Gamma sensitivity of forest plants of Western Ghats

es taka Gamma irradiation s La xb., indi of th obs to r as rad establish under radiation stress. R. minor and A. lindleyana were able to maintain viability up to 100 Gy 6000 tified a f the m tty et suppo al., 201 is, the 170 d lebbek, Zizyphus mauritiana (Selvaraju and Raja, 2001) and Tectona grandis (Bhargava and Khalatkar, 1987). Previous studies have shown that exposure of crop plants to low doses of ionizing radiationmanifests an increased cell proliferation, germination rate, cell growth, enzyme activity, stress resistance 2004; Kovàcs and rk of Sparrow and led that any envi- growth by influ- nsitivity and the e, type of ionizing According to Sparrow et al. (1970), woody species are relatively more sensitive when compared to herbaceous types. Almost all terrestrial plants are exposed to natural background radiation during their growing and dormant periods. The natural background radiation includes cosmic and terrestrial radiation, and plants have developed the natural resistance to the existing radioactivity (Nishiguchi et al., 2012). Apart from pre-operational survey on radiation levels and concentration of artificial * Corresponding author. Tel.: þ91 8242287271; fax: þ91 8242287347. Contents lists availab els Journal of Environmental Radioactivity 132 (2014) 100e107 E-mail address: [email protected] (K.R. Chandrashekar). available on forest tree species such as Acacia leucophloea, Albizia radiation and factors affecting recovery from radiation damage. tional plant breeding and thus directly contributing to the conser- vation and use of plant genetic resources. Approximately, 90% of these released plant varieties were produced using radiation of which more than 64% through gamma irradiation (Ahloowalia et al., 2004; Shu, 2009). There are numerous crop varieties gener- ated by irradiation technology, however, very few reports are depending on the irradiation level (Kim et al., Keresztes, 2002; Wi et al., 2006). Pioneering wo Woodwell (1962) on a forest ecosystem revea ronmental factor which influences the rate of encing the rate of cell division, affect the se sensitivity depends on three factors such as dos more than 60 countries. These plants in different areas of the world increase biodiversity, and provide breeding material for conven- cells. Also, ionizing radiation can affect differentially the morphology, anatomy, biochemistry, and physiology of plants 1. Introduction Western Ghats harbors more than and one third of these have been iden region of India is considered as one o the world (Reddy et al., 2007; She endemic plants, Western Ghats important flora in its core (Bag et Soejarto et al., 2012). Apart from th officially released crop varieties from http://dx.doi.org/10.1016/j.jenvrad.2014.02.006 0265-931X/� 2014 Elsevier Ltd. All rights reserved. species of higher plants s endemic species. This ega diversity centers of al., 2002). Along with rts ethanomedicinally 3; Chopra et al., 1956; re are more than 2700 ifferent plant species in and crop yields in plants (Charbaji and Nabulsi, 1999; Kim et al., 2005). The low-doses of radiation promote cell respiration, enzyme activation and increase the threshold of lethal doses of radiation. In addition, an enhanced production of reproductive structures, higher growth, early maturation, accelerated develop- ment and disease resistance have been reported (Luckey, 1980, 1998, 2003). Conversely, it is well known that a higher dose of ionizing ra- diation has deleterious effects on plants due to generation of free radicals which damage or modify important components of plant Growth Germination dose, however, any further increase in the dose found to have negative effect. � 2014 Elsevier Ltd. All rights reserved. Short communication Gamma sensitivity of forest plants of W Akshatha, K.R. Chandrashekar* Department of Applied Botany, Mangalore University, Mangalagangothri 574199, Karna a r t i c l e i n f o Article history: Received 16 August 2013 Received in revised form 30 January 2014 Accepted 5 February 2014 Available online 12 March 2014 Keywords: Western Ghats plants Sensitivity a b s t r a c t Seeds of Artocarpus hirsutu Pterocarpus marsupium Ro rifolia Roxb. and Oroxylum exposed to different doses ined on germination, grow with increased dose was sensitivity of these plants and generation of leaves w plants were classified as Journal of Environm journal homepage: www. tern Ghats , India m., Garcinia xanthochymus Hook., Saraca asoca Roxb., Rourea minor Gaertn., Terminalia chebula Retz., Aporusa lindleyana (Wt.) bail., Holoptelea integ- cum (L.) Vent. were collected from different regions of Western Ghats and gamma radiation using Co-60 source. The effect of irradiation was exam- and vigor parameters. Decrease in the germination and growth attributes erved in A. hirsutus, G. xanthochymus and S. asoca and thus indicating adiation. In contrast, enhancement in the germination (percentage), vigor observed for P. marsupium, T. chebula, H. integrifolia and O. indicum. These iation tolerant because of the ability of their seedlings to successfully le at ScienceDirect ental Radioactivity evier .com/locate / jenvrad radionuclides in wild plants of Kaiga region of the Western Ghats (Karunakara et al., 2003) none of the reports are available for Western Ghats flora regarding effect of gamma rays on general plant physiology. However, to understand positive and negative effects of gamma irradiation, it is necessary to screen a vast number of plants by subjecting them to various doses of radiation. Radiation hormesis provides a basis for appropriate utilization of ionizing radiation. The low doses of radiation could improve the wild plants by reducing the time of germination and accelerating the growth. The gamma irradiation technology has immense ap- plications in agriculture, industry and medicine. However, its po- tential exploitation is lacking in the field of forestry due to longer of seeds germinated from the first day till the final count. Shoot length, root length and number of leaves were measured on the final day of germination studies of different species as given in the Table 2. Vigor was calculated by using the following equation. Altitude Latitude Elevation Source 13�29 0 49.58 00 N 74�48 0 35.03 00 E 125 ft Ahmedullah and Nayar (1987) 13�11 0 21.40 00 N 74�45 0 14.90 00 E 62 ft Ahmedullah and Nayar (1987) 12�55 0 37.86 00 N 74�54 0 03.14 00 E 3.9 ft IUCN (1998) 13�29 0 36.74 00 N 74�48 0 36.02 00 E 44 ft He et al. (2006) 13�55 0 48.76 00 N 75�33 0 51.02 00 E 180.6 ft IUCN (1998) 14�02 0 14.93 00 N 74�49 0 43.24 00 E 183.6 ft IUCN (1998) 13�03 0 32.04 00 N 75�25 0 51.91 00 E 151 ft Rajkumar and Shivanna (2012) 13�25 0 52.18’’N 74�52 0 58.67 00 E 216 ft Sharma and Singh (2012) 13�25 0 48.70 00 N 74�52 0 51.99 00 E 203 ft IUCN (1998) Table 2 Information on Seed collection, moisture content and the period of observation. Plant species Collection details Sowing dates %Seed moisture Final count (N days) Artocarpus hirsutus Lam. SP April 2011 50.9 100 Garcinia xanthochymus Hook SP April 2011 20 170 Saraca asoca Roxb. DP June 2011 54.207 100 Rourea minor Gaertn. SP April 2011 42.825 100 Pterocarpus marsupium Roxb. DP June 2011 13.2 40 Terminalia chebula Retz SP March 2012 9.19 100 Aporusa lindleyana (wt.) bail DP April 2011 40.34 100 Holoptelea integrifolia (Roxb.) SP April 2011 8.365 50 Akshatha, K.R. Chandrashekar / Journal of Environmental Radioactivity 132 (2014) 100e107 101 life span of species involved compared to agricultural crops (Iglesias-Andreu et al., 2012). The aims of the present study are: (i) evaluating the impact of varying low doses of gamma irradiation on germination and growth of selected wild plant species of Western Ghats; (ii) categorizing species depending on their sensitivity to acute gamma irradiation; (iii) use of irradiation technology in the field of forestry in general. However, assessing presowing radiation effect on entire life cycle of forest tree species is a long termprocess. Therefore, the impact of low doses of gamma radiation on the ju- venile stage of forest plants is discussed in the present study. 2. Materials and methods This study was carried out from April 2011 to May 2013 at Applied Botany Department, Mangalore University, Karnataka, India. 2.1. Collection of seeds Nine species belonging to different families were collected by regular field visits to various localities of Western Ghats. All the species are perennial and tree forms except Rourea minorwhich is a woody climber. The fully ripened fruits were collected, immediately brought to the laboratory, the seeds were separated, cleaned and used for the experiment. Hundred seeds were used for each dose in five replicates for the selected species except in the case of Garcinia xanthochymus where, only 50 seeds were used. Details of the spe- cies and their location of collection are provided in Table 1. 2.2. Irradiation of seed samples Hundred healthy seeds (morphologically intact and free from in- sect or physical damages) from each species were selected and packed in bi-axially oriented polypropylene bags (BOPP, 25 m, 6� 6 cm). Each samplewas subjected to 25 Gy, 50 Gy,100 Gy,150Gy, 200 Gy, 250 Gy and 300 Gy doses of gamma radiation using Co-60 gamma source with a dose rate of 1.386 kGy/hr at BRIT (Board of Research in Radiation & Isotope TechnologyMumbai). Seedswithout any treatment served as control samples. Fricke dosimetry was used Table 1 List of selected species, location and their ecological status. Plant species Family Status Location Artocarpus hirsutus Lam. Moraceae E Mandarthi Garcinia xanthochymus Hook. Clusiaceae E Uchila Saraca asoca Roxb. Caesalpiniaceae V Pilikula Rourea minor Gaertn. Connaraceae WM Mandarthi Pterocarpus marsupium Roxb. Fabaceae V Shivamogga Terminalia chebula Retz. Combretaceae C Sigandoor Aporusa lindleyana (wt.) bail. Euphorbiaceae WM Charmadi Holoptelea integrifolia Roxb. Ulmaceae WM Karje Oroxylum indicum (L.) Vent. Bignoniaceae En, T Karje E, Endemic; C, Critically endangered; En, Endangered; T, Nearly threatened; WM, Wild m to measure the absorbed dose. Seed moisture content (MC) on fresh mass basis was determined by drying of quarterly cut whole seeds at 103 �C for 17 h (ISTA, 1991) in an oven. The difference in initial and final weight was expressed as % moisture content (Table 2). 2.3. Germination and growth All the seeds were kept for germination on sand bed at 2 cm depth in the green house of Applied Botany Department, Mangalore University (13�02 0 27.4 00 N, 75�22 0 52.2 00 E, 146 ft). The levels of soil nitrogen, potassium and sodium were 0.029 � 0.016 mg/g, 0.0823 � 0.98 mg/g and 0.40575 � 119 mg/g soil respectively. The pH of the sandy soil was 6.9 � 1. No fertilizer was used and the seeds/seedlings were irrigated daily. The temperature of the green housewas 28� 5 �Cwith a humidity ranging between 40e60%. The light intensity was 869 � 51 mmol m�2 s�1and the CO2 level in the air was 350 ppm. The seeds were considered as germinated when their radical attained 3 mm in height. Germinating seeds were scored daily from the day of sowing till the final count. The germination speed (S) was determined by using the following equation (Chiapuso et al., 1997). Sðseedling=dayÞ ¼ ðN1 � 1Þ þ ðN2 � N1Þ � 1=2þ ðN3 � N2Þ � 1=3..þ ðNn � Nn�1Þ � 1=n: where N1, N2, N3... Nn�1, Nn. ¼ proportion of germinated seeds observed at 1, 2, 3..up-to n � 1 and nth day. Germination percentage (G) was calculated from total number Oroxylum indicum (L.) Vent SP April 2011 7.275 50 SP seeds were collected from single plant DP; Seeds were collected from different plants. edicinal; V, Vulnerable. V ¼ %G� ðASL þ ARLÞ where V ¼ Vigor index, ASL ¼ Average shoot length, ARL ¼ Average root length, %G ¼ germination percentage. 2.4. Data analysis The data were analyzed using IBM SPSS20 statistical software (SPSS Inc. Chicago, IL, USA) in randomized complete block design. Mean values were compared using Duncan’s Multiple Range Test (Duncun, 1955) at 0.05% level of probability to determine the ho- mogeneity of a set of n values in an analysis of variance. 3. Results and discussion 3.1. Effect of gamma irradiation on germination The effect of gamma irradiation on the germination speed of nine species of plants is given in Fig. 1a and b. The seeds of Arto- carpus hirsutus, Garcinia xanthochymus and Saraca asoca did not germinate above 25 Gy dose. Rourea minor and Aporusa lindleyana showed the germination up to 100 Gy while, the seeds of other species showed germination even at higher doses. An enhancement in the speed of germination was observed in Holoptelea integrifolia and Oroxylum indicum seeds treated with 25 Gy and 200 Gy doses compared to their respective controls. In the case of Terminalia chebula, irradiation with 25 Gy dose showed low-dose hormesis phenomenon. Borzouei et al. (2010) found a significant decrease in mean germination time with increasing dose. Results of present study revealed that gamma irradiation inhibited the germination in A. hirsutus, S. asoca and G. xanthochymus. A higher moisture content in theses seeds might explain the reason for losing the viability after irradiation. The water radiolysis is the predominant effect of ionizing radi- ation in organisms. Thus the reactive oxygen species (ROS) so generated can interfere with the normal functioning of the vital system (De-Vita et al., 1993). The survival of plants to maturity depends on the nature and extent of chromosomal damage (Sax, 1942; Lea, 1947). Increasing the frequency of chromosomal dam- age with increasing dosage may be responsible for reduced germination and reduction in plant survival and growth (Kiong et al., 2008). The increase in germination speed in H. integrifolia and O. indicum at certain low doses of gamma irradiation might be due to its stimulating effects on RNA synthesis or protein synthesis as reported by Kuzin et al. (1976). Dormant seeds are known to be more resistant than the vegetative parts to irradiation and doses up to 300 Gy is useful for the radio sensitivity studies (Geras’kin and Sarapul’tzev, 1993). The material and energy necessary for initial growth are already available in the seed, but some stimulants are required only to activate those already stored substances in the cotyledons. Low doses of gamma radiation may increase the enzymatic activation and awakening of the young embryo which results in stimulating the rate of cell division. In addition to germination, the radiation can also influence vegetative growth (Sjodin, 1962). The seeds of A. hirsutus and G. xanthochymus lost complete viability above 25 Gy dose and found to be very sensitive to ionizing radiation (Fig. 2a and b). In case of R. minor and A. lindleyana, complete inhibition of germination was observed above 100 Gy dose. There was no significant difference in the germination Akshatha, K.R. Chandrashekar / Journal of Environmental Radioactivity 132 (2014) 100e107102 Fig. 1. a and b. Effect of gamma irradiation on germination speed of different plant species. T significantly different at P � 0.05. Letters a, b, c, d, e and f denote significant difference be he data shown are mean � SD of five replicates. Bars showing the same alphabet are not tween the means of different treatments. nviro Akshatha, K.R. Chandrashekar / Journal of E percentage of Pterocarpus marsupium seeds between irradiated and control samples whereas, a significant increase in germination percentage was observed in the seeds of T. chebula, H. integrifolia and O. indicum at different irradiation doses. In Psoralea corylifolia L, a gradual decrease in the germination percentage was observed with increased dose as reported previously (Jan et al., 2012). In these cases, reduction in seed germination with gamma radia- tion might be due to direct DNA or membrane damage. Also, an increased production of reactive radicals can be responsible for seed’s lethality (Maity et al., 2009). Low doses of gamma radi- ation (0.1e0.4 krad) on the seeds of Araucaria angustifollia (Bert.) O. Kuntze. showed the effectiveness of ionizing radiation in improving the seed germination, which was helpful in forest management for the major agronomic trait improvement (Ferreira et al., 1980). 3.2. Effect of varying doses of gamma irradiation on shoot length and root length of different plant species There was no significant difference in the shoot length between the treatment and control groups for the seedlings of R. minor (Fig. 3a and b). Also, no difference in the shoot length was observed for P. marsupium up to 100 Gy, T. chebula up to 150 Gy, H. integrifolia up to 200 Gy and O. indicum up to 250 Gy. The treatment above 250 Gy doses reduced the shoot length compared to the controls. There was a decrease in shoot length with increased doses of ra- diation in A. hirsutus, G. xanthochymus and S. asoca. In two species of Pinus, increased dose of radiation above 100 Gy reduced the shoot epicotyls and the root primordia indicating their sensitivity to gamma rays (Thapa, 2004). In Cicer species, gamma irradiation of Fig. 2. a and b. Effect of gamma irradiation on the germination percentage of different pla alphabet are not significantly different at P � 0.05. Letters a, b, c, d, e and f denote signific nmental Radioactivity 132 (2014) 100e107 103 seeds with 200 Gy dose inhibited the shoot length (Toker et al., 2005) whereas, in Phaseolus vulgaris an enhancement in the shoot length was observed when exposed to 2 krad of gamma irradiation (Beltagi et al., 2006). Shakoor et al. (1978) and Khalil et al. (1986) attributed decreased shoot and root lengths by gamma rays (at 15 krad in wheat and 40 krad in barley) is due to reduced mitotic activity. There was no significant difference in the root length between the treatment and control groups of seeds of A. hirsutus, S. asoca, P. marsupium, T. chebula, H. integrifolia and O. indicum at lower doses, whereas, it decreased at higher doses (Fig. 4a and b). In So- lanum tuberosum L., gamma irradiation completely inhibited the growth of roots as reported previously (Cheng et al., 2010). The stimulating effect of low doses of gamma irradiation on plant growth may be due to stimulation of cell division or cell elongation, along with alteration of metabolic processes which affect synthesis of phytohormones or nucleic acids (Pitirmovae, 1979). In the pre- sent study, a significant enhancement in the root length of P. marsupium was observed at low doses of radiation. Radio- resistance of the plants is the ability of their repair system to overcome the damaging effect of stressors and its successful establishment under adverse condition. In pine forest, the chronic exposure to radionuclide contaminant lead to evolutionary effect of these contaminants and development of population level markers and development of aberrant root cell frequency in the root meri- stem (Geras’kin et al., 2004). The cells which have relatively more chromosomal damage at high irradiation exposures are at a disadvantage due to diplontic selection and cannot compete well with the normal cells and are thus prevented from making any further contribution for the growth (Shah et al., 2008). nt species. The data shown are mean � SD of five replicates. Bars showing the same ant difference between the means of different treatments. Fig. 3. a and b. Effect of gamma irradiation on shoot length of different plant species. The data shown are mean � SD of five replicates. Bars showing the same alphabets are not significantly different at P � 0.05 (p � 0.05). Letters a, b, c and d denote significant difference between the means of different treatments. NS; Not significant. Fig. 4. a and b. Effect of gamma irradiation on root length of different plant species. Bars showing the same alphabets are not significantly different at P � 0.05. The data shown are mean � SD of five replicates. Letters a, b, c and d denote significant difference between the means of different treatments. NS; not significant. Akshatha, K.R. Chandrashekar / Journal of Environmental Radioactivity 132 (2014) 100e107104 3.3. Effect of different doses of gamma irradiation on vigor of different plant species A substantial decrease in vigor in the treated seeds of A. hirsutus, G. xanthochyms and S. asoca was observed compared to controls (Table 3) A negative impact of gamma irradiation on vigor may be due to metabolic changes caused by events such as free radical formation as explained earlier (UNSCEAR, 1996). The vigor was not altered in treated seeds of P. marsupium and O. indicum when compared to control seeds. However, a substantial increase in vigor was noticed in A. lindleyana (38.5%), T. chebula (74.7%) and H. integrifolia (49.32%) when exposed to 25 Gy dose. The increased vigor in the present study is in accordance with our earlier report on Pterocarpus santalinus and Terminalia arjuna (Akshatha and Chandrashekar, 2013; Akshatha et al., 2013). Similarly, an increase in vigor index of gamma irradiated seeds was observed at 10 and 20 krad in tomato and okra seeds respectively (Nargis et al., 1998; assessment of Pinus sylvestris seedlings at contaminated areas revealed the evolutionary trends lead to radio resistance of species by adaptive phenomenon (Geras’kin et al., 2004). In the present study also, enhancement of vigor in H. integrifolia, T. chebula and O. indicum could be due to the stimulatory effect of low doses of radiation along with their adaptive resistance to the radiation stress. 3.4. Effect of varying doses of gamma irradiation on number of leaves of different plant species The radiation either has no effect or it decreased the number of leaves produced in all the plant species except in A. hirsutus, T. chebula and H. integrifolia (Fig. 5a and b). In A. hirsutus and T. chebula, there was a significant increase in the number of leaves up to 25 Gy when compared to controls. In H. integrifolia, maximum leaf number was observed at 300 Gy dose. Mudibu et al. (2012) Table 3 Effect of gamma irradiation on vigor of different plant species. Dose A. hirsutus G. xanthochymus S. asoca R. minor A. lindleyana P. marsupium T. chebula H.integrifolia O. indicum 0 Gy 825 � a 2693 � a 4170 � a 1844 � a 1919 � b 747 � a 340 � c 759 � c 964 � a 25 Gy 219 � a 52 � b 1709 � b 642 � b 2217 � a 559 � ab 1015 � a 1124 � a 1020 � a 50 Gy e e e 139 � c 1345 � c 686 � a 909 � ab 550 � ef 138 � c 100 Gy e e e 370 � bc 696 � d 663�a 756 � b 713 � cd 979 � a 150 Gy e e e e e 411 � b 638 � b 428 � f 551 � b 200 Gy e e e e e 606 � ab 109 � cd 960 � b 1067 � b 250 Gy e e e e e e 66 � d 671 � cde 530 � b 300 Gy e e e e e e e 573 � def 543 � b Columns showing same alphabets are not significantly different at P � 0.05. Letters a, b, c, d, e and f denote significant difference between the means of different treatments. Akshatha, K.R. Chandrashekar / Journal of Environmental Radioactivity 132 (2014) 100e107 105 Arvind-Kumar and Mishra, 2004). The hypothetic origin of these stimulations by irradiation was due to increased cell division rates as well as an activation of growth hormone, e.g., auxin (Gunckel and Sparrow, 1961; Zaka et al., 2004). Environmental impact Fig. 5. a and b. Effect of gamma irradiation on production of leaves in different plant specie shown are mean � SD of five replicates. Letters a, b, c and d denote significant difference b observed that irradiation of Glycine max L with 0.2 kGye0.4 kGy significantly decreased the morpho-agronomic characters like leaf number and size. Similar results were observed in G. xanthochymus, S. asoca, P. marsupium and O. indicum. However, no difference was s. Bars showing the same alphabets are not significantly different at P � 0.05. The data etween the means of different treatments. NS; Not significant. viro observed in treated R. minor and control. In Snap beans, highest number of leaves was recorded when exposed to 30 Gy dose (Abou El-Yazied, 2011). In Vigna unguiculata, plant height and number of leaves produced was shown to be increased (Gnanamurthy et al., 2012). Similar results were obtained in the present study for A. hirsutus and T. chebula. The increased leaf number may be due to the enhanced production of growth hormone, kinetin which stimulates the production of large number of leaves and branches as observed earlier by Minisi et al. (2013). 4. Conclusions The effect of gamma irradiation on seed germination and growth of the nine woody plant species of the Western Ghats forest is being reported for the first time. Based on our findings, A. hirsutus, G. xanthochymus and S. asoca may be classified as sensitive to gamma radiation whereas, R. minor and A. lindleyana as moderately sensitive. Further, P. marsupium, T. chebula, H. integrifolia and O. indicum were observed to be tolerant to lower doses of gamma radiation. Reliance has generally focused on laboratory studies on a few species, thought to be repre- sentatives of biodiversity, hence field experimentation is required for identification of potential environmental effects of low level ionizing radiation to establish safety criteria for nu- clear facilities and its peaceful application. Further work on the monitoring of these species for their reproductive end point is in progress. Acknowledgments Authors are grateful to BRNS (Board of Research in Nuclear Sciences) for financial support and Mangalore University for providing research facilities. Authors also thankful to BRIT (Board of Research in Radiation & Isotope Technology Mumbai) for providing irradiation facility, Karje Manjunathgoli for the collection of samples and the reviewers for their valuable suggestions. References Abou El-Yazied, A., 2011. Growth, biochemical constituents and yield of Snap Bean as influenced by low Gamma Irradiation Doses under Different Sowing Dates. Aust. J. 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Chandrashekar / Journal of Environmental Radioactivity 132 (2014) 100e107 107 Gamma sensitivity of forest plants of Western Ghats 1 Introduction 2 Materials and methods 2.1 Collection of seeds 2.2 Irradiation of seed samples 2.3 Germination and growth 2.4 Data analysis 3 Results and discussion 3.1 Effect of gamma irradiation on germination 3.2 Effect of varying doses of gamma irradiation on shoot length and root length of different plant species 3.3 Effect of different doses of gamma irradiation on vigor of different plant species 3.4 Effect of varying doses of gamma irradiation on number of leaves of different plant species 4 Conclusions Acknowledgments References