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Establishment of a Micropropagation Protocol for Elite Accessions of Bael (Aegle marmelos (L.) Corr.), a Tropical Hardwood Species

Establishment of a Micropropagation Protocol for Elite Accessions of Bael (Aegle marmelos (L.)... Hindawi Advances in Agriculture Volume 2020, Article ID 8840386, 10 pages https://doi.org/10.1155/2020/8840386 Research Article Establishment of a Micropropagation Protocol for Elite Accessions of Bael (Aegle marmelos (L.) Corr.), a Tropical Hardwood Species 1,2 1 1 Chamila Pathirana , Udayanthi Attanayake, Udula Dissanayake, 3 3 2,4 Lakshman Gamlath, Kalyani Ketipearachchi, Terrence Madhujith, 1,2 and Janakie Eeswara Department of Crop Science, Faculty of Agriculture, University of Peradeniya, Peradeniya 20400, Sri Lanka Postgraduate Institute of Agriculture, University of Peradeniya, Peradeniya 20400, Sri Lanka Fruit Crop Research & Development Station, Gannoruwa, Peradeniya, Sri Lanka Department of Food Science and Technology, Faculty of Agriculture, University of Peradeniya, Peradeniya 20400, Sri Lanka Correspondence should be addressed to Chamila Pathirana; ckpathirana0421@gmail.com Received 20 April 2020; Revised 16 July 2020; Accepted 9 September 2020; Published 22 September 2020 Academic Editor: Ga ´bor Kocsy Copyright © 2020 Chamila Pathirana et al. *is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. *e limited availability of high-quality planting material hinders the popularization of bael (Aegle marmelos (L.) Corr.) as a profitable cash crop in Sri Lanka. Five elite bael accessions, namely, Beheth Beli, Paragammana, Mawanella, Rambukkana, and Polonnaruwa-Supun, have been identified and used for mass propagation through budding and grafting. However, this process gets hampered by the limitations in large-scale production of planting material. Micropropagation is an alternative technique to produce clonal plants in large-scale; however, no established protocol is available for the field-grown elite bael accessions in Sri Lanka, and hence, the present study was conducted. *e best sterilization method was found to be the washing of explants in a 2.5% fungicide solution for two hours. *e full MS and ½MS media provided significantly similar performance in shooting, as indicated by the measured parameters. *e twig plants did better than leaf explants concerning shoot extension, in which twig explants yield 1.33 cm taller shoots. *e 1 mg/l of BAP concentration generated the highest number of shoots (6.20) and the most extended shoots (3.83 cm). *e most successful rooting (60% success) was spotted with full MS, 1 mg/ml NAA, and 3% sugar. As previously reported, the successful micropropagation is possible if the explants are harvested from April to June, immediately after the fruiting season of the plant. *e established protocol can mass-produce clonal bael plants from the elite accessions. temples dedicated to ‘God Shiva’ [2]. *e fruits and other 1. Introduction plant parts of bael contain essential phytochemicals such as Aegle marmelos (L.) Corr. of family Rutaceae, commonly tannin, coumarin, aegelinol, and marmelocin, and they exhibit various medicinal properties ranging from laxative, known as bael, is a medicinal fruit tree fruit species [1]. Bael is native to India and found in other South Asian countries antiproliferative, antidiabetic, and anticancerous capabilities such as Sri Lanka and Bangladesh, Southeast Asia, and Egypt [7, 8]. [2]. *e ripened fruit is the economically important plant Bael is a well-established medicinal fruit tree species, and part of bael, and all the plant parts have essential medicinal the selections from the wild germplasm have led to larger properties [3, 4]. *e pulp of the ripened fruits is delicious fruit sizes and superior fruit qualities. *e elite bael plants and can also be processed into value-added products such as have been identified, and attempts have been made to jam, syrup, sweets, and herbal drinks [5, 6]. Bael is con- multiply them for large-scale cultivations in India [9]. sidered as a sacred tree in India and often grown near the However, the propagation using seeds is often hampered by 2 Advances in Agriculture the extreme genetic heterozygosity due to open pollination, mixture and smooth bristles. *e explants were rinsed using lower seed germination rates, and difficulty in collecting and running tap water for 10–15 mins. After washing three to four times, the plant parts were soaked in distilled water processing seeds from mature fruits. Furthermore, it is challenging to produce a large amount of planting material approximately for one hour. *en, subsequent steps were through budding and grafting techniques. *us, plant tissue followed for further sterilization to avoid the bacterial and culture techniques can be used as an alternative to overcome fungal infections. problems associated with the multiplication of superior trees identified in the wild/field. Micropropagation techniques 2.2.2. Fungicide Treatment. *e plant parts were soaked in a have been successfully employed for rapid multiplication of series of fungicide (commercial formulation thiram with the the superior bael plants using cotyledons [10–13], nodes [9], active ingredient dimethyldithiocarbamate (C H N S )) 6 12 2 4 and the axillary buds [14, 15]. However, it is essential to solutions (0.5%, 1.0%, 1.5%, 2.0%, and 2.5%). At each develop a micropropagation protocol for the explants col- concentration of fungicide, the plant parts were subjected to lected directly from the mother plants growing in the field to different exposure times (1 hr, 2 hr, and overnight). multiply the bael accessions. *e establishment of cultures without contamination is one of the major problems when explants are collected from field-grown trees [16]. *us, the 2.2.3. Surface Sterilization. *e plant parts were washed development of a specific sterilization technique for plant carefully with distilled water to remove the fungicides. After materials obtained from the field is an essential requirement that surface sterilization was performed for the explants. *e before identifying the growth stage for the collection of plant leaf explants were transferred into a wash bottle, which materials and perfecting the medium composition for consisted of 10% Clorox (sodium hypochlorite (NaOCl)) multiplication of plants. *erefore, the present study was and a drop (1 ml) of polyethylene glycol sorbitan mono- conducted to develop a protocol for multiplication of bael laurate (Tween 20 (T20)) and shaken for 10 min. *en, from plant materials collected from trees identified as ac- bleach solution was drained off and washed three times cessions by the Fruit Research and Development Station carefully with autoclaved water. *at step was repeated with (FCRDS) at Gannoruwa, Sri Lanka. only 10% NaOCl, and cleaned plant parts were transferred to a vessel containing distilled water. *e nodal segments and 2. Materials and Methods shoot tips (i.e., twigs) were surface-sterilized separately using 10% of Clorox along with two drops of Tween 20. *e 2.1. Plant Material. Five Sri Lankan bael accessions, namely, shaking was continued for ten minutes. *en, the twigs were Beheth Beli (BB), Paragammana (PA), Mawanella (MA), transferred to a 10% NaOCl solution without Tween 20 and Rambukkana (RA), and Polonnaruwa-Supun (PS), which were shaken well for another 10 mins followed by thorough have been selected for large-scale production of planting washing (three times) with sterile, deionized, distilled water materials, were used to collect the explants for developing a to wash off the traces of bleach solution. Each washing protocol for micropropagation. Leaves and twigs were used process lasted for approximately one minute. as the explants. *e study was conducted at the Tissue Culture Laboratory of the Department of Crop Science, Faculty of Agriculture, University of Peradeniya, Sri Lanka. 2.2.4. Culture Establishment. All tissue culture techniques *e explants from the mother plant of BB were collected were conducted under aseptic conditions in a laminar flow from FCRDS (GPS coordinates: 7.277006, 80.595299) during cabinet (Labgard Class II, Type A/B3). *e leaf and twig the vegetative growth stage (April–June, 2018) [17]. All the explants were prepared by trimming the cut surfaces to investigations on sterilization and identification of the about 12–15 mm in length. *e MS [18] medium supple- specific growth stage for culture establishment were con- mented with 1 mg/l of BAP, 3% (w/v) of sucrose, and so- ducted using BB mother plants. *en, the results achieved lidified with 0.2% (w/v) Phytagel (Sigma, UK) was used as were applied to other four accessions when they were at the the medium based on the results obtained for nodal seg- same vegetative growth stage, which are located at distant ments collected from in vitro grown seedlings in the tissue locations from the study site. culture laboratory of the Department of Crop Science. *e pH of the medium was adjusted to 5.8 with 1 M HCl or NaOH before autoclaving at 120 C for 20 mins, 15 psi. *is 2.2. Experiment 1: Effect of Fungicide on Surface Sterilization of medium was used for initiating the cultures. Each culture Twig and Leaf Explants tube contained 10 ml of medium. *e inoculation was 2.2.1. Preparation and Washing of Explants. *e disease-free performed as one explant per culture tube. *e leaf pieces at immature leaves and tender twigs were collected from top the size 1 cm (approximately) holding the midribs were parts of the branches of each mother plant and immediately isolated and placed on the medium. A gentle press was transferred to the laboratory for micropropagation. *e applied to have an even contact between the abaxial (lower) dried/dead and infected portions were removed from the surface of the leaf and the medium. *e single nodal twigs intended explants. *e twigs and leaves were isolated, and were inserted into the medium in the upright position. *e the twigs containing at least three to four internodes were leaf cultures were incubated inside a dark cabinet at 26± 2 C used for culturing. *e leaves and twig explants were until initiating the shoots. *e twig cultures were incubated thoroughly cleaned using a commercially available detergent at 16 hrs of light (flux density) at 25± 2 C. Advances in Agriculture 3 2.2.5. Scanning Electron Microscopy to Detect the Presence of 3. Results Fungi in Leaf and Twig Explants. *e fungicide-treated 3.1. Experiment 1: Effect of Fungicide on Surface Sterilization. (2.5%) and untreated leaf explants were subjected to scan- *e 2.5% fungicide solution in which explants were im- ning electron microscopy (SEM) to visualize the effect on mersed for two hours produced the only successful regen- fungicide treatment on the fungi present on explants. *e eration. When the leaf and twig explants were subjected to leaves and twigs were taken and soaked in the fungicide 2.5% fungicide for two hours of treatment and subsequently solution for two hours. After that, they were cut into explant cultured in 1 mg/l BAP culture, initially till the fourth week, size pieces. *e explants were then sprayed with gold par- substantial amounts of contaminated and few browned ticles for staining purpose and observed using the ZEISS cultures were observed. However, eventually, nearly 60% of scanning electron microscope (SEM) (Jeol SEM 6400, Tokyo, cultures of both leaf and twig explants survived as healthy Japan). *e SEM photographs were taken from the repre- cultures after 5-6 weeks of establishment. sentative places of the explants. 3.2. Effect of Fungicide Treatment on Fungi Present on 2.3. Experiment 2: Effect of N Content in Medium on In Vitro Explants. *e fungal hyphae were observed in upper Multiplication. *e effect of N content on the successful (Figure 1(a)) and lower (Figure 1(b)) surfaces of the leaf establishment of the cultures was assessed. To detect the explants. *e fungicide treatment destroyed the fungal effect of N, full MS and half MS media were prepared. *e hyphae observed in the upper (Figure 1(c)) and bottom parameters such as the number of leaves/shoot, number of surfaces (Figure 1(d)). *e sunken stomata were clearly shoots/cutting, and height of the shoot were measured after visible in both upper and lower surfaces of the leaves eight weeks of establishment and statistically analyzed using (Figures 1(a)–1(d)). In the twig explants under 1000 and the GLM procedure in the Statistical Package SAS 9.4 (SAS 5000 magnifications, fungal hyphae were not observed in Institute, NC, Cary, USA). untreated cases (Figures 1(e) and 1(g), respectively). *e similar appearance was photographed for treated explants under the two magnifications (Figures 1(f ) and 1(h)). 2.4. Experiment 3: Effect of BAP Concentration on Multipli- Compared to the stomata in leaves, the stomata in twigs are cation of Regenerated Shoots. *e effect of five BAP con- smaller in size (Figures 1(e)–1(h)). centrations (0.0, 0.5, 1.0, 2.0, and 4.0 mg/l) on shoot multiplication was investigated. *e subculturing was per- formed at every eight-week intervals. Each proliferated 3.3. Experiment 2: Effect of N Content in the Medium on In adventitious shoot was cut from the basal end and sub- Vitro Multiplication of Regenerated Shoots. *e mean cultured again for further multiple shoot induction. At the number of shoots per cutting and the mean number of leaves end of the 20th week onwards, regenerated multiple shoots per shoot were not significantly different among the two were cut, and the shoots were transferred on to an MS medium compositions and the type of explant (P> 0.05). medium without any plant growth regulators before However, the mean height of the shoot was significantly transferring into a rooting medium. *e number of shoots higher in twig explants than the leaf explants (P< 0.05) per explant and the length of the shoots were measured and (Table 1). Two culture jars of full MS and ½MS are shown in subjected to GLM procedure in SAS. Figure 2 to display the growth differences. 2.5. Experiment 4: Effect of N, Sucrose, IBA, and NAA Con- 3.4. Experiment 3: Effect of BAP Concentration on Multipli- centration on Rooting. *e effects of N (full and ½MS), cation of Regenerated Shoots. *e BAP level of 4 mg/L caused sucrose concentration (3% and 6%), naphthalene acetic acid vitrification of shoots even after subculturing (Figures 3(a) (NAA) (1 mg/L), and indole-3-butyric acid (IBA) (1 mg/L) and 3(b)). *e BAP concentration of 1.0 mg/L provided the on rooting of micropropagated shoots were investigated. *e highest success. *e mean highest number of shoots was experiment was arranged as a three-factor factorial observed for the BAP levels of 0.5, 1.0, and 2.0 mg/l experiment. (5.60–6.20). At the BAP level of 4.0, the mean number of 3.40 shoots was observed, and only two shoots were observed when no BAP was added. *e mean shoot length was sig- 2.6. Experiment 5: Acclimatization of In Vitro-Generated nificantly different among all the BAP treatments. *e sig- Plantlets. *e in vitro rooted plants were maintained in jars nificantly mean highest shoot length was observed at the BAP with ventilation for a month and after that transferred to level of 1.0 mg/L (3.83 cm), whereas the mean lowest shoot small plastic pots (10 cm in diameter) containing a standard length was observed when no BAP was added (2.49 cm) potting mixture and allowed to grow in a greenhouse. *e (P< 0.05) (Table 2). *e shoots generated by BAP treatments plants were covered with polyethylene sheets to maintain the other than the 4 mg/L concentration are displayed in Figure 4. relative humidity. *e artificial lights were provided to have 70% light intensity. *e covers were intermittently removed after one week every day and completely removed after two 3.5. Experiment 4: Effect of N, Sucrose, IBA, and NAA Con- weeks. After four weeks, the survival percentage was centrations on Rooting. Table 3 displays the series of results calculated. obtained for the rooting experiments by altering N (MS or 4 Advances in Agriculture (a) (b) (c) (d) (e) (f) (g) (h) Figure 1: Scanning electron microscopy photographs of the leaf and twig explants (untreated and treated with fungicide). (a) Upper surface of leaves untreated; (b) upper surface of leaves treated; (c) lower surface of leaves untreated; (d) lower surface of leaves treated; (e) twig untreated (×1000); (f ) twig treated (×1000); (g) twig untreated (×5000); (h) twig treated (×5000). *e specific details of each photograph are marked as a label with each photograph. Table 1: *e mean performance of the shoots under full MS and ½MS (N) conditions. Medium Type of explant No. of shoots/cutting Height of the shoot (cm) No. of leaves/shoot a b a Leaf 7.40 2.27 3.67 Full MS a a a Twig 7.25 3.25 6.53 a b a Leaf 6.80 1.93 4.33 ½MS a a a Twig 6.50 3.60 5.57 Means denoted by the same letters within columns are not significantly different at P< 0.05. Advances in Agriculture 5 (a) (b) Figure 2: *e comparison of shoot growth. (a) Full MS and (b) ½MS media. Scale bar, one cm. (a) (b) Figure 3: Vitrification of shoots caused by higher BAP concentrations. (a) Vitrified shoots inside the glass tubes; (b) vitrified shoots taken out from the medium. Scale bars represent one cm separately for panels A and B. ½MS), sugar level (3% and 6%), and hormone (IBA or (Figures 5(a)-5(b) and 6(d)). When the enlarged part was cut NAA). It was clear that NAA was capable of generating roots off and recultured, the enlargement happened once again. required for bael shoots in in vitro cultures. It also high- *e 6% sugar levels also lead to the vitrification of shoots at later stages as in the shoots shown in Figure 3. lighted the fact that growth regulators are needed for rooting as zero hormonal applications did not produce any positive rooting results. Regarding the N content, either full MS or 3.6. Experiment 5: Acclimatization of In Vitro-Generated ½MS was fine in giving positive results; however, full MS provided more rooting success (60%) than ½MS, which Plants. *e plantlets were responsive to the acclimatization treatments slowly and displayed improved tolerance to the displayed only 33% success (Figures 5(a)-5(b) and 6(a)– 6(c)). *e 6% sugar and 1 mg/l of NAA also produced lower relative humidity and 70% light intensity. After two weeks, nearly 80% of the plants survived, and at the end of enlarged ends in the shoot bases proving that 6% sugar level is too much for rooting of bael in in vitro cultures the four weeks, 60% of the plants survived and was ready to 6 Advances in Agriculture Table 2: *e growth of shoots under BAP treatments. BAP (mg/l) Mean number of shoots Mean length of shoots (cm) c e 0.0 2.00 2.49 a d 0.5 5.60 2.99 a a 1.0 6.20 3.83 a b 2.0 6.00 3.34 b c 4.0 3.40 3.14 Within a column, means followed by the same letter are not significantly different at P< 0.05. Figure 4: *e nonvitrified shoots generated by lower BAP concentrations. *e shoots are prepared for measurements and subculturing. Scale bar represents one cm. Table 3: Effect of N, hormone, and sugar levels on rooting of in vitro cultures of bael Medium composition (%) sugar Hormone and concentration (mg/l) Number of shoots produced roots (out of 20) 0 0 3 1 NAA 12 1 IBA 0 Full MS 0 0 6 1 NAA 0 1 IBA 1 0 0 3 1 NAA 6 1 IBA 0 ½ MS 0 0 6 1 NAA 2 1 IBA 1 be used in planting purposes. After two months, 42% of the optimizations were required. *erefore, the present study plants remained in the healthy condition, which is a higher possesses a paramount significance as for the first time in Sri value for woody perennials. Lanka, we have established a sound and easier protocol to clonally multiply the field-grown elite accessions of bael using leaf and twig explants. 4. Discussion Micropropagation techniques have been successfully A well-established micropropagation procedure is essential employed for rapid multiplication of the superior bael using nodal segments [9], cotyledons [10–13], nodes [9], and the for bael if it is to be established as a country-wide cash crop [19]. *e lower percentage of seed germination, the presence axillary buds [14]. *e type of explant and various other of miniature or aborted seeds in the fruits, and highly conditions required for the establishment of micro- heterozygous nature of the species, which is leading to the propagation technique have been extensively discussed and segregation of traits, are making sexual reproduction inef- compared in the study by Vasava et al. [19]. However, for the fective. *e micropropagation of bael has been tried, and elite bael accessions in Sri Lanka, specific sterilization results were obtained in many studies [9, 12, 13, 20]. technique, hormone combination, and nitrogen content However, we noted that in the present study, such protocols required have not been established to come up with a could not be directly translated to our conditions; hence, successful micropropagation protocol. Advances in Agriculture 7 (i) (ii) (iii) (iv) (v) (vi) (a) (vii) (viii) (ix) (x) (xi) (xii) (b) Figure 5: A set of representative cultures showing the effect of N hormone and sugar on rooting. (a) MS (i, no hormone and 3% sugar; ii, 1 mg/l NAA and 3% sugar; iii, 1 mg/l IBA and 3% sugar; iv, no hormone and 6% sugar; v, 1 mg NAA and 6% sugar; and vi, 1 mg IBA and 6% sugar); (b) ½MS (vii, no hormone and 3% sugar; viii, 1 mg/l NAA and 3% sugar; ix, 1 mg/l IBA and 3% sugar; x, no hormone and 6% sugar; xi, 1 mg NAA and 6% sugar; and xii, 1 mg IBA and 6% sugar). *e sterilization of the explants is the most challenging found to be nearly 50%, and it is a practically higher rate in step in bael micropropagation. It was painstaking to carry any form of micropropagation. *us, the fungal species out the necessary sterilization as the phenological growth might be the reason for contaminations in the early stages of stage of the explant always confounded the sterilization the culture establishment. treatment. *us, when the explants were collected from the To verify the presence of fungi in explants, SEM was appropriate phenological growth stage, the effect of steril- carried out, and it was clear that before the fungicide ization can be studied. *e results demonstrated that im- treatment, fungal hyphae were present (Figures 1(a) and mersing explants in 2.5% fungicide solution for two hours 1(c)) in both surfaces of the leaves, and the fungicide provided the highest success, and that was not significantly treatment was able to destroy the fungal hyphae as observed different among leaf and twig explants. *e success rate was in Figure 1(b) and 5.1D. However, in twig explants 8 Advances in Agriculture (a) (b) (c) (d) Figure 6: *e rooting results. (a) *ree shoots just before inserting in the rooting medium; (b) shoots form roots; (c) rooted plantlets removed for subculturing; (d) bottom enlargement observed. examined, no apparent fungal hyphae were observed number of shoots per cutting, taller shoots, and a higher probably due to the falling off during the sample processing number of leaves per shoot (Table 1; P> 0.05) than ½MS for SEM (Figures 1(e)–1(h)). It was also noted that bael medium. Although both leaf and twig explants produced possesses sunken stomata in both surfaces of the leaves. *e successful results in multiplication, the twig explants per- stomata present in twigs are smaller than the ones present in formed significantly better according to the measured pa- rameters (Table 1; P< 0.05). leaves. *e sunken stomata and small stomata are two significant adaptations for drought. *ese observations are In the shooting experiment, the optimum concentration of BAP for shoot development was 1 mg/l that produces the in line with the drought-tolerant trait of the bael. *e phenological growth stage of the tree subjected to mean highest number of shoots and the significantly mean the collection of explants influences the percentage success most extended shoots (Table 2; P< 0.05). *e BAP con- in micropropagation [21]. Bael has marked phenological centration of 4.0 mg/l did not produce successful results and growth difference concerning the rainfall, temperature, and led to the vitrification of shoots, implying that too much fruiting [22]. Rhagu et al. [21] reported that October–De- BAP is not applicable to bael. In some other studies on bael cember is the best time to collect explants as the plant is at its micropropagation, NAA was used in combination with BAP; best phenological stage for a higher success rate of micro- however, we wanted to make sure the appropriate devel- propagation in Kerala, India. *e phenological stage of the opment of shoots before triggering the rooting so that the attempts were made only using BAP. Bindhu [20] reported plants is essential for deciding the effective fruiting season and collecting the vegetative parts as planting material [23]. that the BAP concentration of 2 mg/l is efficient for the shooting of bael over the other combinations/concentrations During flowering and fruiting of the plants, the internal hormonal profiles often hinder the effective regeneration of BAP/kinetin. Pati et al. [9] have obtained successful capabilities. However, if the plant parts are taken from shooting for bael by applying a combination of BAP and actively growing trees, regeneration can be achieved swiftly NAA. with higher success rates [21]. *e phenology of bael is Full MS with 1 mg/l of NAA would provide at least 50% complex, and vegetative and reproductive phases are success in rooting of bael shoots in in vitro cultures (Table 3). overlapped [22]. In India, the bael phenology has been *e comparable images display the variable success as shown correlated with success rates in the micropropagation [21]. in Figures 4 and 5. In a study conducted at *rissur District, *e ideal phenological stage of bael accessions in Sri Lanka Kerala, India, indoleacetic acid (IAA) at the concentration of 0.01 mg/l was found to be effective in inducing rooting [20]. to collect explants for successful micropropagation is be- tween April and June when there is a predominant vegetative Pati et al. [9] observed successful rooting at a combination of growth [17]. IBA + IAA. Raghu et al. [21] obtained successful results with *e N content, as given by the full or ½MS media, has the ex vitro rooting by adding napthoxyacetic acid (NOA) and same effect on the establishment of cultures. *e full MS IBA in a mix, but we did not attempt ex vitro rooting in the medium did not provide better results in terms of a higher present study. *ese contrasting findings demonstrate the Advances in Agriculture 9 fact that the hormonal concentrations for rooting are highly References specific to the given conditions. [1] R. Chanda, A. Ghosh, T. Mitra, J. P. Mohanty, N. Bhuyan, and G. Pawankar, “Phytochemical and pharmacological activity of 5. Conclusions Aegle marmelos as a potential medicinal plant: an overview,” 8e Internet Journal of Pharmacology, vol. 6, no. 1, p. 3, 2008. *e most productive sterilization method at the explant [2] V. K. Singhal, A. Salwan, P. Kumar, and J. Kaur, “Phenology, preparation stage is the washing of explants collected from pollination and breeding system of Aegle marmelos (Linn.) the field in a 2.5% fungicide solution for two hours. Both leaf correa (Rutaceae) from India,” New Forests, vol. 42, no. 1, and twig explants exhibited similar success rates in un- pp. 85–100, 2011. [3] M. K. Seth, “Trees and their economic importance,” 8e dergoing direct organogenesis and axillary bud proliferation. Botanical Review, vol. 69, no. 4, pp. 321–376, 2003. *e full MS and ½MS media provided the same performance [4] J. Benni, R. Suresha, and M. Jayanthi, “Evaluation of the anti- in shooting. *e twig plants outperformed leaf explants in inflammatory activity of Aegle marmelos (Bilwa) root,” Indian terms of shoot extension, in which twig explants on average Journal of Pharmacology, vol. 43, no. 4, pp. 393–397, 2011. yields 1.33 cm taller shoots. *e BAP concentration of 1 mg/l [5] M. S. Baliga, H. P. Bhat, N. Joseph, and F. Fazal, “Phyto- produces the highest number of shoots (6.20) and the most chemistry and medicinal uses of the bael fruit (Aegle marmelos extended shoots (3.83 cm). *e successful rooting was ob- Correa): a concise review,” Food Research International, served with full MS, 1 mg/ml NAA, and 3% sugar with 60% vol. 44, no. 7, pp. 1768–1775, 2011. cultures compared to the other treatments employed. *ese [6] J. F. Morton, Fruits of Warm Climates, Creative Resource results could be successfully applied in the mass production Systems Inc, Winterville, NC, USA, 1987. of clonal bael plants from elite accessions to establish the [7] S. Charoensiddhi and A. Anprung, “Bioactive componds and species bael as a profitable cash crop in Sri Lanka. volatile compounds of *ai bael fruit (Aegle marmelos (Linn.) correa) as a valuable source for functional food ingredients,” International Food Research Journal, vol. 15, no. 3, pp. 287– Data Availability 295, 2008. [8] V. B. Lambole, K. Murti, U. Kumar, B. P. Sandipkumar, and *e data used to support the findings of this study are V. Gajera, “Phytopharmacological properties of Aegle mar- available from the corresponding author upon request. melos as a potential medicinal tree: an overview,” Interna- tional Journal of Pharmaceutical Sciences Review and Research, vol. 5, no. 2, pp. 67–72, 2010. Disclosure [9] R. Pati, R. Chandra, U. K. Chauhan, M. Mishra, and *e funders had no role in the design of the study; in the N. Srivastava, “In vitro clonal propagation of bael (Aegle collection, analyses, or interpretation of data; in the writing marmelos Corr.) CV. CISH-B1 through enhanced axillary branching,” Physiology and Molecular Biology of Plants, of the manuscript; or in the decision to publish the results. vol. 14, no. 4, pp. 337–346, 2008. [10] C. B. Pradeepa Devi, R. M. Gopal, and A. Settu, “Plant re- Conflicts of Interest generation of Aegle marmelos (L.) corr. from cotyledon ex- plants through In vitro studies,” Journal of Natural Product *e authors declare that they have no conflicts of interest. and Plant Resources, vol. 4, no. 2, pp. 52–55, 2014. [11] P. Nayak, P. R. Behera, and T. Manikkannan, “High frequency plantlet regeneration from cotyledonary node cultures of Authors’ Contributions Aegle marmelos (L.) Corr.” In Vitro Cellular & Developmental Biology—Plant, vol. 43, no. 3, pp. 231–236, 2007. C. P., T. M., and J. E. were involved in conceptualization; [12] D. P. Prematilake, H. A. S. Nilmini, and C. Kudagamage, “Es- C. P., U. A., U. D., L. G., K. K., and J. E. were involved in tablishment of an in vitro plant regeneration system for Aegle methodology; C. P. was involved in data curation; L. G., marmelos (L.) Corr. via organogenic callus culture,” Ceylon Journal K. K., and C. P. were involved in collecting plant material; of Science—Biological Science, vol. 35, no. 2, pp. 87–90, 2006. C. P. was involved in writing the original draft and in vi- [13] M. Hossain, R. Islam, M. R. Karim, O. I. Joarder, and sualization; C. P., T. M., and, J. E. were involved in editing, B. K. Biswas, “Regeneration of plantlets from in vitro cultured and reviewed the manuscript; T. M. and J. E. were involved cotyledons of Aegle marmelos Corr. (Rutaceae),” Scientia in supervision and funding acquisition; and J. E. was in- Horticulturae, vol. 57, no. 4, pp. 315–321, 1994. volved in project administration. All authors have read and [14] D. Ajithkumar and S. Seeni, “Rapid clonal multiplication agreed to the published version of the manuscript. through in vitro axillary shoot proliferation of Aegle marmelos (L.) Corr., a medicinal tree,” Plant Cell Reports, vol. 17, no. 5, pp. 422–426, 1998. Acknowledgments [15] K. Gandhi, E. Rajesh, S. Saravanan et al., “Micropropagation of aegle marmelos L. a medicinal tree through culture of *e authors would like to thank the Department of Geology, axillary bud and shoot tip explants from In vitro germinated Faculty of Science, University of Peradeniya, Sri Lanka, for seedlings,” International Journal of Scientific Research in Bi- kindly providing scanning electron microscope facilities. ological Sciences, vol. 5, no. 3, pp. 65–71, 2018. *is research was funded by the National Science Foun- [16] J. P. Eeswara, T. Stuchbury, E. J. Allan, and A. J. Mordue, “A dation, Sri Lanka (research grant no. RG/2015/BT/05). standard procedure for the micropropagation of the neem tree 10 Advances in Agriculture (Azadirachta indica A. Juss),” Plant Cell Reports, vol. 17, no. 3, pp. 215–219, 1998. [17] C. K. Pathirana, A. M. U. R. K. Attanayake, D. M. U. S. K. Dissanayake et al., “Effect of phenological growth stage on establishment of In-vitro cultures of Bael (Aegle marmelos (L.) Corr.),” Tropical Agricultural Research, vol. 29, no. 3, pp. 268–275, 2018. [18] T. Murashig and F. Skoog, “A revised medium for rapid growth and bioassay with tobacco tissue culture,” Plant Physiology, vol. 15, no. 3, pp. 473–797, 1962. [19] D. Vasava, M. M. Kher, M. Nataraj, and J. A. Teixeira da Silva, “Bael tree (Aegle marmelos (L.) Correa): ˆ importance, biology, propagation, and future perspectives,” Trees, vol. 32, no. 5, pp. 1165–1198, 2018. [20] K. B. Bindhu, “In vitro propagation of Aegle marmelos through nodal explants,” International Journal of Science and Research, vol. 4, no. 4, pp. 691–694, 2013. [21] A. V. Raghu, S. P. Geetha, G. Martin, I. Balachandran, P. N. Ravindran, and K. V. Mohanan, “An Improved micropropagation protocol for bael—a vulnerable medicinal tree,” Research Journal of Botany, vol. 2, no. 4, pp. 186–194, [22] K. Kishore, K. K. Mahanti, and D. Samant, “Phenological growth stages of bael (Aegle marmelos) according to the extended Biologische Bundesantalt, Bundessortenamt und Chemische industrie scale,” Annals of Applied Biology, vol. 170, no. 3, pp. 425–433, 2017. [23] M. C. Salinero, P. Vela, and M. J. Sainz, “Phenological growth stages of kiwifruit (Actinidia deliciosa ‘Hayward’),” Scientia Horticulturae, vol. 121, no. 1, pp. 27–31, 2009. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Advances in Agriculture Hindawi Publishing Corporation

Establishment of a Micropropagation Protocol for Elite Accessions of Bael (Aegle marmelos (L.) Corr.), a Tropical Hardwood Species

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Hindawi Advances in Agriculture Volume 2020, Article ID 8840386, 10 pages https://doi.org/10.1155/2020/8840386 Research Article Establishment of a Micropropagation Protocol for Elite Accessions of Bael (Aegle marmelos (L.) Corr.), a Tropical Hardwood Species 1,2 1 1 Chamila Pathirana , Udayanthi Attanayake, Udula Dissanayake, 3 3 2,4 Lakshman Gamlath, Kalyani Ketipearachchi, Terrence Madhujith, 1,2 and Janakie Eeswara Department of Crop Science, Faculty of Agriculture, University of Peradeniya, Peradeniya 20400, Sri Lanka Postgraduate Institute of Agriculture, University of Peradeniya, Peradeniya 20400, Sri Lanka Fruit Crop Research & Development Station, Gannoruwa, Peradeniya, Sri Lanka Department of Food Science and Technology, Faculty of Agriculture, University of Peradeniya, Peradeniya 20400, Sri Lanka Correspondence should be addressed to Chamila Pathirana; ckpathirana0421@gmail.com Received 20 April 2020; Revised 16 July 2020; Accepted 9 September 2020; Published 22 September 2020 Academic Editor: Ga ´bor Kocsy Copyright © 2020 Chamila Pathirana et al. *is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. *e limited availability of high-quality planting material hinders the popularization of bael (Aegle marmelos (L.) Corr.) as a profitable cash crop in Sri Lanka. Five elite bael accessions, namely, Beheth Beli, Paragammana, Mawanella, Rambukkana, and Polonnaruwa-Supun, have been identified and used for mass propagation through budding and grafting. However, this process gets hampered by the limitations in large-scale production of planting material. Micropropagation is an alternative technique to produce clonal plants in large-scale; however, no established protocol is available for the field-grown elite bael accessions in Sri Lanka, and hence, the present study was conducted. *e best sterilization method was found to be the washing of explants in a 2.5% fungicide solution for two hours. *e full MS and ½MS media provided significantly similar performance in shooting, as indicated by the measured parameters. *e twig plants did better than leaf explants concerning shoot extension, in which twig explants yield 1.33 cm taller shoots. *e 1 mg/l of BAP concentration generated the highest number of shoots (6.20) and the most extended shoots (3.83 cm). *e most successful rooting (60% success) was spotted with full MS, 1 mg/ml NAA, and 3% sugar. As previously reported, the successful micropropagation is possible if the explants are harvested from April to June, immediately after the fruiting season of the plant. *e established protocol can mass-produce clonal bael plants from the elite accessions. temples dedicated to ‘God Shiva’ [2]. *e fruits and other 1. Introduction plant parts of bael contain essential phytochemicals such as Aegle marmelos (L.) Corr. of family Rutaceae, commonly tannin, coumarin, aegelinol, and marmelocin, and they exhibit various medicinal properties ranging from laxative, known as bael, is a medicinal fruit tree fruit species [1]. Bael is native to India and found in other South Asian countries antiproliferative, antidiabetic, and anticancerous capabilities such as Sri Lanka and Bangladesh, Southeast Asia, and Egypt [7, 8]. [2]. *e ripened fruit is the economically important plant Bael is a well-established medicinal fruit tree species, and part of bael, and all the plant parts have essential medicinal the selections from the wild germplasm have led to larger properties [3, 4]. *e pulp of the ripened fruits is delicious fruit sizes and superior fruit qualities. *e elite bael plants and can also be processed into value-added products such as have been identified, and attempts have been made to jam, syrup, sweets, and herbal drinks [5, 6]. Bael is con- multiply them for large-scale cultivations in India [9]. sidered as a sacred tree in India and often grown near the However, the propagation using seeds is often hampered by 2 Advances in Agriculture the extreme genetic heterozygosity due to open pollination, mixture and smooth bristles. *e explants were rinsed using lower seed germination rates, and difficulty in collecting and running tap water for 10–15 mins. After washing three to four times, the plant parts were soaked in distilled water processing seeds from mature fruits. Furthermore, it is challenging to produce a large amount of planting material approximately for one hour. *en, subsequent steps were through budding and grafting techniques. *us, plant tissue followed for further sterilization to avoid the bacterial and culture techniques can be used as an alternative to overcome fungal infections. problems associated with the multiplication of superior trees identified in the wild/field. Micropropagation techniques 2.2.2. Fungicide Treatment. *e plant parts were soaked in a have been successfully employed for rapid multiplication of series of fungicide (commercial formulation thiram with the the superior bael plants using cotyledons [10–13], nodes [9], active ingredient dimethyldithiocarbamate (C H N S )) 6 12 2 4 and the axillary buds [14, 15]. However, it is essential to solutions (0.5%, 1.0%, 1.5%, 2.0%, and 2.5%). At each develop a micropropagation protocol for the explants col- concentration of fungicide, the plant parts were subjected to lected directly from the mother plants growing in the field to different exposure times (1 hr, 2 hr, and overnight). multiply the bael accessions. *e establishment of cultures without contamination is one of the major problems when explants are collected from field-grown trees [16]. *us, the 2.2.3. Surface Sterilization. *e plant parts were washed development of a specific sterilization technique for plant carefully with distilled water to remove the fungicides. After materials obtained from the field is an essential requirement that surface sterilization was performed for the explants. *e before identifying the growth stage for the collection of plant leaf explants were transferred into a wash bottle, which materials and perfecting the medium composition for consisted of 10% Clorox (sodium hypochlorite (NaOCl)) multiplication of plants. *erefore, the present study was and a drop (1 ml) of polyethylene glycol sorbitan mono- conducted to develop a protocol for multiplication of bael laurate (Tween 20 (T20)) and shaken for 10 min. *en, from plant materials collected from trees identified as ac- bleach solution was drained off and washed three times cessions by the Fruit Research and Development Station carefully with autoclaved water. *at step was repeated with (FCRDS) at Gannoruwa, Sri Lanka. only 10% NaOCl, and cleaned plant parts were transferred to a vessel containing distilled water. *e nodal segments and 2. Materials and Methods shoot tips (i.e., twigs) were surface-sterilized separately using 10% of Clorox along with two drops of Tween 20. *e 2.1. Plant Material. Five Sri Lankan bael accessions, namely, shaking was continued for ten minutes. *en, the twigs were Beheth Beli (BB), Paragammana (PA), Mawanella (MA), transferred to a 10% NaOCl solution without Tween 20 and Rambukkana (RA), and Polonnaruwa-Supun (PS), which were shaken well for another 10 mins followed by thorough have been selected for large-scale production of planting washing (three times) with sterile, deionized, distilled water materials, were used to collect the explants for developing a to wash off the traces of bleach solution. Each washing protocol for micropropagation. Leaves and twigs were used process lasted for approximately one minute. as the explants. *e study was conducted at the Tissue Culture Laboratory of the Department of Crop Science, Faculty of Agriculture, University of Peradeniya, Sri Lanka. 2.2.4. Culture Establishment. All tissue culture techniques *e explants from the mother plant of BB were collected were conducted under aseptic conditions in a laminar flow from FCRDS (GPS coordinates: 7.277006, 80.595299) during cabinet (Labgard Class II, Type A/B3). *e leaf and twig the vegetative growth stage (April–June, 2018) [17]. All the explants were prepared by trimming the cut surfaces to investigations on sterilization and identification of the about 12–15 mm in length. *e MS [18] medium supple- specific growth stage for culture establishment were con- mented with 1 mg/l of BAP, 3% (w/v) of sucrose, and so- ducted using BB mother plants. *en, the results achieved lidified with 0.2% (w/v) Phytagel (Sigma, UK) was used as were applied to other four accessions when they were at the the medium based on the results obtained for nodal seg- same vegetative growth stage, which are located at distant ments collected from in vitro grown seedlings in the tissue locations from the study site. culture laboratory of the Department of Crop Science. *e pH of the medium was adjusted to 5.8 with 1 M HCl or NaOH before autoclaving at 120 C for 20 mins, 15 psi. *is 2.2. Experiment 1: Effect of Fungicide on Surface Sterilization of medium was used for initiating the cultures. Each culture Twig and Leaf Explants tube contained 10 ml of medium. *e inoculation was 2.2.1. Preparation and Washing of Explants. *e disease-free performed as one explant per culture tube. *e leaf pieces at immature leaves and tender twigs were collected from top the size 1 cm (approximately) holding the midribs were parts of the branches of each mother plant and immediately isolated and placed on the medium. A gentle press was transferred to the laboratory for micropropagation. *e applied to have an even contact between the abaxial (lower) dried/dead and infected portions were removed from the surface of the leaf and the medium. *e single nodal twigs intended explants. *e twigs and leaves were isolated, and were inserted into the medium in the upright position. *e the twigs containing at least three to four internodes were leaf cultures were incubated inside a dark cabinet at 26± 2 C used for culturing. *e leaves and twig explants were until initiating the shoots. *e twig cultures were incubated thoroughly cleaned using a commercially available detergent at 16 hrs of light (flux density) at 25± 2 C. Advances in Agriculture 3 2.2.5. Scanning Electron Microscopy to Detect the Presence of 3. Results Fungi in Leaf and Twig Explants. *e fungicide-treated 3.1. Experiment 1: Effect of Fungicide on Surface Sterilization. (2.5%) and untreated leaf explants were subjected to scan- *e 2.5% fungicide solution in which explants were im- ning electron microscopy (SEM) to visualize the effect on mersed for two hours produced the only successful regen- fungicide treatment on the fungi present on explants. *e eration. When the leaf and twig explants were subjected to leaves and twigs were taken and soaked in the fungicide 2.5% fungicide for two hours of treatment and subsequently solution for two hours. After that, they were cut into explant cultured in 1 mg/l BAP culture, initially till the fourth week, size pieces. *e explants were then sprayed with gold par- substantial amounts of contaminated and few browned ticles for staining purpose and observed using the ZEISS cultures were observed. However, eventually, nearly 60% of scanning electron microscope (SEM) (Jeol SEM 6400, Tokyo, cultures of both leaf and twig explants survived as healthy Japan). *e SEM photographs were taken from the repre- cultures after 5-6 weeks of establishment. sentative places of the explants. 3.2. Effect of Fungicide Treatment on Fungi Present on 2.3. Experiment 2: Effect of N Content in Medium on In Vitro Explants. *e fungal hyphae were observed in upper Multiplication. *e effect of N content on the successful (Figure 1(a)) and lower (Figure 1(b)) surfaces of the leaf establishment of the cultures was assessed. To detect the explants. *e fungicide treatment destroyed the fungal effect of N, full MS and half MS media were prepared. *e hyphae observed in the upper (Figure 1(c)) and bottom parameters such as the number of leaves/shoot, number of surfaces (Figure 1(d)). *e sunken stomata were clearly shoots/cutting, and height of the shoot were measured after visible in both upper and lower surfaces of the leaves eight weeks of establishment and statistically analyzed using (Figures 1(a)–1(d)). In the twig explants under 1000 and the GLM procedure in the Statistical Package SAS 9.4 (SAS 5000 magnifications, fungal hyphae were not observed in Institute, NC, Cary, USA). untreated cases (Figures 1(e) and 1(g), respectively). *e similar appearance was photographed for treated explants under the two magnifications (Figures 1(f ) and 1(h)). 2.4. Experiment 3: Effect of BAP Concentration on Multipli- Compared to the stomata in leaves, the stomata in twigs are cation of Regenerated Shoots. *e effect of five BAP con- smaller in size (Figures 1(e)–1(h)). centrations (0.0, 0.5, 1.0, 2.0, and 4.0 mg/l) on shoot multiplication was investigated. *e subculturing was per- formed at every eight-week intervals. Each proliferated 3.3. Experiment 2: Effect of N Content in the Medium on In adventitious shoot was cut from the basal end and sub- Vitro Multiplication of Regenerated Shoots. *e mean cultured again for further multiple shoot induction. At the number of shoots per cutting and the mean number of leaves end of the 20th week onwards, regenerated multiple shoots per shoot were not significantly different among the two were cut, and the shoots were transferred on to an MS medium compositions and the type of explant (P> 0.05). medium without any plant growth regulators before However, the mean height of the shoot was significantly transferring into a rooting medium. *e number of shoots higher in twig explants than the leaf explants (P< 0.05) per explant and the length of the shoots were measured and (Table 1). Two culture jars of full MS and ½MS are shown in subjected to GLM procedure in SAS. Figure 2 to display the growth differences. 2.5. Experiment 4: Effect of N, Sucrose, IBA, and NAA Con- 3.4. Experiment 3: Effect of BAP Concentration on Multipli- centration on Rooting. *e effects of N (full and ½MS), cation of Regenerated Shoots. *e BAP level of 4 mg/L caused sucrose concentration (3% and 6%), naphthalene acetic acid vitrification of shoots even after subculturing (Figures 3(a) (NAA) (1 mg/L), and indole-3-butyric acid (IBA) (1 mg/L) and 3(b)). *e BAP concentration of 1.0 mg/L provided the on rooting of micropropagated shoots were investigated. *e highest success. *e mean highest number of shoots was experiment was arranged as a three-factor factorial observed for the BAP levels of 0.5, 1.0, and 2.0 mg/l experiment. (5.60–6.20). At the BAP level of 4.0, the mean number of 3.40 shoots was observed, and only two shoots were observed when no BAP was added. *e mean shoot length was sig- 2.6. Experiment 5: Acclimatization of In Vitro-Generated nificantly different among all the BAP treatments. *e sig- Plantlets. *e in vitro rooted plants were maintained in jars nificantly mean highest shoot length was observed at the BAP with ventilation for a month and after that transferred to level of 1.0 mg/L (3.83 cm), whereas the mean lowest shoot small plastic pots (10 cm in diameter) containing a standard length was observed when no BAP was added (2.49 cm) potting mixture and allowed to grow in a greenhouse. *e (P< 0.05) (Table 2). *e shoots generated by BAP treatments plants were covered with polyethylene sheets to maintain the other than the 4 mg/L concentration are displayed in Figure 4. relative humidity. *e artificial lights were provided to have 70% light intensity. *e covers were intermittently removed after one week every day and completely removed after two 3.5. Experiment 4: Effect of N, Sucrose, IBA, and NAA Con- weeks. After four weeks, the survival percentage was centrations on Rooting. Table 3 displays the series of results calculated. obtained for the rooting experiments by altering N (MS or 4 Advances in Agriculture (a) (b) (c) (d) (e) (f) (g) (h) Figure 1: Scanning electron microscopy photographs of the leaf and twig explants (untreated and treated with fungicide). (a) Upper surface of leaves untreated; (b) upper surface of leaves treated; (c) lower surface of leaves untreated; (d) lower surface of leaves treated; (e) twig untreated (×1000); (f ) twig treated (×1000); (g) twig untreated (×5000); (h) twig treated (×5000). *e specific details of each photograph are marked as a label with each photograph. Table 1: *e mean performance of the shoots under full MS and ½MS (N) conditions. Medium Type of explant No. of shoots/cutting Height of the shoot (cm) No. of leaves/shoot a b a Leaf 7.40 2.27 3.67 Full MS a a a Twig 7.25 3.25 6.53 a b a Leaf 6.80 1.93 4.33 ½MS a a a Twig 6.50 3.60 5.57 Means denoted by the same letters within columns are not significantly different at P< 0.05. Advances in Agriculture 5 (a) (b) Figure 2: *e comparison of shoot growth. (a) Full MS and (b) ½MS media. Scale bar, one cm. (a) (b) Figure 3: Vitrification of shoots caused by higher BAP concentrations. (a) Vitrified shoots inside the glass tubes; (b) vitrified shoots taken out from the medium. Scale bars represent one cm separately for panels A and B. ½MS), sugar level (3% and 6%), and hormone (IBA or (Figures 5(a)-5(b) and 6(d)). When the enlarged part was cut NAA). It was clear that NAA was capable of generating roots off and recultured, the enlargement happened once again. required for bael shoots in in vitro cultures. It also high- *e 6% sugar levels also lead to the vitrification of shoots at later stages as in the shoots shown in Figure 3. lighted the fact that growth regulators are needed for rooting as zero hormonal applications did not produce any positive rooting results. Regarding the N content, either full MS or 3.6. Experiment 5: Acclimatization of In Vitro-Generated ½MS was fine in giving positive results; however, full MS provided more rooting success (60%) than ½MS, which Plants. *e plantlets were responsive to the acclimatization treatments slowly and displayed improved tolerance to the displayed only 33% success (Figures 5(a)-5(b) and 6(a)– 6(c)). *e 6% sugar and 1 mg/l of NAA also produced lower relative humidity and 70% light intensity. After two weeks, nearly 80% of the plants survived, and at the end of enlarged ends in the shoot bases proving that 6% sugar level is too much for rooting of bael in in vitro cultures the four weeks, 60% of the plants survived and was ready to 6 Advances in Agriculture Table 2: *e growth of shoots under BAP treatments. BAP (mg/l) Mean number of shoots Mean length of shoots (cm) c e 0.0 2.00 2.49 a d 0.5 5.60 2.99 a a 1.0 6.20 3.83 a b 2.0 6.00 3.34 b c 4.0 3.40 3.14 Within a column, means followed by the same letter are not significantly different at P< 0.05. Figure 4: *e nonvitrified shoots generated by lower BAP concentrations. *e shoots are prepared for measurements and subculturing. Scale bar represents one cm. Table 3: Effect of N, hormone, and sugar levels on rooting of in vitro cultures of bael Medium composition (%) sugar Hormone and concentration (mg/l) Number of shoots produced roots (out of 20) 0 0 3 1 NAA 12 1 IBA 0 Full MS 0 0 6 1 NAA 0 1 IBA 1 0 0 3 1 NAA 6 1 IBA 0 ½ MS 0 0 6 1 NAA 2 1 IBA 1 be used in planting purposes. After two months, 42% of the optimizations were required. *erefore, the present study plants remained in the healthy condition, which is a higher possesses a paramount significance as for the first time in Sri value for woody perennials. Lanka, we have established a sound and easier protocol to clonally multiply the field-grown elite accessions of bael using leaf and twig explants. 4. Discussion Micropropagation techniques have been successfully A well-established micropropagation procedure is essential employed for rapid multiplication of the superior bael using nodal segments [9], cotyledons [10–13], nodes [9], and the for bael if it is to be established as a country-wide cash crop [19]. *e lower percentage of seed germination, the presence axillary buds [14]. *e type of explant and various other of miniature or aborted seeds in the fruits, and highly conditions required for the establishment of micro- heterozygous nature of the species, which is leading to the propagation technique have been extensively discussed and segregation of traits, are making sexual reproduction inef- compared in the study by Vasava et al. [19]. However, for the fective. *e micropropagation of bael has been tried, and elite bael accessions in Sri Lanka, specific sterilization results were obtained in many studies [9, 12, 13, 20]. technique, hormone combination, and nitrogen content However, we noted that in the present study, such protocols required have not been established to come up with a could not be directly translated to our conditions; hence, successful micropropagation protocol. Advances in Agriculture 7 (i) (ii) (iii) (iv) (v) (vi) (a) (vii) (viii) (ix) (x) (xi) (xii) (b) Figure 5: A set of representative cultures showing the effect of N hormone and sugar on rooting. (a) MS (i, no hormone and 3% sugar; ii, 1 mg/l NAA and 3% sugar; iii, 1 mg/l IBA and 3% sugar; iv, no hormone and 6% sugar; v, 1 mg NAA and 6% sugar; and vi, 1 mg IBA and 6% sugar); (b) ½MS (vii, no hormone and 3% sugar; viii, 1 mg/l NAA and 3% sugar; ix, 1 mg/l IBA and 3% sugar; x, no hormone and 6% sugar; xi, 1 mg NAA and 6% sugar; and xii, 1 mg IBA and 6% sugar). *e sterilization of the explants is the most challenging found to be nearly 50%, and it is a practically higher rate in step in bael micropropagation. It was painstaking to carry any form of micropropagation. *us, the fungal species out the necessary sterilization as the phenological growth might be the reason for contaminations in the early stages of stage of the explant always confounded the sterilization the culture establishment. treatment. *us, when the explants were collected from the To verify the presence of fungi in explants, SEM was appropriate phenological growth stage, the effect of steril- carried out, and it was clear that before the fungicide ization can be studied. *e results demonstrated that im- treatment, fungal hyphae were present (Figures 1(a) and mersing explants in 2.5% fungicide solution for two hours 1(c)) in both surfaces of the leaves, and the fungicide provided the highest success, and that was not significantly treatment was able to destroy the fungal hyphae as observed different among leaf and twig explants. *e success rate was in Figure 1(b) and 5.1D. However, in twig explants 8 Advances in Agriculture (a) (b) (c) (d) Figure 6: *e rooting results. (a) *ree shoots just before inserting in the rooting medium; (b) shoots form roots; (c) rooted plantlets removed for subculturing; (d) bottom enlargement observed. examined, no apparent fungal hyphae were observed number of shoots per cutting, taller shoots, and a higher probably due to the falling off during the sample processing number of leaves per shoot (Table 1; P> 0.05) than ½MS for SEM (Figures 1(e)–1(h)). It was also noted that bael medium. Although both leaf and twig explants produced possesses sunken stomata in both surfaces of the leaves. *e successful results in multiplication, the twig explants per- stomata present in twigs are smaller than the ones present in formed significantly better according to the measured pa- rameters (Table 1; P< 0.05). leaves. *e sunken stomata and small stomata are two significant adaptations for drought. *ese observations are In the shooting experiment, the optimum concentration of BAP for shoot development was 1 mg/l that produces the in line with the drought-tolerant trait of the bael. *e phenological growth stage of the tree subjected to mean highest number of shoots and the significantly mean the collection of explants influences the percentage success most extended shoots (Table 2; P< 0.05). *e BAP con- in micropropagation [21]. Bael has marked phenological centration of 4.0 mg/l did not produce successful results and growth difference concerning the rainfall, temperature, and led to the vitrification of shoots, implying that too much fruiting [22]. Rhagu et al. [21] reported that October–De- BAP is not applicable to bael. In some other studies on bael cember is the best time to collect explants as the plant is at its micropropagation, NAA was used in combination with BAP; best phenological stage for a higher success rate of micro- however, we wanted to make sure the appropriate devel- propagation in Kerala, India. *e phenological stage of the opment of shoots before triggering the rooting so that the attempts were made only using BAP. Bindhu [20] reported plants is essential for deciding the effective fruiting season and collecting the vegetative parts as planting material [23]. that the BAP concentration of 2 mg/l is efficient for the shooting of bael over the other combinations/concentrations During flowering and fruiting of the plants, the internal hormonal profiles often hinder the effective regeneration of BAP/kinetin. Pati et al. [9] have obtained successful capabilities. However, if the plant parts are taken from shooting for bael by applying a combination of BAP and actively growing trees, regeneration can be achieved swiftly NAA. with higher success rates [21]. *e phenology of bael is Full MS with 1 mg/l of NAA would provide at least 50% complex, and vegetative and reproductive phases are success in rooting of bael shoots in in vitro cultures (Table 3). overlapped [22]. In India, the bael phenology has been *e comparable images display the variable success as shown correlated with success rates in the micropropagation [21]. in Figures 4 and 5. In a study conducted at *rissur District, *e ideal phenological stage of bael accessions in Sri Lanka Kerala, India, indoleacetic acid (IAA) at the concentration of 0.01 mg/l was found to be effective in inducing rooting [20]. to collect explants for successful micropropagation is be- tween April and June when there is a predominant vegetative Pati et al. [9] observed successful rooting at a combination of growth [17]. IBA + IAA. Raghu et al. [21] obtained successful results with *e N content, as given by the full or ½MS media, has the ex vitro rooting by adding napthoxyacetic acid (NOA) and same effect on the establishment of cultures. *e full MS IBA in a mix, but we did not attempt ex vitro rooting in the medium did not provide better results in terms of a higher present study. *ese contrasting findings demonstrate the Advances in Agriculture 9 fact that the hormonal concentrations for rooting are highly References specific to the given conditions. [1] R. Chanda, A. Ghosh, T. Mitra, J. P. Mohanty, N. Bhuyan, and G. Pawankar, “Phytochemical and pharmacological activity of 5. Conclusions Aegle marmelos as a potential medicinal plant: an overview,” 8e Internet Journal of Pharmacology, vol. 6, no. 1, p. 3, 2008. *e most productive sterilization method at the explant [2] V. K. Singhal, A. Salwan, P. Kumar, and J. 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Fazal, “Phyto- produces the highest number of shoots (6.20) and the most chemistry and medicinal uses of the bael fruit (Aegle marmelos extended shoots (3.83 cm). *e successful rooting was ob- Correa): a concise review,” Food Research International, served with full MS, 1 mg/ml NAA, and 3% sugar with 60% vol. 44, no. 7, pp. 1768–1775, 2011. cultures compared to the other treatments employed. *ese [6] J. F. Morton, Fruits of Warm Climates, Creative Resource results could be successfully applied in the mass production Systems Inc, Winterville, NC, USA, 1987. of clonal bael plants from elite accessions to establish the [7] S. Charoensiddhi and A. Anprung, “Bioactive componds and species bael as a profitable cash crop in Sri Lanka. volatile compounds of *ai bael fruit (Aegle marmelos (Linn.) correa) as a valuable source for functional food ingredients,” International Food Research Journal, vol. 15, no. 3, pp. 287– Data Availability 295, 2008. [8] V. B. Lambole, K. Murti, U. Kumar, B. P. 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Samant, “Phenological growth stages of bael (Aegle marmelos) according to the extended Biologische Bundesantalt, Bundessortenamt und Chemische industrie scale,” Annals of Applied Biology, vol. 170, no. 3, pp. 425–433, 2017. [23] M. C. Salinero, P. Vela, and M. J. Sainz, “Phenological growth stages of kiwifruit (Actinidia deliciosa ‘Hayward’),” Scientia Horticulturae, vol. 121, no. 1, pp. 27–31, 2009.

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Advances in AgricultureHindawi Publishing Corporation

Published: Sep 22, 2020

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