Journal of Agricultural Science and Technology A 4 (2014) 219-223 Earlier title: Journal of Agricultural Science and Technology, ISSN 1939-1250

D

DAVID

PUBLISHING

Effects of Different Natural Organic Additives on in Vitro Shoot Regeneration of Raphanus sativus L. Var. Beeralu Indeewari Manawadu, Nilanthi Dahanayake and Senanayakage Gamini Nonis Senanayake Department of Agricultural Biology, Faculty of Agriculture, University of Ruhuna, Mapalana, Kamburupitiya, Sri Lanka

Received: January 25, 2014 / Published: March 20, 2014. Abstract: Raphunus sativus L. commonly known as “radish” is a popular vegetable crop used by people all over the world for its culinary and medicinal properties. Enhancement of in vitro regeneration system for radish variety beeralu is needed to further tissue culture studied. Therefore, the present study was carried out to find out the effects of different organic additives on in vitro shoot regeneration of Radish (Raphanus sativus L.) Var. Beeralu. Hypocotyl explants of aseptic plantlets were cultured on MS basal medium supplemented with 2.5 mg/L BAP (benzyl adenine) and 0.1 mg/L NAA (1-nphthaleneacetic acid) with different natural additives; 20% coconut water, 20% coconut milk, 10% grind spinach leaves, 10% grind potato tubers, 10% grind carrot, 5% rice flour, 10% green gram, 10% grind pumpkin, 10% banana fruit, 10% orange and control (without any additives). Complete randomized design (CRD) with five replicates was used. After one month the numbers of regenerated shoots were counted and statistical analysis was carried out using the Student Newman-Kuells Means Separation Test of SAS program (9.1.3). The highest mean number of shoots (12 shoots/explant) from Radish (Raphanus sativus L.) Var. Beeralu observed in MS basal medium with 2.5 mg/L BAP and 0.1 mg/L NAA with 10% orange juice whereas the 2nd highest shoots were obtained with 20% coconut water. The lowest number of shoots (0 shoot/explant) was observed from medium with carrot juice and pumpkin juice, but they induced callus formation. Media with grind spinach leaves, rice flour, green gram, grind potato tubers and banana inhibit the shoot regeneration. Key words: Raphunus sativus L., natural additives, orange juice, in vitro regeneration.

1. Introduction Various factors should be considered in developing plant tissue culture technique. In vitro regeneration ability was greatly influenced by medium composition of culture media which deliver nutrients into in vitro plant growth as well as in vivo plant growth. In addition to inorganic salts, a carbon and energy source, vitamins, and plant growth regulators, in case of specific needs of particular species or tissues, other components, including organic nitrogen compounds, organic acids, and a wide variety of complex natural extracts, can be important. The in vitro cell and tissue cultures of higher plants are characterized by the use of isolated parts of plants obtained from an intact plant

Corresponding author: Nilanthi Dahanayake, Ph.D., research fields: developmental physiology of plant and molecular biology. E-mail: [email protected].

body and kept on, or in a suitable nutrient medium. This nutrient medium functions as replacement for the cells, tissue, or conductive elements originally neighboring the explant and usually maintained under aseptic conditions. The composition of a medium is the main task for the establishment of a successful plant cell and tissue culture technique. The exact conditions required to regenerate intact plants from cultured cells, are different for each plant species. The essential nutrients consist of inorganic salts, a carbon and energy source, vitamins, and plant growth regulators. In addition to these standard components, in case of specific needs of particular species or tissues, other components, including organic nitrogen compounds, organic acids, and a wide variety of complex natural extracts, can be important [1]. The basic Murashige and Skoog (MS) medium [2] is the most widely used media. However, the amounts of the

220

Effects of Different Natural Organic Additives on in Vitro Shoot Regeneration of Raphanus sativus L. Var. Beeralu

various ingredients in the medium vary for cultures of different plant species. Much number of researchers influenced to find alternatives materials to substitute alternatives to gelling agents, use of household sucrose, and some medium components objectively to reduce cost in media culture preparation. A large number of complex additives like coconut water, banana pulp, peptone, tomato juice, slap honey and beef extract, can be very effective in providing undefined mixture of organic nutrients and growth factors. From the previous study, demonstrated that the addition of potato, corn and papaya extract at various concentrations able to enhanced the growth of the calli of the Doritaenopsis orchid, and promoted the regeneration of protocorm-like bodies (PLBs) from the calli and plant extracts such as coconut milk, banana extract, and tomato juice can be very effective in providing undefined mixture of organic nutrients and growth factors [3]. Radish (Raphanus sativus L., 2n = 18; Brassicaceae) is a common vegetable in Asia and in most parts of the world. Radish produces a red or white colored edible root with different shapes. Apart from culinary purposes; radish has diverse medicinal properties as well. Enhancement of shoot [4] regeneration ability of radish variety beeralu, a Sri Lankan variety is needed to further tissue culture techniques. Thus, the present study was aimed to compare the potentiality of in vitro regeneration ability of Radish, variety Beeralu in Murashige and Skoog’s (MS) media modified with various natural additives.

2. Materials and Methods 2.1 Plant Source Seeds of radish were purchased from the Seed and Planting Material Division, Department of Agriculture, Sri Lanka. 2.2 Establishment of Aseptic Cultures Seeds were surface sterilized by washing tap water, soapy water, immersing in 70% ethanol for 3 min,

three times from distilled water and soaking in a 20% clorox for 20 min respectively. Sterilized seeds were then rinsed three times in sterilized distilled water and inoculated on a medium comprised of half strength MS salts, 3% sucrose and the medium was solidified with 0.8% agar and all the media used were adjusted to a pH value of 5.8-6.0 with 1 N NaOH or 1 N HCl solution, prior to autoclaving (1.4 kg/cm2 for 20 min). The seeds were cultured under light for 15 days [5]. 2.3 Regeneration Ability in MS Media Supplemented with Different Natural Additives Hypocotyl explants of aseptic plantlets were cultured on MS basal medium (100 mL) supplemented with natural additives (Table 1) with 2.5 mg/L benzyl adenine (BAP) and 0.1 mg/L 1-naphthaleneacetic acid (NAA) to investigate the regeneration ability in the 15 days old radish plantlets [6]. Hypocotyl was cut into about 5 mm and cultured by laying randomly on the media. Five replicates were used from each explant and cultures were kept under light. All the media used were adjusted to a pH value of 5.8-6.0 with 1n NaOH or 1n HCl solution, gelled with 0.8% agar prior to autoclaving at 1.4 kg/cm2 for 20 min [5] and cultured under light for 30 days. 2.4 Roots from Regenerated Shoots After 30 days in regeneration media, shoots were isolated and introduced to rooting media containing indole-3-butyric acid (IBA) 0.25 mg/L [7]. 2.5 Data Collection and Analysis Experiment was arranged according to the complete randomized design (CRD). Regeneration was evaluated 30 days after initiation. Number of explants with buds in different treatment was recorded. All experiments had five replicates, each with five explants per bottle. Statistical analysis was carried out using the Student Newman-Kuells Means Separation Test of SAS program (9.1.3).

Effects of Different Natural Organic Additives on in Vitro Shoot Regeneration of Raphanus sativus L. Var. Beeralu Table 1 Amount of different additives which add to 100 mL of MS medium + 0.1 mg/L NAA + 2.5 mg/L BAP. Additives Coconut water (Cocos nucifera) Coconut milk (Cocos nucifera) Grind spinach leaves (Spinacia oleracea L.) Grind potato tubers (Solanum tuberosum) Grind carrot (Daucus carota) Rice flour (Oryza sativa) Green gram seeds (Vigna radiate) Grind pumpkin fruit (Cucurbita pepo) Banana fruit (Musa sp.) Orange juice (Citrus sp.) Control (Agar)

Concentration 20 mL 20 mL 10 g 10 g 10 g 5g 10 g 10 g 10 g 10 mL 0.8 g

3. Results and Discussion Highest mean numbers of regenerated shoots (12 shoots/explant) were observed on MS basal medium with 2.5 mg/L BAP, 0.1 mg/L NAA and 10% orange juice while lowest mean number of shoots (0.0 shoot/explant) observed on MS basal medium with 2.5 mg/L BAP, 0.1 mg/L NAA with grind carrot, grind pumpkin and coconut milk. When added carrot juice and pumpkin juice to the medium induce callus formation without development of shoots. Second highest mean numbers of regenerated shoots (8 shoots/explant) were obtained in the treatment which supplemented with coconut water.MS with 2.5 mg/L BAP + 0.1 mg/L NAA with rice flour offered 6 shoots/explant. Furthermore one shoot/explant was gained by grind potato tubers and green gram while two shoots with roots were obtained in media with banana juice. Medium with grind spinach leaves gained five shoots/explant and it was less than number of shoots which gained by control (agar) (Table 2 and Fig. 1). As reported in Ref. [1], plant cell and tissue cultures are characterized by the use of isolated parts of plant obtained from an intact plant body and kept on, or in a suitable nutrient medium. This nutrient medium functions as replacement for the cells, tissue, or conductive elements originally neighboring the explant. The exact conditions required to initiate and sustain plant cells in culture, or to regenerate intact plants from cultured cells, are different for each plant

221

Table 2 Shoots/explant which were regenerated in different treatments. Additive Shoots/explant Coconut water 8b Coconut milk 0gf Grind spinach leaves 5d Grind potato tubers 1ef Grind carrot 0g Rice flour 6d Green gram 1ef Grind pumpkin 0g Banana 2e Orange 12a Control 7c Means fallowed by the same lowercase letters in each column are not significantly different at 5% level in Duncan’s multiple range test.

(b)

(a)

(e)

(f)

(i)

(d)

(c)

(g)

(j)

(h)

(k)

Fig. 1 Number of shoots/explant in MS medium, 2.5 mg/L BA, 0.1 mg/L NAA with: (a) coconut water, (b) coconut milk, (c) grind spinach leaves, (d) grind potato tubers, (e) grind carrot juice, (f) rice flour, (g) green gram, (h) grind pumpkin, (i) banana, (j) orange and (k) control.

species. The empirical approach has shown that three factors, namely explant choice, medium composition, and control of the physical environment are important in successful cultures. When the completely defined plant culture media did not give the desired results, employing natural substances have beneficial effects on in vitro plant cell and tissue cultures. The present studies revealed the medium with 10% orange juice enhance the number of shoots than the control treatment.

222

Effects of Different Natural Organic Additives on in Vitro Shoot Regeneration of Raphanus sativus L. Var. Beeralu

As reported by Prades et al. [8], coconut water contains a growth factor, cytokinin and micronutrients that stimulate in vitro culture of plants. As an example a preliminary study has shown that the addition of 15% (v/v) coconut water to the culture medium significantly improved callus growth, shoot regenerative capacity, and shoot growth in leaf disk cultures of spinach [9]. Likewise the present study revealed shoot regeneration ability was increased by adding coconut water. Coconut milk was used in orchid tissue culture to enhance orchid seed germination and auxin-like and gibberellin-like activity was detected in coconut milk [10, 11]. Auxins were the first class of growth regulators discovered; their major function is promoting elongation of emerging shoots. However, shoots were not emerged in the present study even coconut milk has auxin like activities. There was no any report spinach juice used as natural additives in tissue culture and those was not increased the shoot regeneration ability in the present study. Furthermore, there was no any report pumpkin juice used as natural additives and the media supplemented with pumpkin juice enhanced the callus growth. This is most probably due to the presence of abscisic acid (ABA) [12] in pumpkin juice which is negatively affecting the plant growth. ABA is known to slow down growth and to moderate the effects of auxins and cytokinins in in vitro culture. Starch is the main constituent in potato tubers and it was act as gelling agent to medium except agar [13, 14] and it may be the reason for inhibit the shoot regeneration in the medium with potato juice. The juice of the carrot when expressed contains crystallizable and uncrystallizable sugar [15]. Higher concentrations of carbon sources (sugars) in the medium resulted callus proliferation than shoot differentiation [16]. It may be the reason of callus formation from explants in medium with carrot juice. Rice flour act as gelling agent for the medium and

therefore shoot growth was not positively affected by the rice flour in the present study [17]. Green gram contains protein as major component and protein is not directly influence to the growth of plants [18]. However, unknown substituent may be inhibit the regeneration ability. Vacin and Went (VW) media with banana juice was enhanced the seedling growth in Dendrobium lituiflorum Lindl because of banana contained cytokinin, auxin and gibberellins which can act as buffer in culture media [19]. Moreover ½MS medium and banana pulp interaction performed best for in vitro regeneration of Dendrobium orchid [20]. However, the regeneration ability of Radish was not enhanced by the banana juice in the present study even contain cytokinin. Furthermore banana juice could promote root formation and growth [21] as resulted in the present study. Callus growth of orange (Citrus sinensis L. Osbeck) was enhanced medium supplemented with orange juice and it supported to their vigorous growth [22] and in vitro growth of explant (juice vesicle or albedo tissues) from citron (Citrus medica), lemon (C. limon), grapefruit (C. paradisi), sweet orange (C. sinensis), and mandarin (C. reticulata) fruits was stimulated by addition of orange juice in MS medium [23]. The present results was revealed shoot regeneration was enhanced by orange juice correspondingly. There were no any previous studies related to the radish with these organic additives. This experiment showed natural additives has a potential to apply in micro-propagation of Raphanus sativus to enhance the shoot regeneration, callus growth and root induction. The protocol developed in this study can be applied with other types of radish as appropriate.

4. Conclusions The best natural additive is 10% orange juice to enhance the regeneration ability of radish variety beeralu in MS basal medium with 2.5 mg/L BAP and 0.1 mg/L NAA from hypocotyl explant.

Effects of Different Natural Organic Additives on in Vitro Shoot Regeneration of Raphanus sativus L. Var. Beeralu

References [1]

Z. Molnár, E. Virág, V. Ördög, Natural substances in tissue culture media of higher plants, Acta Biologica Szegediensis 5 (1) (2011) 123-127. [2] T. Murashige, F. Skoog, A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol. Plant. 15 (1962) 473-497. [3] N. Daud, R.S. Taha, N.N.M. Noor, H. Alim, Potential of Alternative Gelling Agents in Media for the in vitro micro-propagation of Celosia sp., International Journal of Botany 7 (2) (2011) 183-188. [4] A. Mohammad, B.H. Mohammad, The Effects of different culture media on the callus production of radish (Raphanus sativus L.), Romanian Biotechnological Letters 4 (14) (2009) 4519-4523. [5] N. Dahanayake, X.L. Chen, F.C. Zhao, Y.S. Yang, H. Wu, An efficient in vitro propagation system for purple cone-flower (Echinacea purpurea L.), Journal of Tropical Agricultural Research & Extension 13 (2) (2010) 29. [6] I.P. Manawadu, N. Dahanayake, S.G.J.N. Senanayake, Effects of different explants and hormones on callus growth and regeneration of radish (Raphanus sativus L.) Var. Beeralu, in: 3rd International Symposium, South Eastern University, 2013, pp. 73-78. [7] I.P. Manawadu, N. Dahanayake, S.G.J.N. Senanayake, Effect of indole-3-butyric acid on in vitro root development in radish (Raphanus sativus L. Variety Beeralu), in: Third YSF Symposium, Young Scientists Forum, Sri Lanka, 2014, p. 99 [8] A. Prades, M. Dornier, N. Diop, J. Pain, Coconut water uses, composition and properties: A review, Fruits 67 (2011) 87-107. [9] M.J. Al-Khayri, F.H. Huang, T.E. Morelock, T.A. Busharar, Spinach tissue culture improved with coconut water, Hortscience 27 (4 ) (1992) 357-358 . [10] R.J. Griesbach, Orchid tissue culture, Current Plant Science and Biotechnology in Agriculture 2 (1986) 343-349. [11] L. Dix, J.V. Staden, Auxin and gibberellin-like substances in coconut milk and malt extract, Plant Cell, Tissue and Organ Culture 1 (1) (1981) 239-246. [12] Rhonda Allison Pumpkin Series, 2012, https://www.google.lk/url?sa=t&rct=j&q=&esrc=s&sourc e=web&cd=1&cad=rja&ved=0CCkQFjAA&url=https%3 A%2F%2Fwww.rhondaallison.com%2Fdownloads%2FP

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

223

umpkin_Series_Ingredients_2012.pdf&ei=ONW1UsqKC 469iAe6jYH4DA&usg=AFQjCNHXsdsKHTC7zvwF-S8 QvNrrTjQOGg&sig2=AS5Hx-XbhNYL0-tDNGb63w&b vm=bv.58187178,d.dGI (accessed Nov. 10, 2013). C.A. Brautlecht, A.S. Getchell, The chemical composition of white potatoes, American Potato Journal 28 (3) (1951) 531-550. R.H. Zimmerman, S.V. Bhardwaj, I.M. Fordham, Use of starch-gelled medium for tissue culture of some fruit crops, Plant Cell, Tissue and Organ Culture 43 (3) (1995) 207-213. Robert Hook, CARROT, 2012, fromhttp://www.herballegacy.com/Hook_Chemical.html (accessed Nov. 10, 2013). K.M. Sudipta, M. Kumara Swamy, M. Anuradha, Influence of various carbon sources and organic additives on in vitro growth and morphogenesis of Leptadenia reticulata (Wight & Arn): A valuable medicinal plant of India, Int. J. Pharm. Sci. Rev. Res. 21 (2) (2013) 174-179. N. Daud, R.S. Taha, N.N.M. Noor, H. Alim, Potential of alternative gelling agents in media for the in vitro micro-propagation of Celosia sp., International Journal of Botany 7 (2) (2011) 183-188. A.S. Mesallam, M.A. Hamz, Studies on green gram (Phaseolus aureus) protein concentrate and flour, Plant Foods for Human Nutrition 37 (1) (1987) 17-27. S. Prasertsongskun, R. Awaesuemae, Effect of additive substances and planting substrateon growth development of Aerides houlletiana Rchb.f. seedling by tissue culture, KKU Sci. J. 37 (3) (2009) 320-324. S. Aktar, S.K.M. Nasiruddin, K. Hossain, Effects of different media and organic additives interaction on in vitro regeneration of Dendrobium Orchid, Journal of Agric. Rural Development 6 (1&2) (2008) 69-74. L. Jiang, P. Ding, Y. Zheng, Effects of additives on tissue culture and rapid propagation of Dendrobium canducum, Journal of Chinese Medicinal Materials 26 (8) (2003) 539-541. Y. Erner, Partial purification of a growth factor from orange juice which affects citrus tissue culture and its replacement by citric acid, Plant Physio. 56 (1975) 279-282. J.W. Einset, Stimulation of fruit explant cultures with orange juice, Plant Physiology 62 (6) (1978) 885-888.