated control.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Internal as well as external factors are indispensable for various metabolic activities of plants during growth and development. Since 1950, lasers have been in use in various biological experiments (51,52). A number of recent reports on application of different types of lasers in agriculture with improved photosynthetic efficiency (53), alleviation of drought stress by electromagnetic treatment (54), increase in biochemical, physiological and yield characteristics (55) and laser induced water acclimation and protective effect from drought stress are available in the literature (27). However, the effect of laser energy under in vitro environment is not well studied in crop plants. In the present study we report the influence of 632.8 nm He–Ne laser on in vitro seed germination, physiological and biochemical characteristics of a unique variety of brinjal which is only being cultivated in the aforementioned region with distinct flavor.

Germination and growth characters

The laser irradiation positively and significantly affected the seed germination and time to 50% germination at lower doses of laser rays. The enhanced percentage of germination due to laser irradiation has positive correlation with induced internal energy level of seeds. A similar result of laser biostimulation was reported by Vasilevski (9) and Li and Feng (56) with 20–35% of improved seed germination in Atractylodes macrocephala. Similarly in sunflower, Perveen et al. (18) also observed the significant and considerable biological effects on physiology of seed germination with low power continuous wave He–Ne laser treatment. The enhanced germination percentage of seeds may be due to the fact that the alterations in the physiological state of seeds and plants are due to different intensities of laser irradiation which may stimulate or inhibit the seed physiology at lower and higher doses respectively (13). The different intensities of laser irradiation (λ = 675 nm) stimulated seed germination in wheat, maize, amaranth and some vegetables (13,14,57). For example, Svetleva and Aladjadjian (58) have observed that soaking in distilled water before irradiation leads to higher stimulation of development of bean. Evenari (59) has suggested that He-Ne laser irradiation breaks the seed dormancy and showed significant effect even in slow-germinating viable seeds. In general, the different levels of laser energy irradiation has induced the internal energy of seeds which leads to increase in percentage of germination through enhanced activity of amylases and proteases involved in the physiology of seed germination, growth and development of seedlings.

Similarly, Chen et al. (25) have reported the positive effects of the spectral influence of laser irradiation on seed germination and suggested that the laser energy induces alterations in the enzyme activities during germination and triggers the rate of cell division, which ultimately leads to enhanced rate of growth and development. Hence, the impacts of laser energy on seed germination shown in this study might be due to an increased rate of cell division during seed germination. Furthermore, Dziwulska (60), and Dziwulska and Koper (61) have reported that the stimulation by the laser energy is a physical phenomenon which includes absorption and radiant energy storage by cells and tissues of plant systems. The seeds also have the same phenomenon; first they absorb the laser energy and then convert it into chemical energy which will be utilized for germination and growth.

The growth characters, viz., length, fresh and dry weight of shoots and roots, number of leaves and roots were higher at lower doses of laser irradiation was found to be decreased at higher doses. Similarly, the dry weight of roots, number of leaves and roots were also significantly higher in lower doses of laser radiation compared with un-irradiated control. Among the growth characteristics, length, fresh weight of shoot, dry weight of root, number of leaves and roots were higher at 25 J cm−2 whereas the fresh weight of root and dry weight of shoot was higher at 20 J cm−2 laser irradiation. The laser irradiation induced significant improvement in seed germination and subsequent growth of seedlings. Our results are in conformity with Chen et al. (25) who have reported the significant improvement in biomass and leaf area of the seedlings of Isatis. Similarly, 7.6% and 5.7% increase in phytomass was noted with laser irradiation in tomato and cucumber respectively (62). Cholakov and Petkova (63) have reported higher quality seedlings with laser irradiation. Furthermore, Govil et al. (64) reported the maximum increase in shoot and root length, dry weight of the seedlings from laser irradiated (30 min) green gram seeds. Laser also influenced 63% gain of dry mass of maize seedlings (65), and enhanced emergence, early flowering and maturity, plant height due to the presowing He–Ne laser treatment than the control plants of faba bean (66).

Physiological and biochemical characteristics

The effect of laser irradiation on pigment content (chlorophylls, carotenoids and anthocyanin) was studied and it significantly increased at lower doses of laser and decreased at higher doses (35 and 40 J cm−2). The total chlorophyll content (chlorophyll a and b) were increased gradually over control. Similarly, the carotenoids and anthocyanin contents were also higher than un-irradiated controls. Among the pigments, levels of total chlorophyll, chlorophyll a and carotenoids was increased significantly, whereas levels of chlorophyll b and anthocyanin were not significant with respect to the controls, as has been reported for the kidney bean, cabbage and beet cultivars by Kacharava et al. (17). The increased chlorophyll, carotenoid and anthocyanin contents in leaves of experimental plants correspond with the existing results of other authors (67). Chen et al. (25) have observed the significant increase in the concentration of total chlorophyll compared with that of control in Isatis indigotica. Husainov et al. (68) have observed that laser radiation stimulates the pigment content. Recently, Perveen et al. (55) have noted the considerable increment in chlorophyll a, b and a/b ratio due to presowing laser treatment at lower energies.

Several reports have demonstrated that laser irradiation at appropriate dose significantly enhanced the activities of enzymes which are indispensable during germination of seeds and further growth of seedlings (15,20,21,25,26,69,70). In the present study, we have noted the significant improvement in both amylases and protease activity in shoot and root of laser irradiated seedlings. The α-amylase activity was increased over control in both shoot and root and maximum activity was observed at 25 J cm−2. The β-amylase activity also shows similar trends with α-amylase activity and the maximum activity was noted at 20 J cm−2 in shoots and roots. The protease activity also showed increasing trends over control. Chen et al. (25) have demonstrated increase in amylases, transaminase and proteinases from the cotyledon of Isatis indigotica treated with He–Ne laser and microwave irradiation. Similarly, Perveen et al. (18) reported that increased activities of amylases and proteases from He–Ne laser treated sunflower seeds. This improved biological activity may be due to increased entropy and intrinsic energy of seeds during the germination process. Danie (71) has suggested that the He–Ne laser irradiation might trigger the rate of cellular reactions through the enhancement of essential enzyme activities.

The results obtained for the protein content of laser irradiated seedlings demonstrated clearly that significant increase in shoots and roots over un-irradiated control. There was gradual increase in protein content and it showed maximum at 25 J cm−2 in shoots and roots. Our observation is consistent with those of previous reports (15,18,21,23,25,72,73). Govil et al. (64) noted the improved protein content in green gram with 20 min laser irradiation at 337.1 nm, Khalifa and Ghandoor (21) reported the higher concentration of 55 kDa protein in soybean treated with 532 nm laser for 120 min when compared with the control. Similarly, total soluble sugar content also showed increasing trend at the lower doses of laser irradiation and we have observed a sharp decline in total soluble sugar at 15 and 20 J cm−2 in both shoot and roots. The maximum total soluble sugar was noted at 25 J cm−2 (< 0.001) in shoots and roots. The results of the present study are in conformity with Chen et al. (25) where they have reported the laser induced significant increase in soluble protein, pyruvic acid and soluble saccharides of the seedlings. Furthermore, Koper et al. (74) have documented the effect of triple laser treatment with elevated sugar content in sugar beet. Significant increase in RNA, DNA and protein contents up to 5-min exposure of Vigna radiata with Argon+ laser and the enhancement was evidence of the definite relationship between the laser irradiation and their biological responses (75).

Based on the findings of the present study, it can be concluded that physical factors are beneficial to a number of physiological and biochemical responses during seed germination and it subsequently led to enhanced growth and development of brinjal seedlings. The enhanced biochemical response observed in terms of improved activities of amylases and proteases which are responsible for digestion of the stored carbohydrates and to supply the energy to growing radicle and plumule. Similarly, the physiological responses (pigment contents) are responsible for enhanced photosynthetic rate which eventually leads to overall increase in length, fresh and dry weight, number of leaves and roots of the seedlings from irradiated seeds. A further experiment on influence of laser irradiation on in vitro culture responses of different explants and efficiency of plantlet regeneration via direct and indirect organogenesis is in progress. The laser irradiation to the seeds of brinjal which is unique variety of Mattu Village, Udupi District, Karnataka, India, is very useful to support the farmers with enhanced germination and growth and development of plants. It is also expected to withstand disease resistance and stress tolerance to plants for managing crop establishment in the field and subsequent contribution to the farmers with improved yield. Although the positive effects of laser rays was studied in detail with major crops, the molecular mechanisms of laser irradiation on positive and significant influence on plant characters are not yet fully known and it needs further investigation.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Acknowledgements— The authors are grateful to Manipal University, Manipal, Karnataka, India and TIFAC-CORE Pharmacogenomics for their encouragement and financial support respectively. The Patronage of DRDO-LSRB, GOI (Grant DLS/81/48222/LSRB-164/BDB/2008, 2008-2012) is also gratefully acknowledged. We are grateful to Mrs. Shashikala Tantry and Mr. Subhash Chandra for their experimental assistance. The authors also would like to thank Shri Lakshmana Rao, Mattu Village, Udupi District, Karnataka, India for the seeds of Mattu Gulla.

References

  1. Top of page
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  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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