Introduction

The genus Lilium comprises of about 85 species, all of which belonging to Liliaceae family, majorly comprising of various ornamental species. As it is derived from interspecific hybridization, holds a great importance in the commercial market being a monocot bulbous crop. Apparently, 80 species of Lilium are found in the temperate and subtropical zones of northern hemisphere [1]. The plants within Lilliacae family have high value due to their scented nature, wild range of colors resistance and their effective adaptation to their imperfect surroundings. Tresor belongsto Asiatic hybrid Lilium varieties, is a perennial plant with erect stems bearing lance-shaped, spirally-arranged, glossy, dark green leaves and, in summer, large, fragrant, upward-facing, orange flowers with dark orange-speckled, yellow-flushed throats. It goes to a height of 1m and spread of 0.5m in 2-3 years having a very strong supporting stem [2].

Flower bulbs have been appreciated and cultivated for thousands of years and long before they were widely grown commercially or extensively researched. Previous literature suggests that explants from flower organs to bulb scales of Lilium could be easily manipulated and regenerated using tissue culture techniques [2]. Lilium propagation is usually done by vegetative means which produces 3-4 bulbs per bulb scale depending on size and variety. The multiplication efficacy by bulb is low and the plantlets are more susceptible to diseases. Therefore, there is a need to develop a protocol for its mass propagation. Through tissue culture, there is not only a continuous supply of bulblets but true-to-type and disease free plants can be obtained. Therefore the objective of the present research was to establish a protocol for standardization of sterilization time and Plant bioregulators for callus formation in Lilium hybrid Cv. Tresor.

Materials and Methods

The present investigation was carried out during the year 2013-14, at Biotechnology-cum-Commercial Tissue Culture Centre, OUAT, Bhubaneswar.

Source of Explants

For this study, Healthy and disease free bulbs of  hybrid Lilium Cv. Tresor was collected from fresh plants grown under poly-house in the Biotechnology-cum-Commercial Tissue Culture Center, OUAT, Bhubaneswar, maintained as Mother plant for conducting in vitro in the laboratory.

The bulb scale was taken for in vitro culture.

Stock solution, Media Preparation and Sterilization

The chemicals used for the present study were analytical reagents of excel R grade of Titan Biotech Ltd., Ranbaxy Laboratory Ltd., Merck (India), Qualigen Fine Chemicals, and Himedia Laboratories Ltd. (India). Auxins, Cytokinins, myo-inositol and Fe-EDTA that were supplied by Sigma (USA) and Agar from Ranbaxy Laboratory Limited. MS Medium (Murashige and Skoog, 1962)[3] was used throughout the investigation, required quantities of macronutrients, micronutrients, Fe-EDTA, vitamins and plant bioregulator’s were taken from the stock solution and required quantity of sucrose dissolved in distilled water was added fresh to the medium. The pH of the solution was adjusted to 5.7+ 0.1 using 0.1N NaOH or 0.1 N HCL. Then volume was made up to 1 L with distilled water. Agar (0.6% w/v) was added to the medium boiled and poured into the culture tube and plugged. Culture tube containing culture medium was autoclaved for the 20 minutes at 121⁰C and 15 Psi pressure. The autoclaved medium was kept in laminar air flow bench for cooling. All the glassware were dipped in the detergent solution for overnight and washed under running tap water. They were rinsed with distilled water and then dried in an oven for 2hrs at 150⁰C. Forceps, petridish and scalpel were thoroughly cleaned with iso-propanol,  rapped with paper and kept in a clean sterilized autoclave at 15 psi and 121⁰C for 20 minutes. The working chamber of laminar air flow cabinet was wiped with iso-propanol . Filtered air (80-100 cft/min) to ensure that particles do not settle in working area was blown for 5 minutes. The sterilized materials to be used (except living tissue) were kept in the chamber and exposed to UV light for 30 minutes.

Inoculation

The sterilized explants were then  prepared by removing the rhizome and separating the scales into individuals Continuing with the above conditions these explants were then subjected to further  30 min with tap water washing followed by 15 minute tween 20 solution washing,  further the explants was treated with 2 % bavistin for 20 min and as per the treatment plan the explants was treated with  0.1% HgCl₂ (3 min, 4min, 5min, 6 min, 7min, 8min and 9 min) and a control (without treatment). These sterilized explants were then cultured on Murashige and Skoog (1962) [3] medium supplemented with growth hormones BAP (1.0 mg/l) and NAA (0.5mg/l) with 8% (w/v) agar, 30% (w/v) sucrose with three replications. The data recorded for different parameters i.e. fungal %, bacterial %, death %, aseptic % and survival % of explants were recorded from 15 DAI (Days after inoculation), 30 DAI and 45 DAI. The treatment having the maximum survival percentage with minimum infection and death percentage were taken as the standard time for disinfecting explants before inoculating them for in vitro propagation.

For callus formation study, explants were treated with the best surface sterilizing time (5 minute) among the surface sterilization treatments taken into consideration. After the disinfection each section of the bulb scale (7 × 7 mm), with the dorsal side in contact with the medium, was placed in a culture tube with MS (Murashige and Skoog,1962) [3] medium containing BAP (0.5, 1.0 mg/l) in combination with 2,4-D (0.5,1.0,1.5,2.0, and 2.5 mg/l) and 2,4-D (0.5,1.0,1.5,2.0, and 2.5 mg/l)  alone and a control. Observation on days to callus initiation, % of callus produced, callus spread were recorded from 30 DAI and callus spread, colour of the callus and nature of callus recorded at 60 DAI.

Establishment of Culture

After inoculation, the culture were kept at 25±2⁰ C in an air conditioned room with a 16 hours light period (3000-3200 lux) supplied by fluorescent tubes and 80% relatively humidity [4].

Statistical Analysis

The raw data obtained during the experimental observations were subjected to statistical analysis as per method by Gomez and Gomez, (1984) [5]. The significance and non- significance of the treatment effect were judged with the help of ‘F’ variance ratio test. Calculated ‘F’ value was compared with the table value of ‘F’ at 5% level of significance. The data were transferred from where ever required before suitability of Analysis of Variance (ANOVA) analyzed in statistical package SAS version 7.0.

Results and Discussion

Sterilization time response of bulb scale explants of hybrid Lilium Cv. Tresor in response to the time of disinfection is depicted in table.1 to 3. The data revealed that among the different timing for the tissue cultured bulb scale explants at 15 DAI (Days after inoculation), the maximum fungal contamination % recorded at T₁ (36.67%) in control (Tap-water)_, with minimum fungal contamination % recorded at T₇ (8 min) and T₈ (9 min) with (0.00%). Bacterial contamination was recorded maximum at T₃ (4 min) (13.33%) and minimum was recorded at T₆ (7 min) and T ₇ (8 min) with (0.00%). In case of explants death, maximum was recorded throughout all treatments (3.33%). In case of aseptic culture, maximum was recorded at T₇ (8 min)(100%) followed by T₈ (9 min) (93.33%) and the minimum were obtained in T₁ (53.33%). Maximum survival percentage of explants were obtained at T₇ (8min) (96.67%) and minimum at T₁ (50.00%).

Table 1: Effect of Surface sterilization time on level of contamination, aseptic culture and survival of bulb scale explants of hybrid Lilium Cv. Tresor after 15 DAI (Days after inoculation).

After 30 DAI (Table.2) it was observed that the fungal contamination % was maximum at T₁ (40.00%) and minimum at T₇ (8 min), T₈ (9 min) (0.00%). In case of bacterial contamination % maximum was at T₂(3 min) ,T₃(4 min) (16.67%) and minimum at T₆ (7min), T₇ (8 min) (0.00%). In Case of Death % of explants was recorded maximum at T₈ (9 min) (6.67%) and the minimum at T₂ (3 min) (3.33%), similar results were also obtained from T₂ and T₇. In case of aseptic culture maximum was recorded at T₇ (6 min) (100%) and the minimum was recorded at T₁ (tap water) (46.66%). Survival % was maximum at T₇ (8 min) (96.67%) and minimum was recorded at T₁ (43.33%).

Table 2: Effect of Surface sterilization time on level of contamination, aseptic culture and survival of bulb scale explants of hybrid Lilium Cv. Tresor after 30 DAI.

At 45 DAI (Table.3) fungal contamination % was maximum recorded at T₁ (tap water) with (60.00%) and minimum was recorded at T₇ (8 min) and T₈ (9 min) (0.00%). The maximum bacterial contamination % was recorded at T₁ (tap water) (36.67%) and the minimum was at T₇ (8 min) with (0.00%). The explants that did not survive the whole process were seen maximum in T₈ (9 min) (13.33%) with a minimum death % from T₁ (tap water) to T₅ (6 min) (3.33%). In case aseptic culture % maximum was at T₇ (8 min) with (100%) and a minimum at T₁ (tap water) with (3.33%). The maximum survival % was recorded at T₇ (8 min) (93.33%) followed by T₈ (9 min) (80.02%) and the minimum survival %  was at  T₁(Tap water) (0.00%).

Table 3: Effect of Surface sterilization time on level of contamination, aseptic culture and survival of bulb scale explants of hybrid Lilium Cv. Tresor after 45 DAI.

The increase in the exposure of timing  to sterilants, lead to the death of explants which may be due to the heavy metal contamination of HgCl₂ proving phytotoxics that is against the survival of explants. Some explants in our experiment did not survive because of damage during sterilization procedure as HgCl₂ are toxic to the plant tissues, thus proper concentration and time duration of sterilizing agents should be carefully selected .Here it is observed that some bulb explants did not survive, it was probably due to damage occurred during the sterilization procedure. The sterilizing agent should be used for appropriate duration to control contamination. However, HgCl₂, which has mainly anti-bacterial action, was more efficient and showed more decontamination percentage. However, HgCl₂ was phytotoxic to hybrid Lilium bulb explants with the increase in the time duration [6].

Callus Induction

The sterilized bulb scale subjected to various treatments for callus emergence, development, proliferation and spread produced calli mass satisfactorily in MS  basal media (Control) only (14.33), and  with 2.5 mg/l 2-4-D significantly induced callus in 15 DAI (Days after inoculation) followed by 0.5 mg/l BAP and 2.5 mg/l 2-4-D (15.33) DAI. A high concentration of Cytokinin in the medium promotes abundant callus proliferation (Chawala, 2002). A combination of 1.0 mg/l BAP and 1.5 mg/l 2-4-D induced maximum percentage of callus (90.00) followed by 1.0 mg/l BAP and 1.0mg/l 2-4-D (80.00). Minimum callus percentage was recorded at the 2.5 mg/l 2-4-D followed by 0.5 mg/l BAP and 2.5 mg/l 2-4-D (10.00) (Table.4). Callus spread maximum was recorded at 1.0 mg/l BAP and 1.0 mg/l 2-4-D at 30 days and minimum were recorded in the 0.5 mg/l BAP and 0.5 mg/l 2-4-D. In case of 60 days similar trends were observed in the same treatment for maximum spread while minimum spread was seen in 0.5 mg/l BAP and 1.5 mg/l 2-4-D. The results are in alignment with the findings of Aswath and Choudhry, (2002) [7]; Patnaik  and Beura, (2008)[8] due to the synergetic effect of BAP was significant in maximum callus production.

Table 4: Effect of Plant bioregulators on callus emergence, development, nature of callus, callus spread and callus colour of bulb scale explants of Hybrid Lilium Cv. Tresor.

Conclusion

It was concluded that hybrid Lilium Cv. Tresor  bulb scale explants can be used to produce callus in vitro which could in turn be used to produce healthy plantlets under the aseptic condition on large a number and less time. Hence this investigation opens the way for uses of biotechnological were for mass propagation and improvement of Hybrid Lilium Cv. Tresor.

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