Asian Pacific Journal of Reproduction 2012; 1(1):38-41 38 Contents lists available at ScienceDirect Asian Pacific Journal of Reproduction Journal homepage: www.elsevier.com/locate/apjr Document heading 10.1016/S2305-0500(13)60045-7 Micropropagation of Tigridia pavonia (L.f) DC-a potential floricultural plant from twin scale explants Lekha Kumar*, Sincy Joseph, Narmatha Bai Department of Botany, Bharathiar University, Coimbatore-46, Tamilnadu, India ARTICLE INFO ABSTRACT Article history: Objective: The presentTigridia study pavonia was performed to standardize Methods: an effective protocol for Received 15 February 2012 micropropagation of using tissue culture. The explants were cultured Received in revised form 16 February 2012 on Murashige and Skoog (MS) medium supplemented with cytokinins like thidiazuron (TDZ), Accepted 15 March 2012 zeatin, kinetin and auxins such as indole-3-acetic acid (IAA), 1-naphthalene acetic Results: acid Available online 20 March 2012 (NAA) and indole-3-butyric acid (IBA), individually at different concentrations. Multiple shoots were obtained on MS medium containing either 2.0 mg/L TDZ or 2.0 mg/L IAA or 0.5 mg/L IBA and in the same medium for a long period (120 d) produced tiny bulbs Keywords: at the base of the senescent leaves. TDZ favored only multiple shoots without roots, whereas Tigridia pavonia IAA or IBA individually or in combination with TDZ produced rooted shoots. Shoots developed on MS medium supplemented with TDZ were rooted on MS medium containing either IBA or Twin scale explant NAA at 0.5 mg/L. The plantlets were acclimatized in pots containing gardenConclusions: soil. Regenerated Murashige and Skoog medium plantlets developed into normal plants. The plants showed 99% survival. The Growth regulator highest number of bulblets obtained in the present study represents an effective alternative to the conventional method. 1. Introduction Additionally production of storage organs (Corms, tubers or bulbs) during micropropagation can reduce the losses The world floriculture market is estimated to be worth US incurred during acclimatization and decrease the time to $ 60 billion, and constituent increase in demand for cut flower[4]. and potted flower has made floriculture one of the important Successful micropropagation in Iridaceae has been commercial trades in Indian agriculture. achieved using explants such as anthers, bulbs, corms, The Iridaceae is a large family with over 2 000 species flowers, hypocotyl, leaves, ovaries, roots, shoots and twin many of which are endemic to South Africa and produce scales[1]. Many bulbous species successfully produce attractive flowers and foliage. Approximately 40 species adventitious shoots from the tissue at the base of bulb and hybrids from 12 genera have been successfully scales[5,6], and from the junction of the scales on the basal [1] Tigridia pavonia [7] micropropagatedT. pavonia by tissue culture . ‘ plate , so the present study was conducted to develop a (L.f) DC’ ( ‘ ) commonly’ ‘ known ’as Mexican shell feasible and reliable method for multipleT. pavonia shoot regeneration flower or Peacock flower or Tiger lily plant is a native using twin bulb scale explants in . of Mexico. The name alludes to coloration and spotting of flowers. The bulb of the plant is edible and the plant is 2. Materials and methods known to promote fertility. Tissue culture technique is a powerful tool which can 2.1. Surface sterilization, explants preparation and medium be employed as an alternative to the conventional method preparation of vegetative propagation with the objective of enhancing [2,3] the rate of multiplication of desired genotypes . Tissue T. pavonia culture procedures allow the rapid production of superior The bulbs of were collected from Government disease free clonal material for nurseries and growers. Botanical Garden, Udhagamandalam, Tamilnadu, India. The root, leaves and the brown coating of the bulbs were removed. Twin scales with basal meristem was used as an *Corresponding author: K Lekha, Department of Botany, Bharathiar University, explant. The explant was washed several times with tap Coimbatore-46, Tamilnadu, India. water along with teepol for 30 min to remove the dirt and E-mail: [email protected] debris. Lekha Kumar et al./Asian Pacific Journal of Reproduction (2012)38-41 39 3. Results The explant was then treated with a systemic fungicide Bavistin (2%, v/v) for 30 min and washed thoroughly. 3.1. Effect of NAA, kinetin, zeatin and IBA on multiple shoot An additional treatment of 20 min soaking in antibiotic formation gentamycin sulphate (0.01%, v/v) was essential to eliminate contamination. The explants were then thoroughly washed and surface sterilized with one or two drops of Tween 80 The explants did not respond when cultured on MS medium in ethanol (70%, v/v) for 20 min and then treated with devoid of plant growth regulators (control). Shoots were mercuric chloride (0.02%, v/v) and washed repeatedly prior initiated on the twin scales on all the media irrespective of to inoculation. the growth regulators tested individually. Multiple shoots Explants were cultured on Murashige and Skoog medium were formed between the twin scales containing the basal (MS medium) containing various° growth regulators. tissue and single scale explants were not suitable for shoot Autoclaving was done at 121 C for 20 min. All± the° initiation. In the present study, IBA at 0.5 mg/L regenerated cultures were maintained at a temperature of (25 2) C maximum number of shoots (3.6/twin scale) (Table 1). NAA and at relative humidity of 65%-70%. The cultures were failed to induce multiple shoots (Table 1). The effect of kept under white light at intensity of 2 000 lux provided kinetin and zeatin in bulblet initiation was negligible. Also, from white fluorescent lamps (PHILIPS, India) with 12 h the development of the shoots was very slow. Significant photoperiodic duration. F Pvariation existed between concentrationsF ( 0.01(1,16)P =48.19; 2.2. Shoot multiplication 0.01(3,16) <0.01), among hormones ( =9.06;F <0.01) and Phormone concentration interactions ( 0.01(3,16)=17.51; <0.01) in number of shoots. Maximum bulblet diameter For culture initiation, twin scales with the basal meristem were cultured in MS basal medium containing various (1.43 cm) and bulblet length (1.43 cm) was recorded in concentrations of thidiazuron (TDZ, 0.5-3.0 mg/L), zeatin IBA rather than NAA at 0.5 mg/L.F Significant variationP also (0.5-1.0 mg/L), kinetin (0.5-1.0 mg/L), 1-naphthalene existed between concentrationsF ( 0.01(1,16)P =14.58; <0.01), acetic acid (NAA, 0.5-1.0 mg/L), indole-3-acetic acid among hormones ( 0.01(3,16)F=5.18; <0.01)P and hormone (IAA, 0.5-3.0 mg/l) and indole-3-butyric acid (IBA, concentration interactions ( 0.01(3,16)=10.01; <0.01) in shoot 0.5-1.0 mg/L) individually with 30 g/L sucrose, activated length. charcoal (2 mg/L) solidified with 0.8% agar. The pH was 3.2. Effect of IAA and TDZ on multiple shoot formation adjusted to 5.8. 2.3. Root formation IAA at 2 mg/L regenerated maximum number of shoots (4.67/twin scale) (Table 2). The highest number of shoots The multiple shoots were cultured in MS medium per explant was recorded as 6.4 in MS medium containing supplemented with NAA (0.5 mg/L) and IBA (0.5 mg/L). MS TDZ at 2 mg/L (Table 2). Tiny bulbs appeared at the base of medium containing TDZ (2.0 mg/L) in combination of IAA the developing shoots after 2 month period. One bulb/shoot and IBA was also employed to induce rooting. was observed. The maximum bulb diameter (1.90 cm) was 2.4. Acclimatization recorded on MS medium with TDZ at 3 mg/L. The maximum bulb length (2.87 cm) was obtained on MS with TDZ at 2F mg/L. SignificantP variations existed between hormone The bulblets were washed thoroughly in distilled water to (F0.01(1,24)=27.97; P <0.01), hormone concentration interaction remove the traces of medium and then acclimatized in pots (F0.01(5,24)=8.09; <0.01) and no variation in concentration containing garden soil for hardening. ( 0.01(5,24)=1.00) (Table 2). 2.5. Statistical analysis 3.3. Effect of growth regulators on rooting Each treatment consisted of five replicates and the Twin scales when cultured on MS medium supplemented experiment was repeated thrice. After 2 weeks of culture, with auxins such as IAA or IBA individually or in data was recorded for shoot initiation and thereafter combination with a cytokinin like TDZ produced shoots and periodically for shoot development and rooting. Analysis roots in the same medium. Maximum number of roots (4.34) of variance (ANOVA) was performed on all the data to was produced from medium containing IBA at 0.5 mg/L. compare concentration effects of growth regulators on The highest root length (12.50 cm) was recorded on medium multiple’ shoot regeneration. Means were separated using containing TDZ (2.0 mg/L) and IBA (0.5 mg/L). MS medium Duncan s multiple range test. Table 1 Effect of growth regulators on the formation of multiple shoots and lengths. Number of shoots Shoot length (cm) Growth regulator 0.5 mg/L regulator 1.0 mg/L regulator 0.5 mg/L regulator 1.0 mg/L regulator c a b a NAA 1.40 1.00 5.40 6.30 b b b b Kinetin 2.30 0.00 5.70 0.00 d ab c ab Zeatin 0.30 0.67 1.37 2.77 a ab a b IBA 3.60 0.30 10.87 0.80 P IAA, indole-3-acetic’ acid; IBA, indole-3-butyric acid.