328 American-Eurasian Journal of Sustainable Agriculture, 3(3): 328-331, 2009 ISSN 1995-0748 © 2009, American Eurasian Network for Scientific Information This is a refereed journal and all articles are professionally screened and reviewed
O RIGINAL A RTICLE Varietal Differences in Epicuticular Wax Content in Peanut Aman Verma, C P Malik, and Varsha Chaturvedi Institute of Life Sciences, Jaipur National University, Jaipur Aman V erma, C P Malik, and Varsha Chaturvedi: Varietals Differences in Epicuticular W ax Content in Peanut, Am.-Eurasian J. Sustain. Agric., 3(3): 328-331, 2009 ABSTRACT The intent of present study was to assess the varietal differences in epicuticular wax (ECW ) content in peanut (Arachis hypogaea L.) genotypes M-13 and PBS24030. Complete plants were regenerated from in vitrosectioned embryonated cotyledons i.e. coteledonary node (CN). Multiple shoots arose on varying concentrations of 0.1% Brassinolides (BR) of G odrej Agrovet (0.5, 1.0 and 2.0 ml L -1 ) alone and in combination with BAP (3 mg L -1 ). The control set was devoid of any PGR. Large number of shoots was secured from single whole embryonated cotyledon explants cultured on Brassinolides-supplemented medium for periods up to 45 days. Shooting potential as well as epicuticular wax was calculated. Excised shoots developed roots in vitro upon transfer to medium supplemented with NAA at 1 mg L -1 for 30 d or 1 mg L -1 NAA along with 0.5 mg L -1 indole butyric acid (IBA). The two genotypes differed significantly in their ECW . The seedling subjected to BR or BAP or in combination, however, demonstrated variable responses in the two genotypes. PBS24030, which is a drought resistant variety, accumulated a greater ECW than M -13 which is a drought susceptible genotype. The two genotypes also differed in their response to BR under in vitro conditions. The ECW content varied in the in vitro plantlets leaves and those sampled from the field at comparable stages of growth. Interestingly leaves of in vitro plantlets showed poor activities in stomatal functioning and formation of ECW . Presumably low survival rates during the acclimatization stages may be attributed to these characters. Key words:Arachis hypogaea, epicuticular wax, Brassinolide, plant growth regulators, thin layer chromatography Introduction Peanut (Arachis hypogaea L.) is one of the most important oilseed crops of the world. Besides income for the farmers, groundnut provides an inexpensive source of high quality nutrition. Groundnut seeds contain 44-56% oil and 22-30% protein on a dry seed basis (Savage and Keenan, 1994). Due to its drought tolerant nature, peanut crop is grown under rain-fed conditions (Sharma et al., 1993). Consequently, the crop is well accepted by the marginal farmers of semi-arid tropics. However, at times the region experiences erratic and low precipitation and hence subjected to mild to severe drought. Large number of morphological and physiological adaptations is shown to impart drought tolerance to crop plants in general and peanut in particular. Some of the adaptive traits are rooting, accumulation of osmolytes, leaf/leaflet folding, reduced leaf area and regulation of transpiration. One such adaptive feature is closure of stomata to reduce water loss under decreased water availability. In recent years several reports have appeared where epicuticular wax is shown to help leaves in the retention of water by reducing cuticular transpiration. In a species where stress tolerant varieties are evolved have low rate of cuticular transpiration by furnishing advantage during drought environments due to efficient water use efficiency (Bengston et al., 1978). In fact high load of leaf epicuticular wax is correlated with changes in environmental factors (Voleti and Rajagopal, 1991) in coconut. Recently it has been shown in Tridax that the leaf epicuticular wax content varied with the season; in the months when soil has low environmental moisture content, ECW was more ( W adhwani and Malik, 2009). Corresponding Author: Aman Verma, Institute of Life Sciences, Jaipur National University, Jaipur E-mail:
[email protected]
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In order to boost yield in this crop, there is a considerable interest in the development of tissue culture technique for this species. Plant growth regulators are known to enhance the yield , oil and fatty acids content in peanut (Malik et al., 1993; 88; Verma et al., 2008). Vakharia et al (1997) observed that following water deficit imposition, the ECW enhanced and this was correlated with a decline in leaf moisture and relative water content. The objectives of the present study were to compare the contents of ECW in the two genotypes (resistant and sensitive to drought) and also to determine the influence of brassinosteroids on ECW contents in controlled micro propagated plants. M aterials and methods Seeds of groundnut (Arachis hypogaea L.) varieties such as M-13 and PBS24030 (obtained from ARS, Durgapura, and Jaipur) were selected as the experimental material. Freshly collected healthy seeds were washed with few drops of liquid soap (Labolene) for 5 min, after which the surface was disinfected by 90% ethanol (v/v) for 2 minutes, followed by treatment with 0.1% (w/v) mercuric chloride solution for 5–6 min and finally washed repeatedly with sterile distilled water. The seeds were then aseptically germinated on a moistened cotton bed in a 250 ml conical flask and incubated in the culture room for germination and subsequent development into complete seedlings. From the 12–15 day- old seedlings cotyledonary nodes (CNs) were excised and utilized for the initiation of cultures. CNs were inoculated into culture tubes each containing MS semisolid basal medium (Murashige and Skoog, 1962) solidified with 0.8% (w/v) agar (Himedia) and supplemented with various concentrations of 0.1% Brassinolides of Godrej Agrovet (0.5, 1.0 and 2.0 ml L -1 ) alone and in combination with BAP (3 mg L -1 ). The control set was devoid of any PGR. The pH of the medium was adjusted to 5.6–5.8 prior to autoclaving. The cultures were incubated at 25 ± 2 °C under a 10 h photoperiod of 37.5 m mol m – 2 s – 1 light intensity. After 30 days of incubation apical fully opened leaves were used for the estimation of epicuticular wax. Healthy and elongated shoots of approximately 3 cm in length were excised and cultured on MS medium supplemented with á- Naphthalene acetic acid (NAA) at 0.5, 1.0, 1.5 and 2.0 mg L -1 concentrations with Indole butyric acid (IBA) at concentrations 0.5, 1.0, 1.5 and 2.0 mg L -1 respectively and without IBA for root induction. Rooted shoots were observed till the formation of secondary roots. They were then washed with running tap water to remove the traces of medium from the roots and transferred to plastic cups containing a mixture of soil: sand: farmyard manure/vermicompost (3:1:1 w/w) in the growth chamber for 7-10 days. Colorimetric methods were used for the estimation of contents of epicuticular wax by the methods of Ebercon et al., (1977) , Malik and Singh (1994) and M alik et al., (2008). Twenty five discs (16 cm 2 ) each were taken for the estimation of wax. Purified wax was used as a standard and contents were compared with it. W ax components were quantified through TLC on silica gel-G plates using the method of Holloway and Baker (1968) and as described by Malik and Singh (1994).The silica gel plates were activated at high temperature and spots were developed with benzene solvent. Plates were sprayed with 5% K 2 Cr 2 O 7 in 40 per cent H 2 SO 4 and heated at 150°C. The components were individually identified. For comparison leaves were collected from the plants during the months of December. Results and Discussion Levels of ECW in control plants of the two varieties are given in table 1. M-13 had ECW contents much less (13.64±0.54 mg/ g FW ) than PBS24030 (35.86±0.35 mg/ g FW ). In the latter variety with increasing concentrations of BR in the medium total ECW content showed a progressive decrease though higher concentrations of BR i.e. up to 2.0 ml, leaves were able to accumulate the ECW (28.07±0.09) but failed to show any increase in combination with BAP (16.21±0.18). The same applies for M-13 also. The addition of BAP (3 mg L -1 ) caused a decline in both the genotypes. W hen both B R and BAP were used together, ECW load decreased over control. In M-13 BAP enhanced ECW slightly though BR stimulated the enhancement. The increase was complemented with increased concentrations of BR (Table 1). W hen the two hormones were added to the medium together, ECW content enhanced in M-13 (19.29±0.08 mg/ g FW ) only. The two varieties differed in regard to total ECW contents; PBS24030 having 140% more than M-13 genotype. The two genotypes also registered variable responses to BAP and BR, when used individually and in combination. W e also noticed increase in ECW contents with increased leaf age (47.78 mg/ g FW in PBS24030 and M-13 16.28 mg/ g FW ).On an average, there was an increase of 34% in PBS34020. On the contrary in M-13, with age, contents of ECW did not increase significantly (16%).
Am.-Eurasian J. Sustain. Agric, 3(3): 328-331, 2009 Table 1: S.N o.
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Content of ECW (m g/ g FW ) in leaves of in vitro plantlets after various treatm ents in two peanut genotypes. The values given in the table are m ean ±SE. Treatm ents/Genotypes Genotypes -------------------------------------------------------------PB S24030 M -13 Control 35.86±0.35 13.64±0.54 0.5 m l / L BR 21.98±0.17 17.22±0.22 1.0 m l L BR 18.49±0.21 21.78±0.34 2.0 m l / L BR 28.07±0.09 23.35±0.27 3 m g L -1 BAP 17.31±0.27 15.17±0.12 -1 3 m g L BAP + 2 m l BR 16.21±0.18 19.29±0.08
Discussion The result of the present study, which was conducted in the micro propagated seedlings, showed that after 45 DAC, peanut genotypes differed significantly in their ECW . The seedling subjected to BR or BAP or both, however, demonstrated variable responses in the two genotypes. PBS24030, which is a drought resistant variety, accumulated a greater ECW than M-13 which is a drought susceptible genotype. The two genotypes also differed in their response to BR under the cultural conditions. The extent of response was minimal in PBS 240390 genotype whereas M-13, the drought sensitive genotype exhibited high accumulation of ECW with added BR and BAP or with added BAP + BR. M aximum ECW was estimated with BR ( 2 ml). Reverse trend was noticed in PBS24030. Turunen and Huttunen (1990) have reported that ECW s are the first targets of a variety of air pollutants, and hence could be used as an early indicator of air pollution effects. The primary function of ECW deposition is to reduce water loss through the epidermis, a feature which ensures drought tolerance. Additionally this outer layer has a pivotal function in the interaction with pathogenic fungi. The wax biosynthesis starts with hexadecanoic acid and long chain fatty acids with an even carbon number are produced by elongation. These fatty acids are then reduced to fatty aldehydes and primary alcohols and primary alcohols or reduced and decarbonylated to yield alkanes with an uneven carbon number. The latter can be further converted to secondary alcohols and ketones. W e also attempted to compare the ECW contents in the in vitro plantlets leaves and those sampled from the field at comparable stages of growth. Interestingly leaves of in vitro plantlets showed poor activities in stomatal functioning and formation of ECW . Presumably low survival rates during the acclimatization stages may be attributed to these characters. It may be stated that during establishing the protocols for the two genotypes M-13 and PBS24030, we noticed marked differences in the survival rate of the two during acclimatization. PBS24030 plantlets derived from rooted shoots exhibited high rate of survival while M-13 plantlets showed poor survival rate. Yokota et al., (2007) reported similar results in micro propagation stages in Aralia elata and Phellodendron amurense. It may be concluded that the morphological and histological changes occurring in the leaves of in vitro plantlets may be crucial during the micro propagation stages. However, more detailed studies need to be carried out. References Bengston, C., S. Larsson and C. Liijenberg, 1978. Effect of water stress on cuticular transpiration rate and amount and composition of epicuticular wax in seedling of six oat varieties. Physiologia Plantarum., 44: 319-325. Ebercon, A., A. Blum and W .R. Jordan, 1977. A rapid colorimetric method for epicuticular wax content of sorghum leaves. Crop Science, 17: 179-180. Holloway, P.J., E.A. Baker, 1968. Isolation of plant cuticles with zinc chloride-hydrochloric acid solution. Plant Physiol., 43: 1878. Malik, C.P., M. Grewal U. Parmar D.S. Bhatia and P. Parmil, 1988. Bioregulators enhancement of yield and modification of oil, fatty acids composition of peanut; In Hormonal Regulation of Plant Growth and Development IV; 123-153 Ed. SS Purohit (Bikaner India: Agro-Botanical Publishers). Malik, C.P., R.C. Setia and N. Setia, 1993. Role of plant growth regulators in increasing crop productivity and the Indian Scenario; In Hormonal Regulation of Plant Growth and Development IV; 161-167 Ed. SS Purohit (Bikaner India: Agro-Botanical Publishers). Malik, C.P., A. Verma and A. Chaudhary, 2008. Influence of NaCl on content and composition of epicuticular wax and rate of cuticular transpiration in Arachis hypogaea L. Journal Plant Sc Res., 24: 223-224. M alik, C.P. and M.B. Singh, 1994. Plant Enzymology and Histoenzymology. Kalyani Publishers, New Delhi.
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