IN VITRO MASS MULTIPLICATION OF NONI (Morinda citrifolia L.) THROUGH NODAL SEGMENT EXPLANTS

Transcription

1 Journal of Cell and Tissue Research Vol. 15(1) (2015) (Available online at ISSN: ; E-ISSN: Original Article IN VITRO MASS MULTIPLICATION OF NONI (Morinda citrifolia L.) THROUGH NODAL SEGMENT EXPLANTS? SAINI, M. K. AND PATEL, R. M. Department of Plant Molecular Biology and Biotechnology, ASPEE College of Horticulture and Forestry, Navsari Agricultural University, Navsari , Gujarat. E. mail: Cell: Received: December 3, 2014; Revised: December 13, 2014; Accepted: January 1, 2015 Abstract: Noni (Morinda citrifolia L.) fruits are widely used for blood pressure, respiratory problems, immune deficiencies, antibacterial, anti-inflammatory, analgesic and anti-congestive therapies. To produce large scale true to type plant a tissue culture protocol was developed. Maximum establishment of nodal segment explants observed in Murashige and Skoog (MS) medium containing 2.0 mg/l 6- benzylaminopurine (BAP). However, MS medium fortified with 2.0 mg/l BAP mg/l kinetin exhibited maximum multiplication rate in second and third sub-cultures. The maximum frequency of multiple shoots in nodal segment explants (84.33 %) was observed on treatment MS mg/l BAP mg/l kinetin. In vitro rooting of regenerated shoot developed in half strength MS medium supplemented with 1.0 mg/l IBA, which produced maximum number of roots per shoot (10) and length of root (3.10). In vitro grown plantlets having 6.23 cm length of shoot were transferred to coco peat media under green house, which showed better survival of plantlets (95 %). Key words: Morinda citrifolia, Tissue culture INTRODUCTION Indian Noni (Morinda citrifolia L.) is popularly known as Indian mulberry. It belongs to the family Rubiaceae. The genus name for Morinda has been derived from two latin worlds; morus which means mulberry and indicus which means India. In reference to similarity of fruits of noni to that of true mulberry (Morus alba), noni has been named so. The species name indicates resemblance of plant foliage to that of citrus species. Noni is a small evergreen tree or shrub growing to a height of 3-6m at maturity of years. Noni can be grown in almost all types of soil under wide climatic conditions from dry to humid area of tropics to subtropical zones. This non-conventional under-utilized fruit is much valued in today s emerging health conscious societies for its therapeutic attributes like antibacterial, antiinflammatory, analgesic and anti-congestive. Noni fruit and its products are used as a health panacea against high blood pressure, respiratory problems and immune deficiencies [1]. Although, noni is mainly propagated by seeds but seedling raised plants are not similar to parent tree in fruit quality. Hence, at present to get good quality planting material, noni is propagated by air layering or cutting. However, it has several limitations like average success and very slow propagation, which do not produces plantlets on mass scale throughout the year. Therefore, it is warranted to search another alternate method which can help to solve the problems associated with the conventional method of propagation to multiply planting material on large 4877

2 J. Cell Tissue Research scale rapidly in short time and available around the year. In vitro culture technique is becoming increasingly popular as alternative and feasible means in vegetative propagation of some commercially important plants [2,3]. Hence, to meet the demand of large scale planting material of desirable genotype, micro propagation technique is only the way to fulfil the requirements round the year. Micropropagation is one of the major areas in plant tissue culture which has begun to manifest potential for mass production of sapling material in short period of time. This technique has been shown to have definite and indispensable advantage over the former, as it ensures extremely rapid rate of multiplication. It is not season dependent and requires only a limited quantity of plant tissue as a source of initial explants. MATERIALS AND METHODS Preparation and establishment of explants: Explants were collected from nursery area of Department of Horticulture, Navsari Agricultural University, Navsari. Nodal segment explants from newly emerged shoots containing one node each were collected from 5-6 years old plants and leaves were removed leaving the petiole. They were swabbed with cotton and then dipped in 70 per cent alcohol and washed thoroughly in running tap water for about 30 minutes to remove traces of alcohol and dirt. The nodal segment was pre-treated with bavistin 0.2% (Carbendazim 50 per cent WP) and streprocyline 0.07 per cent for one hour. Thereafter, explants were washed with 10 per cent solution of detergent (Teepol) for 10 minutes. All traces of detergent were removed by repeated washing in double distilled water. Further sterilization procedure was carried out under aseptic condition in laminar air flow cabinet. The nodal segments were subjected to surface sterilization using 0.05 to 0.1 per cent HgCl 2 solution for 5 minutes and 10 per cent for minutes duration. They were then, thoroughly rinsed at least three times with autoclaved de-ionized distilled water. The sterilized nodal Table 1: Effect of different media on establishment of nodal segment explants of noni var. Local Incubation: 4 Weeks Explants: Nodal segment * Figures in paretheses are arc sin transformed value Media Composition/Treatmentno. Establishment (%) MS + BAP 2.0 mg/l (63.90) SH + BAP 2.0 mg/l (43.83) WPM + BAP 2.0 mg/l (58.46) MS+ BAP 2.0 mg/l + NAA 0.5 mg/l (53.71) SH + BAP 2.0 mg/l+ AA 0.5 mg/l (36.85) Days taken for establishment Length of longest shoot(cm) Number of shoots/ explant WPM+ BAP 2.0 mg/l + NAA 0.5 mg/l (48.62) S.Em CD at 5% CV% Treatment No. Frequency of multiple shoots/explants (%) Nodal segments MS + BAP 1.0 mg/l (25.83) MS + BAP 2.0 mg/l 31.67(34.23) MS + BAP 4.0 mg/l 57.00(49.00) MS + BAP 1.0 mg/l + KIN 1 mg/l 66.67(54.72) MS + BAP 2.0 mg/l + KIN 1 mg/l 84.33(66.67) MS + BAP 4.0 mg/l + KIN 1 mg/l 50.67(45.36) MS + BAP 1.0 mg/l + NAA 0.5mg/l 1.33(27.50) Table 2: Effect of plant growth regulators on frequency of shoot multiplication of nodal explants of noni var. Local. Medium : MS Medium. Incubation : 4 Weeks. * Figure in paratheses are arc sin transformed value MS + BAP 2.0 mg/l + NAA 0.5mg/l 9.00(38.63) MS + BAP 4.0 mg/l + NAA 0.5mg/l 4.33(41.73) Em CD at 5% 1.23 CV%

3 Saini and Patel Tale 3: Effect of IBA, combination of IBA with NAA and strength of media induction of rooting of noni var. Local. Medium : MS Medium, Incubation : 4 Weeks. * Figure in paratheses are arc sin transformed value Treatment No. Rooting % Days taken for root initiation Length of longest root (cm) No. of roots per shoot ½ MS + IBA 0.5 mg/l (18.39) ½ MS + IBA 1.0 mg/l (71.60) ½ MS + IBA 2.0 mg/l (47.85) ½ MS + IBA 0.5 mg/l + NAA 0.5 mg/l (22.77) ½ MS + IBA 1.0 mg/l + NAA 0.5 mg/l (52.52) ½ MS + IBA 2.0 mg/l + NAA 0.5 mg/l ( Full MS + IBA 0.5 mg/l 8.00 (16.40) Full MS + IBA 1.0 mg/l (40.96) Full MS + IBA 2.0 mg/l (31.29) Full MS + IBA 0.5 mg/l+ NAA 0.5 mg/l (38.04) Full MS + IBA 1.0 mg/l+ NAA 0.5 mg/l (58.68) Full MS + IBA 2.0 mg/l+ NAA 0.5 mg/l (29.99) S.Em CD at 5% CV% Table 4: Effect of different potting mixtures on hardening of noni var. Local. * Figures in paratheses are arc sine transformed value Treatment no. Survival of plantlets (%) Days taken for establishment of plantlets Length of longest shoot(cm) Vermicompost 35.50(36.55) Soil (47.85) Coco peat 95.25(77.43) Vermicompost: Soil. (1:1 v/v) 66.00(54.31) FYM : Soil : Sand (1:1:1 v/v) 74.50(59.65) S.Em CD at 5% CV% segment explants were excised and inoculated into 250 ml screw caped glass bottle. Initially three different media MS (Murashige and Scoog, [4], WPM (Lloyd and McCown [5], and SH (Schenk and Hildebrandt [6]) were tested for culture establishment. The media was supplemented with 2.0 mg/l BAP alone and in combination with 2.0 mg/ l BAP mg/l NAA. Sucrose was added at 30.0 g/l and media was autoclaved at pressure of 15 lb/ inch2 for 20 minutes at approximately 121 o C. The ph of the medium was adjusted at 5.8 prior to autoclaving. Cultures were incubated for four weeks. Best medium found in culture establishment was further used for multiplication. Shoot multiplication: For multiplication 1.5 to 2.0 cm long raised plantlets from nodal segment explants were aseptically isolated and transferred to MS medium supplemented with various concentration of growth substances BAP (1.0, 2.0 and 4.0 mg/l) alone and in combination with kinetin 1.0 mg/l and NAA 0.5 mg/l. Subculture was made on the same medium after four weeks of culture. In vitro rooting and acclimatization: After four weeks of sub-culture individual shoots were transferred into the rooting medium. For rooting stage MS full and half MS strength with different concentration of IBA alone and in combination with 4879

4 J. Cell Tissue Research IBA (0.5, 1.0 and 2.0 mg/l) + NAA (0.5 mg/l) were used. The nutrient medium was gently removed and washed thoroughly in tap water ensuring that all the adhering agar particles were completely removed without damaging the roots. For acclimatization different media like vermicompost, soil, coco peat, vermicompost : Soil. (1:1 v/v) and FYM : Soil: Sand (1:1:1 v/v) in 300 ml plastic cups were used. In vitro rooted plants were placed in a culture room at o C. The rooted plantlets were then dipped in 0.05% bavistin (carbendazim 50% WP) and plantlets in 300 ml plastic cup. They were covered with a plastic cup continuously for 6 to 7 days and kept in an air conditioned room. The cover was gradually removed after seven days initially for 2 hours followed by 4 hours and 8 hours in next four days. The cover was removed during night and light put-off for next three days. Subsequently the period of keeping the plantlets without any cover was gradually increased and after 15 days they were brought outside the room in shade. Within 10 days by gradually exposing them to sun they were acclimatized to natural environment. Statistical analysis: Statistical methods were used for comparison of treatment means for micropropagation. Completely randomized design (CRD) was used for the experiment. The data were subjected to analysis of variance (ANOVA) and treatment means were compared [7]. RESULTS AND DISCUSSION Establishment of explants: The results presented in Table 1 reveal that medium type influenced establishment of explants significantly. Maximum establishment (80.67%) was achieved on treatment MS medium supplemented with 2.0 mg/l BAP (Fig. 2) followed by WPM medium supplemented with 2.0 mg/l BAP. Minimum days taken for establishment of explants (4.33 days) were recorded in treatment MS medium supplemented with 2.0 mg/ l BAP). This treatment also showed significantly maximum length of shoots (2.33 cm) and number of shoots per explants (2) followed by MS medium supplemented with 2.0 mg/l BAP mg/ NAA. Earlier workers were used MS and WPM medium and achieved success for establishment of explants and growth in noni var [8,9]. Local. The multiplication rate of the MS medium was superior as compared to the WPM and SH medium. Frequency of shoot regeneration and multiplication: In order to study the multiplication rate, trial was conducted with treatments involving different levels of BAP, NAA and kinetin. In all, 9 treatments were tested and results are presented in Table 2. The sub culturing was done at 4 weeks interval. Each shoots of cultures were cut into nodal segments (2.0 cm size) and were used for multiplication. The treatment MS fortified with 2.0 mg/l BAP mg/ l kinetin was found significantly superior for producing high frequency of multiple shoots (84.33%) in culture from nodal segment explants (Fig. 3). The dominant buds of vegetative apex are stimulated to grow and elongated into presence of cytokinin and also produce new axes. For the shoot proliferation BAP was found more effective when used in combination with auxin in pomegranate [9]. Similarly, Subramani et al. [10] also reported high frequency of shoot multiplication from nodal segment and shoot tip of noni. Effects of serial sub culturing on shoot multiplication: The result for shoot multiplication with serial sub culturing of nodal segment explants is shown in (Fig. 1). Regeneration of multiple shoots at the rate of 6 folds and highest length of shoots were observed in treatment MS mg/l BAP mg/l kinetin) up to second subcultures then, declined. The declined trend has also been reported in papaya [11] and grape [12]. Effect of IBA, NAA and strength of the medium on rooting of in vitro shoots: Results on in vitro Explanation of figures: Fig. 1: Effect of serial sub culturing on multiplication of nodal segment explants of noni var. Local (Murashige and Skoog medium). M 1 W + BA 1.0 mg/l, M 2 W + BA 2.0 mg/l, M 3 W + BA 4.0 mg/l, M 4 W + BA 1.0 mg/l + KIN 1 mg/l, M 5 W + BA 2.0 mg/l + KIN 1 mg/l, M 6 W + BA 4.0 mg/l + KIN 1 mg/l, M 7 W + BA 1.0 mg/l + NAA 0.5mg/l, M 8 W + BA 2.0 mg/l + NAA 0.5mg/l, and M 9 W + BA 4.0 mg/l + NAA 0.5mg/l Fig. 2 Establishment of explants on MS medium mg/l BAP. Fig. 3. Shoot multiplication on MS medium mg/l BAP mg/l kinetin. Fig. 4 In vitro rooting on half MS mg/l IBA. Fig. 5 Hardening of plantlet in potting mixture of coco peat. 4880

5 Saini and Patel No. of shoots per culture Fig. 1 1st 2nd 3rd 4rth (Subculture) M1 M2 M3 M4 M5 M6 M7 M8 M9 Treatment Fig. 2 Fig. 3 Fig. 4 Fig

6 J. Cell Tissue Research rooting response to different level of IBA and combination of IBA with NAA supplemented in half and full strength of MS medium is presented in Table 3. The maximum rooting (90.00 %), minimum days taken for root induction (8.0 days), number of roots/ shoot (10.0) and length of rooted shoot (3.10 cm) was found in treatment half MS mg/l IBA (Fig. 4) followed by Full MS mg/l IBA mg/l NAA. Response of rooting was reduced either with increased or decreased level of IBA. Reduction in rooting response at higher concentration of auxin was observed in pear [13] and black plum [14]. Similar findings were reported in papaya [15,16]. Effect of different potting mixtures on survival of in vitro raised plantlets: The maximum per cent survival of plantlets (95.25%) was reported in coco peat (Table 4). The potting mixtures used in the present investigation helped in giving better grip for the roots and ample aeration. Coco peat was found as the best hardening medium, which gave maximum survival per cent of in vitro raised plantlets of noni var. Local (Fig. 5). These results are consistent with earlier finding of Subramani et al. [10] in noni. [6]. Schenk, R.U. and Hildebrandt, A.: Can J. Bot., 50: (1972). [7] Panse, V. G. and Sukhatme, P. V.: Statistical methods for Agricultural works. 4ed., ICAR, New Delhi (1985). [8] Selvaraj, S., Subramani, J., Vijay, D. and Kamath, L.: Intl. J. Noni Res. 1(2) (2006). [9] Singh S. K. And Khawale R. N.: Recent advances in Plant Biotechnology and its applications in tissue culture. 12: (2006). [10]Subramani, J; Selvaraj, S; Vijay, D and Sakthivel, M.: Intl. J. Noni Res. 2(1-2) (2007). [11]Patel, J. R.: M.Sc. (Hort.) thesis submitted to Navsari Agricultural University, Navsari Gujarat. (2008). [12]Shinde, K. A.: M.Sc. (Hort.) thesis submitted to Navsari Agricultural University, Navsari Gujarat (2008). [13]Bannok; Mayashi, S and Tanabe, K.: J. Jap. Soc. Hort. Sci., 58: 37-42(1989) [14]Yadav U, Lal M, Jaiswal V. S.: Plant Cell Tiss Org Cult 21:87-92 (1990). [15]Suthamathi, S. J.; Haripriya, K. and Kamalakannan, S.: Indian J. of Hort., 59 : (2002). [16]Beniwal, V. S; Sehrawat, S. K.; Dahiya, D. S.; Benival, L. S. and Singh, S.: Haryana J. Hort. Sci., 53 (1 & 2): (2006). CONCLUSION The present investigation on in vitro mass multiplication of noni cv. Local through nodal segment explants clearly demonstrated its potentiality for rapid clonal propagation. It appeared that using the present protocol of in vitro propagation, large number of plantlets can be produced in a year starting from single nodal segment explants. REFERENCES [1] Shah, A. and Gupta, D. K.: Proceedings of National symposium on production, utilization and export of underutilized fruits with commercial potentialities. Bidhan Chandra Krishi Viswavidyalaya, West Bengal, November (2006). [2] Murashige, T.: Annual Rev. Pl. Physiol., 25: (1974). [3] Morel, G. M.: Amer. Orchid Soc. Bulle., 29: (1960). [4] Murashige, T and Scoog, F.: A revised medium for rapid growth and bioassays with tobacco cultures. Plant physiol., 15: (1962). [5] Lloyd, G. and McCown, B.C.: Proceeding of International Plant Propagation Society., 30: (1981). 4882