Indian Journal of Biotechnology Vol 11, April 2012, pp 197-204
In vitro plant regeneration of wintergreen (Gaultheria fragrantissima Wall.): Assessment of multiple nutrient formulations and cytokinin types R A Ranyaphi1*, A A Mao1 and S K Borthakur2 1
Plant Tissue Culture Laboratory, Botanical Survey of India, Eastern Regional Centre Laitumkhrah, Shillong 793 003, India 2 Department of Botany, Gauhati University, Guwahati 781 014, India Received 26 August 2010; revised 11 May 2011; accepted 20 July 2011
A rapid clonal micropropagation protocol of Gaultheria fragrantissima Wall. (Ericaceae), an important medicinal plant from which wintergreen oil is extracted, has been developed. Mature plants collected from the field and in vitro raised juvenile seedling were used as source, and node and apical shoot were used as explants. Different basal media (MS, WPM, RM & MRM) supplemented with cytokinins (BA, Kin and 2iP) at a range of various concentrations have been investigated for multiple shoot induction. The 3 cytokinins induce shoot regeneration from nodal cuttings of in vitro raised seedlings in all the media. Optimum shoot induction occurred on MRM (modified rhododendron medium) supplemented with 2iP. After the first 3 cycle of culture with cytokinin, the shoots proliferated on cytokinin free basal medium. Subculturing the multiple shoots by separating into clumps of 5 shoots on MRM produced an average of 25 new shoots per culture within 8 wk. The in vitro raised microshoots were successfully rooted on half-strength basal MRM. Of the different substrates (sand, soil and leaf mold) studied for weaning, sand was the ideal substrate with 80% survival. Irrigation with half-strength MRM increased the survival rate and promoted the growth of the plantlets. Over 1000 plantlets were successfully weaned. Keywords: Gaultheria fragrantissima, medicinal plant, rapid clonal propagation
Introduction The genus Gaultheria (Ericaceae) comprises of about 200 species and G. fragrantissima is a bushy evergreen shrub of higher elevation, growing in shaded woodland and margin of forests1. The plant grows in sandy (light), loamy (medium) and acidic soils. This aromatic plant has long been valued for its wintergreen oil. Besides Betula lenta (the sweet birch), G. fragrantissima is a main natural source of wintergreen oil2. The bruised leaves have powerful camphor like smell. The essential oil rich in methyl salicylate is extracted by distillation of leaves. The oil has high demand in pharmaceutical and perfumery industries. The plant has been used as an antiseptic, carminative, flavouring agent (confectionaries, herbal tea, toothpaste, etc.) and condiment, and also in rheumatic and arthritis treatments3-5. Methyl salicylate is a the natural precursor of pharmaceutical aspirin. In the US, there are more than 40 products that contain ———————— *Author for correspondence: Mobile: +91-9436163731; Fax: +91-364-2224119 E-mail:
[email protected]
methyl salicylate as an active ingredient to treat various kinds of external pains6. The oil has a powerful antiseptic property and is a constituent for several insecticidal and insect repellent preparations7,8. The yield of wintergreen oil from the plants of Northeast region is reported to be higher than the other parts of India2,7. Hence, there is a great potential for commercial cultivation in the Northeastern hill states of India. The plantation can be taken up like tea gardens and the leaves can be harvested for oil extraction. There are reports on vegetative propagation and cultivation of wintergreen in Nepal9. However, our studies have shown that rooting of cuttings is moderately successful only in semi-hardwood cuttings and is extremely limited by the effect of season. Seed setting and germination rate is high but the seedlings are very slow in growth. Moreover, plants are vulnerable to damping off (Pythium sp.) at the early stages of growth and as such it makes difficult to raise the seedlings. Micropropagation has been an alternative technique to conventional methods of propagation and has found
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wide commercial application in the propagation of many commercially viable medicinal plants. However there is no proper study on the micropropagation of wintergreen, except a report of preliminary studies10. Hence, present study was taken up to develop a protocol for rapid micropropagation of wintergreen for large-scale propagation and conservation without any threat to local biodiversity. Materials and Methods Plant Material and Culture Establishment
Young and healthy shoots of G. fragrantissima were collected from the natural habitat at Shillong peak (1100 m in altitude), Meghalaya. All the leaves were removed and the stems were cut into about 2 inches long. They were washed thoroughly in Teepol solution (0.1%, v/v) and rinsed in running tap water for about 20 min. The explants were then disinfected with (10%, v/v) sodium hypochlorite (4.0-6.0% available chlorine) solution with 2-3 drops of Tween-20 for 20 min, followed by (0.1%, w/v) mercuric chloride solution for 5 min under aseptic condition in a laminar air flow bench. They were then washed 3-4 times with sterile distilled water. All subsequent manipulations were performed under aseptic conditions. Single nodal cutting possessing a single axillary bud or apical shoot were excised and used as explants. Single explant was inoculated in culture tubes (20×150 mm2) on semi-solid rhododendron medium (RM)11, Murashige and Skoog medium (MS)12 and woody plant medium (WPM)13, supplemented with BA (N-6-benzyladenin), Kin (kinetin) and 2iP (2-iso-pentenyladenine) (2, 5, 10, 15, 20 mg/L) separately for multiple shoot induction. Each culture tube contained 20 mL of the medium. The growth regulator free basal medium (BM) was use as control in all the studies. The medium contained sucrose (3%), activated charcoal (AC, 0.1%) and Difco-Bacto agar (0.8%). The pH of the medium was adjusted to 5.8 before autoclaving and the medium was autoclaved at 121°C and 1.06 kg/cm2 pressure for 20 min. All cultures were maintained under warm white fluorescent light at an irradiance of 50 mmol m-2 s-1, with a 16 h photoperiod and at 24+2°C. The newly developed shoots were excised from the mother explants and subcultured after every 6 wk for 4 times. In the 4th subculture, the percentage and the number of multiple shoots produced, along with shoot length, from different explants were evaluated. Similarly, nodal explants from 6-month-old in vitro raised
seedlings were studied for multiple shoot induction following the same procedure as mentioned above for mature explants. Standardization of Medium
Of the 3 media tested, RM was superior to MS and WPM for multiple shoot induction; however, the shoots produce on RM were hyperhydric. Anderson11 reported better shoot growth of rhododendron on reduce strength of RM. Hence, similar investigation for better growth of G. fragrantissima was carried out in the present study. Clumps having 5 shoots raised in medium supplemented with 10 mg/L 2iP were subcultured on different strength, i.e., full strength, ½ , ⅓ and ¼ strength of RM. Culture on ½ strength RM improved the shoot growth but the shoots were difficult to root as the shoots were still hyperhydric. To overcome this problem, modification of RM was made by reducing Fe2SO4 content in the medium for multiple shoot induction and healthier growth of the plant. In the original RM, the iron content, i.e., Fe2SO4.7H2O (55.6 mg), was chelated with Na2EDTA (74.6 mg) for 1 L of medium. These contents were reduced to half, i.e., Fe2SO4.7H2O (27.8 mg) chelated with Na2EDTA (37.3 mg), in modified rhododendron medium (MRM), while the other constituens remain same. Thereafter, all the subsequent experiments were carried out on MRM. Multiple shoot induction from nodal explants of 6-month-old in vitro raised seedlings was studied on MRM supplemented with Kin or 2iP (2, 5, 10 & 15 mg/L) separately or in combination with IAA and NAA (0.5, 1 & 2 mg/L) for synergistic effect. Also, to study the effect of cytokinin treatment for continuous proliferation of shoots, the multiple shoots were subcultured after every 8 wk by dividing each treatment into two sets: (i) subculture on the same cytokinin supplemented media, and (ii) subculture on basal media without any cytokinin. The multiple shoots produced from MRM containing 2iP (10 mg/L) were subcultured onto growth regulator free medium by separating them into group of 5, 10, 15, 20 shoots to study the effect of initial shoot number on multiple shoot proliferation in the subculture. Similarly, the effect of reduced strength of the modified medium (MRM) was also studied by culturing a clump of 5 shoots on different strength, i.e., full strength, ½, ⅓ and ¼ strength of medium. Rooting occurred freely on basal ½ strength MRM. Hence, there was no need of setting up experiment for rooting.
RANYAPHI et al: IN VITRO PLANT REGENERATION OF G. FRAGRANTISSIMA WALL.
Weaning
The rooted plantlets (2-month-old) were removed from the culture vessels, washed thoroughly in tap water to remove the agar medium on roots, treated with Bavistin fungicide (0.4%, w/v) for 15 min and then planted in coarse sand, soil and leaf mould separately in nursery root trainers. The plantlets were hardened for 4 months in greenhouse, where 80% relative humidity and 25˚C temperature with no additional lighting was maintained. The effect of nutrient supplement on weaning was studied by watering the plantlets with different strength of MRM (full, ½ & ⅓) nutrient at 1 wk interval for the plantlets grown on sand, as sand has poor nutrient content. Control was maintained by irrigating the plantlets with distilled water. After 4 month, the plantlet were transferred to pot containing garden soil and leaf mould (1:1) for further growth and development. Experimental Design and Measurement
In all the experiments, each treatment consisted of 15 replicates and each experiment was repeated 4 times by using a completely randomized design. Number of days taken for shoot initiation was recorded, and number of shoots/roots produced per explant along with shoot lengths were recorded after 8 wk of culture. For acclimatization experiment, shoot length and number of new leaves developed were recorded after 2, 3 and 4 months. Collected data were subjected to ANOVA and the treatment means were compared by least significant difference (LSD) at the 5% level of significance. Result and Discussion The treatment of explants with sodium hypochlorite alone for 20 min resulted in 80-90% decontamination, while the treatment for a longer time killed the explants due to bleaching. Similarly, when explants were treated with mercuric chloride solution (0.1% w/v) alone for 5 min gave 80-90% decontamination and extension of treatment for 10 min killed the explants. However, sterilizing explants with sodium hypochloride for 20 min, followed by mercuric chloride for 5 min gave 70-80% aseptic explants. Of the initial 3 media tested, RM was found to be superior for shoot regeneration from mature node explant. About 38% of nodal explants on 2iP supplemented medium responded in shoots sprouting on RM, whereas only 23 and 13% response was
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achieved on MS and WPM, respectively. The shoots produced on WPM were stunted in growth with small leaves, showing symptoms of NPK deficiency. On MS, the shoots were tender and weak, which may be due to considerably high content of NPK in comparison to RM and WPM. Thus, RM was found to be more suitable medium for G. fragrantissima culture, which is probably because both the species belong to Ericaceae family. Of the 3 cytokinins (BA, Kin and 2iP) studied for multiple shoot induction on mature node explants, 23-38% explants showed shoot sprout on medium supplemented with 2iP and Kin, whereas in control only 13% response was obtained. This clearly shows that 2iP and Kin had significant effect on the shoot proliferation. In medium supplemented with 2iP, the explants sprouted into new shoots within 4 wk at higher concentrations (15, 20 mg/L); whereas, in control, Kin and lower concentration of 2iP, it took 6 wk to sprout into new shoots. Moreover, no response was observed in BA treatment and the explants started dying within 2 wk. Multiple shoot induction started after 3 cycles of subculture in the medium supplemented with 2iP or Kin with an average of 4 shoots within 8 wk. In the subsequent subcultures, profuse multiple shooting (20-50 shoots) occured within 2 months. Similar multiplication of plants through sequential subculture of nodal explants from mature plants has been reported in olive14 and teak15. Induction of multiple shoots after 3 cycles of subcultures in matured explants could be due to requirement for a period of rejuvenation before active production of multiple shoots can start. Apical shoot and nodal explants resulted in direct shoot formation. There was no significant difference in the percentage of response between the two explants in the initial culture. In nodal explants, 15 mg/L 2iP and 10 mg/L Kin gave the highest number of shoot induction and shoot length. Whereas, in apical explants, the percentage of multiple shoot induction as well as the number of shoots was found to be more in the supraoptimal concentrations (20 mg/L 2iP & 15 mg/L Kin), indicating that higher concentration of cytokinin treatment is required to induce multiple shoot from apical explants (Table 1). Similar requirement of higher cytokinin concentration to induce shoot proliferation from shoot tip explant was reported in Juglans nigra16. The chances of somaclonal variation is less in multiple shoot induction from nodal explants rather than the apical
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Table 1—Axillary shoot regeneration of G. fragrantissima from apical and node explants of mature plant on rhododendron medium (RM) with different cytokinin treatment 4th subculture
Initial culture % of response Growth regulators Control 2iP
Conc. (mg/L) 0 2 5 10 15 20 2 5 10 15 2-15
Kin
BA SE LSD
Apical 16e* 27bc 30ab 33a 33a 33a 27bc 33a 33a 23cd 0 + 2.87 4.69
Apical
% multiple Av. no. of shoot formation shoots 13e 11j 1.2gh 27c 17h 1.4fg 27c 32f 1.6ef 33ab 41d 1.9cd 37a 48b 2.3b 33ab 54ba 2.6a 23cd 15hi 1.4fg 33ab 29g 1.7de 33ab 38de 1.9cd 23cd 47bc 2c 0 0 0 + 2.67 + 2.86 + 0.18 4.52 5.23 0.24 Nodal
Nodal Av. shoot % multiple Av. no. of length (cm) shoot formation shoot 1.3fg 38h 1.8ghi 1.7cdef 69f 2.10g 1.9abcde 76de 3.27c 2.3a 89bc 4.28a 2.2ab 96a 4.62ab 2.1abc 91abc 3.23cd 1.2fgh 57g 1.9gh 1.6defg 78d 3.68ef 2.0abcd 93ab 4.12cde 1.8bcde 86c 3.2e 0 0 0 + 0.28 + 2.64 + 0.21 0.42 5.61 0.33
Av. shoot length (cm) 2.1fg 2.3ef 2.6d 3.3ab 3.6a 3.1bc 2.2efg 2.5de 3.1bc 3.3ab 0 + 0.23 0.37
*Observations made after 8 wk of culture; Means followed by common letters are not significantly different (P
