Crop Protection 30 (2011) 1425e1429

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Crop Protection
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Efcient shoot regeneration from direct apical meristem tissue to produce virus-free purple passion fruit plants
Siripatr Prammanee a, b, *, Sarut Thumjamras a, b, Pissawan Chiemsombat c, Narongchai Pipattanawong d
a

Faculty of Liberal Arts and Science, Kasetsart University, Kamphaeng Saen, Nakhon Pathom 73140, Thailand Center for Advanced Studies in Tropical Natural Resources, Kasetsart University, Bangkok 10900, Thailand c Department of Plant Pathology, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Kamphaeng Saen, Nakhon Pathom 73140, Thailand d Agro-Ecological System Research and Development Institute, Kasetsart University, Bangkok 10900, Thailand
b

a r t i c l e i n f o
Article history: Received 1 February 2011 Received in revised form 3 June 2011 Accepted 9 July 2011 Keywords: Apical meristem culture Purple passion fruit Virus-free plant Shoots regeneration Root formation

a b s t r a c t
Purple passion fruit is an important fresh table fruit. At present, the production of passion fruit is decreasing because of the spread of viral diseases throughout the planting area. The aim of this research was to propagate virus-free plants using a tissue culture technique involving the apical meristem of purple passion fruit. Shoot tips were excised to a length of 2 mm and the shoots were regenerated by culturing on Murashige and Skoog medium supplemented with different concentrations of benzyladenine (BA) (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mg/l) and 1-naphthaleneacetic acid (NAA) (0.2, 0.5, 1.0, 1.5 and 2.0 mg/l). Root formation was promoted using different concentrations of indole-3-butyric acid (IBA) (0.0, 0.2, 0.4, 0.6, 0.8 and 1.2 mg/l). A greater number of shoots were produced with BA concentrations of 1.0 and 1.5 mg/l than with any other BA concentrations tested (less than 1.0 mg/l or greater than 1.5 mg/ l). However, when NAA at any concentration was included in the medium, no shoots were produced in culture. The cultures including 1.0 mg/l and 1.5 mg/l BA were then subcultured four times every two weeks. Initially, the tissue cultured in the 1.5 mg/l BA medium grew faster than that cultured in the 1.0 mg/l BA medium. The tissue cultured with 1.5 mg/l BA generated many short shoots, whereas the tissue cultured with 1.0 mg/l BA, generated long shoots that could be subcultured into individual plants. These regenerated shoots were assayed for the presence of the passion fruit woodiness virus using ELISA or a test strip kit; only virus-free shoots were used for further propagation. Root formation was very good in IBA concentrations of 0.4 and 0.6 mg/l. Thus, virus-free plants could be successfully regenerated directly from the apical meristem. 2011 Elsevier Ltd. All rights reserved.

1. Introduction Passion fruit is an important crop and is grown commercially in several countries, including Thailand, Australia, the United States of America (Hawaii), Brazil and South Africa. The two most popular varieties are the purple-skinned Passiora edulis and the yellow-skinned P. edulis forma avicarpa. The purple P. edulis has a much sweeter juice, reduced acid content and a richer aroma. The fresh fruits are either consumed fresh locally or sold to the juice industry. Unfortunately, growing the P. edulis purple variety in Thailand has major constraint as it is susceptible to a woodiness disease caused by a species of potyvirus. The virus was designated as Passion fruit woodiness potyvirus Pangda15 isolate (AM409188),
* Corresponding author. Faculty of Liberal Arts and Science, Kasetsart University, Kamphaengsean, Nakorn Pathom 73140, Thailand. E-mail address: faasspp@ku.ac.th (S. Prammanee). 0261-2194/$ e see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2011.07.008

which was shown to be homologous to some isolates of potyviruses previously reported to infect passion fruit,such as East African passiora virus (EAPV) and soybean mosaic virus (SMV) at 74 and 72% identity, respectively (Chiemsombat et al., 2007). The virus causes severe disease resulting in great yield losses and reduced fruit quality. In practice, most planting stocks are prepared by using the purple passion fruit as the scion grafted onto the yellow variety stock to control soil-borne diseases, such as Fusarium wilt caused by Fusarium. oxysporum f. sp. passiorae and viral diseases (Nakasone and Paull, 1998; Amugune et al., 1993). Plant propagation using infected shoots as the grafting material produces grafted plants that are also infected. Several methods have been suggested to address this problem. Isutsa (2004) developed a micropropagation protocol for generating healthy yellow passion fruit plants in which proliferating the yellow variety on a medium containing 6benzylaminopurine (BAP) resulted in ex vitro, rooting (96% rooting, three roots per shoot, 92% survival) that was signicantly better

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Fig. 1. Steps in the technique used to cut and culture the meristem tip of the purple passion fruit plant (a). The shoot development and propagation of the apical meristem in culture containing 1.0 mg/l BA (b) or 1.5 mg/l BA (c). Shoot propagation on MS medium supplemented with 1.0 mg/l BA (d). An individual shoot preparation for root formation (e).

in vitro than rooting (62% rooting, one root per shoot on IBA medium, 50% survival). The purple variety proliferated satisfactorily only on a medium containing both BAP and gibberellic acid 3 (GA3). Consistent with its restricted proliferation conditions, purple passion fruit rooting was difcult and survival was poor when compared with those of the yellow passion fruit. A multiplication rate of 4.5 per month can be achieved using Murashige and Skoog medium supplemented with 2% sucrose and 2 mg/l BAP, no callus is produced. Problems of chlorosis and a method for avoiding subsequent death after prolonged culture were reported by Kantharajah and Dodd (1990). Plant regeneration of passion fruit through leaf disc in vitro culture. Greatest bud formation was found with 0.6 mg/l of BA when the shoots were placed in MS media without hormones for rooting, resulting in an average of ve roots per shoot (Otahola, 2000). Trevisan et al. (2006) suggested the use of transgenic plants, but the recovery of transgenic shoots cultured on selective media was reported to be difcult due to the very low frequency of in vitro bud elongation. Unfortunately, these methods could not eliminate PWV from the purple passion fruit growing areas. Another classic method utilises the shoot apical meristem, which is known to be a part of plants that is virus-free (Mori and Hosokawa, 1977). Katoh et al. (2003) obtained virus-free sweet pepper plants using the shoot-tip excised from virus-infected plants grown in a greenhouse that were grafted onto in vitro rootstock seedlings. This method was further developed for use on some fruit trees, including oranges (Navarro et al., 1974) and peaches (Navarro et al., 1982). After grafting, some tips turned brown and did not grow further. The aim of the present study was to propagate virus-free passion fruit plants by regenerating the shoot directly from the meristem tip of virus-infected purple passion fruit parent explants to avoid their genetic variations and produce a virus-free planting stock.

2.2. Apical meristem culture and shoot regeneration The shoots of purple passion fruit samples from the eld were cut and cleaned with water before removing small leaves that covered shoot tips. The shoot tips were soaked in a solution of 0.5% carbendazim for 15 min, surface sterilised in 5% (v/v) and 10% (v/v) sodium hypochlorite with 0.1% (v/v) Tween-20 for 15 min, followed by rinsing three times with sterile, distilled water for 5 min each. The apical meristem was cut to a length of approximately 2 mm and cultured on MS medium (Murashige and Skoog, 1962). The surviving meristems were transferred to MS medium supplemented with various concentrations of benzyladenine (BA) (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mg/l) and 1-naphthaleneacetic acid (NAA) (0.2, 0.5, 1.0, 1.5 and 2.0 mg/l) to determine the composition of the optimal growth medium. Growth was determined by weight, using ve replicates per treatment. The steps in the process used to cut the meristem tip are shown in Fig. 1a. 2.3. Virus detection in the shoots regenerated from apical meristems using ELISA and an immune strip kits Leaves from healthy yellow seedlings and diseased purple passion fruit plants were served as the negative and positive controls, respectively. These samples were tested for woodiness virus using the ELISA technique described by Clark and Adams (1977). The polyclonal antiserum raised against puried preparation of Pangda15 isolate of PWV was locally produced. Immunoglobulin G (IgG) was extracted using ammonium sulphate
Table 1 Passion fruit woodiness virus (PWV) detection by ELISA. The following samples were analysed by ELISA: PWV-inoculated leaves from the purple passion fruit variety; regenerated shoot from meristem culture; healthy leaf of a yellow passion fruit seedling. Sample PWV-inoculated leaf Regenerated shoot Healthy seedling leaf Average O.D. 405 nma 0.504 0.261 0.190 PWVb Positive Negative Negative

2. Materials and methods 2.1. Plant material Shoots from purple passion fruit plants were collected in the Samoeng district, Chiang Mai province, located in northern Thailand. The samples were naturally infected by PWV. The plants showed various symptoms of the disease, including leaf and fruit mosaic, leaf mottle mosaic, leaf curl and abnormal formations.

a Average O.D. was a mean O.D. from two replicate wells per sample reading taken 60 min after adding the substrate solution. b Tested samples with an average O.D. 405 ELISA value at least two times higher than the healthy sample are considered to be positive for PWV.

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Fig. 2. Root formation of regenerated shoots using different concentrations of IBA: 0.0 (a), 0.2 (b), 0.4 (c), 0.6 (d), 0.8 (e) and 1.2 mg/l (f). Virus-free purple passion fruit plants propagated in large numbers (g and h).

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precipitation procedure and adjusted to 1.5 mg/ml concentration before being coated with 40 nm colloidal gold particles. An immunostrip kit was developed according to gold-labelled IgG ow technique as described by Chiemsombat et al. (2009), and was used for virus assay in the passion fruit plant parts. 2.4. Shoot propagation and root formation The regenerated shoots were tested for PWV prior to being used for propagation. The virus-free shoots were propagated by subculturing every two weeks on MS medium supplemented with 1.0 mg/l BA. Individual shoots were cut and transferred to the root formation medium, which contained MS supplemented with IBA at different concentrations (0.0, 0.2, 0.4, 0.6, 0.8 and 1.2 mg/l). 3. Results 3.1. Apical meristem culture using plant growth regulators to induce shoots directly from the meristem The apical meristems were cultured on two plant growth regulators, BA and NAA, at various concentrations. BA (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mg/l) and NAA (0.2, 0.5, 1.0, 1.5 and 2.0 mg/l) were used to induce the shoot directly from the meristem cells. The growth and average weight of the cultures on BA at concentrations of 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mg/l were 0.0385, 0.0663, 0.1354, 0.0055, 0.0059 and 0.0272 g, respectively. Thus, the concentrations of BA at 1.0 and 1.5 mg/l produced a greater number of shoots than any other concentration. The cultures from the 1.0 and 1.5 mg/l concentrations were then subcultured every two weeks for a total of four times (Fig. 1b,c). Initially, the tissue cultured in the 1.5 mg/l BA medium grew faster than that cultured in the presence of 1.0 mg/l BA. The tissue cultured with 1.5 mg/l BA generated many short shoots. In contrast, the tissue cultured with 1.0 mg/l BA generated long shoots that could be subcultured for individual plants. The shoots were propagated on 1.0 mg/l BA medium (Fig. 1d, e), but no shoots were produced in the presence of any concentration of NAA. 3.2. PWV Assays of the regenerated shoot by ELISA and the immunostrip kit The shoots regenerated from the apical meristems of purple passion fruit were assayed for PWV. Leaves from healthy yellow seedlings and diseased purple passion fruit plants were used as the negative and positive controls, respectively. Four randomly selected regenerated shoots were shown to be PWV-free by ELISA (Table 1). The immunostrip kit for PWV detection revealed that sap samples from these same regenerated shoot samples were also virus-free. PWV was readily detected in diseased leaf sap (positive control) as shown by a purple band of the test line on the strip within a minute. 3.3. Root formation using the plant growth regulator IBA to complete the propagation of a virus-free plant The plant growth regulator IBA was used to promote root formation. Various concentrations (0.0, 0.2, 0.4, 0.6, 0.8 and 1.2 mg/l) were added to the MS medium to determine the optimal concentration of IBA to culture the regenerated shoot (Fig. 2aef). After an approximately one-month subculture, IBA concentration of 0.4 or 0.6 mg/l were shown to induce better root formation than the other concentrations. Thus, all shoots were cultured on a medium supplemented with 0.4 mg/l IBA to produce whole, virus-free purple passion fruit plants (Fig. 2 g, h).

4. Discussion The viral concentration measure in the shoot apex of virus-infected plants differ among plant and virus species. Tobacco mosaic virus (TMV), potato virus X (PVX) and potato virus S (PVS) could be eliminated using a culture from a very small meristem tip (0.1e0.3 mm), but cucumber mosaic virus (CMV), potato virus Y (PVY), potato leaf roll virus (PLRV) and internal cork virus (ICV) were eliminated using a larger size of meristem tip (1.0e3.0 mm) (Mori and Hosokawa, 1977). In the present experiment, PWV was eliminated by cutting the meristem tip at a length of approximately 2 mm. The larger size was able to develop more easily, with the addition of 0.1 mg/l BA inducing direct shooting from the meristem tissue. Verma et al. (2004) observed that a smaller meristem (0.1e0.2 mm) resulted in callus development and that a larger meristem size (greater than 0.3 mm) decreased the efciency of the viral elimination. Virus elimination through tissue culture techniques requires an efcient plant regeneration system. Plant growth regulators used for shoot regeneration and root formation vary depending on the plant. For example, pumpkin meristem tip culture initially using MS medium containing 8 mg/l Indole-3acetic acid (IAA), produced maximum shoot proliferation with the addition of 1.0 mg/l BA and maximum root formation on a medium containing 8 mg/l IAA (Pink and Walkey, 1984). Regeneration of the garden chrysanthemum shoot meristem used MS medium supplemented with BAP and IBA (Verma et al., 2004). Kumar et al. (2009), however, reported that 3.0 mg/l BAP and 0.5 mg/l NAA were suitable for multiple shoot regenerations, but the best response of buddleia cultivars was obtained using 5.0 mM thidiazuron (TDZ), resulting in more than 101 shoots per explant. BA was less effective (Phelan et al., 2009). In the present experiment, the apical meristem was cut and successfully developed into whole, virus-free plants using IBA as the plant growth regulator. The results show that shoots can be regenerated directly from apical meristems. Acknowledgements The authors thank the Royal Project Foundation for the research grant funding this study. This experiment was conducted in the cell biology laboratory of the Faculty of Liberal Arts and Science, Kasetsart University, Kamphaneg Saen, Nakhon Pathom, Thailand. We also thank the Kasetsart University Research and Development Institute (KURDI) for supporting the grant for manuscript preparation. References
Amugune, N.O., Gopalan, H.N.B., Byterbier, B., 1993. Leaf disc regeneration of passion fruit. Afric. Crop Sci. J. 1, 99e104. Chiemsombat, P., Keeratiya-angool, S., Prammanee, S., Pipattanawonge, N., 2007. Production of antibody and test kit for the detection of passion fruit woodiness virus. In: Annual Report Meeting on Research Project of Royal Project Foundation, (14e15 November 2007) in Chaing Mai Thailand, pp.132e141. Chiemsombat, P., Hongprayoon, R., Larprom, A., 2009. Development of a simple strip test for rapid diagnosis of sugarcane mosaic virus infecting sweet corn in Thailand. Proceedings of The First International Conference on corn and sorghum research. 8e10 April 2009, Pattaya City, Thailand, pp. 216e222. Clark, M.F., Adams, A.N., 1977. Characteristics of the microplate method of enzymelinked immunosorbent assay for the detection of plant viruses. J. Gen. Virol. 34, 475e483. Isutsa, D.K., 2004. Rapid micropropagation of passion fruit (Passiora edulis Sims.) varieties. Sci. Hortic. 99, 395e400. Kantharajah, A.S., Dodd, W.A., 1990. In vitro micropropagation of Passiora edulis (purple passionfruit). Annals. Bot. 65, 337e339. Katoh, N., Yui, M., Sato, S., Shiral, T., Yuasa, H., Hagimori, M., 2003. Production of virus-free plants from virus-infected sweet pepper by in vitro grafting. Sci. Hortic. 100, 1e6.

S. Prammanee et al. / Crop Protection 30 (2011) 1425e1429 Kumar, S.S., Khan, M.S., Raj, S.K., Sharma, A.K., 2009. Elimination of mixed infection of cucumber mosaic and tomato aspermy virus from Chrysanthemum morifolium Ramat. cv. Pooja by shoot meristem culture. Sci. Hortic. 119, 108e112. Mori, K., Hosokawa, D., 1977. Localization of viruses in apical meristem and production of virus-free plants by means of meristem and tissue culture. Acta Hortic. 78, 389e396. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassay with tobacco tissue cultures. J. Plant Physiol. 15, 473e497. Nakasone, H.Y., Paull, R.E., 1988. Tropical Fruit. CAB International, Wallingford, UK. Navarro, L., Llacer, G., Cambera, M., Arregui, J.M., Juarez, J., 1982. Shoot-tip grafting in vitro for elimination of viruses in peach plants (Prunus persica Batsch). Acta Hortic. 130, 185e192. Navarro, L., Roistacher, C.N., Murashige, T., 1974. Improvement of shoot-tip grafting in vitro for virus-free citrus. J. Am. Soc. Hort. Sci. 100, 471e479.

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Otahola, V., 2000. Regeneracion de plantas de parchita (Passiora edulis f. Flavicarpa) a partir del cultivo in vitro de discos de hojas. Bioagro 12, 71e74. Phelan, S., Hunter, A., Douglas, G.C., 2009. Effect of explants source on shoot proliferation and adventitious regeneration in 10 Buddleia cultivars. Sci. Hortic. 120, 518e524. Pink, D.A.C., Walkey, D.G.A., 1984. Rapid propagation of Cucurbita pepo L., by culture of meristem tips. Sci. Hortic. 24, 107e114. Trevisan, F., Mendes, B.M.J., Maciel, S.C., Vieira, M.L.C., Meletti, L.M.M., Rezende, J.A.M., 2006. Resistance to passion fruit woodiness virus in transgenic passion ower expressing the virus coat protein gene. Plant Dis. 90, 1026e1030. Verma, N., Ram, R., Hallan, V., Kumar, K., Zaidi, A.A., 2004. Production of cucumber mosaic virus-free chrysanthemum by meristem tip culture. Crop Prot. 23, 462e473.