In vitro multiplication of Wthnia somnifera auxiliary buds for mass propagation

Wthnia somnifera of family Solanaceae commonly known as Amukkara or Ashwaganda is an important medicinal perennial herb with long tapering roots. The roots are widely used in ayurvedic medicine and prescribed for hiccup, female disorders, cough and rheumatism. Wild and cultivated forms are available and cultivated plants differ in morphological and therapeutical action from wild ones. The annual requirement of 42347 kg is imported from India spending Rs. 2, 840, 449 (IUCN , 2005). Development of a method for in vitro mass production of this valuable species would substantially reduce the import cost and generate employment opportunities. This study was conducted to develop an in vitro protocol for mass production of W. somnifera.

In vitro grown seedlings of W. somnifera were used to excise single nodal cuttings and cultured on MS (Murashige & Skoog, 1962) medium. The effect of NAA (0, 0.1, 0.2 mg/l) in combination with BAP (0.5, 1.0, 1.5 mg/l) on shoot proliferation was tested in solid and liquid MS media containing two levels of sucrose (3% and 4%). Proliferation (number of plantlets produced) was observed at weekly intervals.

Highest shoot proliferation rate (1: 20) was observed in solid MS medium containing 3% sucrose and 1.5mg/l BAP after two months. In solid MS medium with 4% sucrose and 1.0 mg/l BAP 1: 10 shoot proliferation rate was observed after two months. During the same time period 1:15 shoot proliferation rate was observed in solid MS medium with 3% sucrose, 0.5 mg/l BAP and 0.2 mg/l NAA, where 1: 12 proliferation rate was observed when BAP concentration increased up to 1.0 mg/l. Solid MS media both with 3% and 4% sucrose levels and 0.5 mg/l BAP showed some proliferated shoots while other solid cultures were not proliferated. However cultures containing NAA in addition to BAP enhance rooting of proliferated shoots before transferring the separated shoots to rooting media. The explants in liquid MS medium with 4% sucrose and 1.0 mg/l BAP, proliferation initiated in one week and gave 1:40 shoot multiplication rate after two months. Rest of the liquid cultures with different BAP and NAA combinations were not proliferated within the same time duration.

Finally it can be concluded that highest shoot proliferation of W. somnifera through single nodal cuttings can be obtained on liquid MS medium with 4% sucrose and 1.0 mg/l BAP.

P S Warakagoda, D L C Kumari and S Subasinghe
Department of Crop Science, Faculty of Agriculture, University of Ruhuna, Sri Lanka

Effect of physiological status on rooting of Masbedda (Gymnema sylvestre) cuttings

Vegetative propagation by means of cuttings is an important method for starting new plants identical to the parent plants. Many plants can be propagated with good results by cutting, though the success depends upon the propagator’s circumstances, the time of year, and the plant to be propagated. The present study was carried out to investigate the effect of physiological stage on rooting of Gymnema sylvestre stem cuttings.

Healthy, double nodded cuttings were made from the mature plant stock established at the Faculty of Agriculture, University of Ruhuna. The cuttings taken from pre-flowering (T1), flowering (T2) and post-flowering (T3) stages were stuck into preformed holes in poly bags filled with moistened rooting medium which consisted of sand, top soil and compost (1:1:1 by volume). They were placed in a shade house and watered once a day. The Completely Randomized Design (CRD) was used with ten replicates. Assessment was done 75 days after for rooting. The percentage survival was not significantly (p £ 0.05) different between cuttings taken from the pre-flowering (92%) and post-flowering (87%) stages. No significant (p £ 0.05) differences also in the percentage of callused and rooted cuttings were recorded between T1 and T3. However, number of roots and length of the longest root per cutting were significantly (p £ 0.05) higher in T1 than any other. Furthermore, T2 showed the lowest figures for all the parameters assessed, indicating that the physiological status of the stock plant at the time the cuttings are excised is of great importance for the rooting process.

K K I U Arunakumara1, U Wickramasinghe1, B C Walpola2 and S Subasinghe1
1Department of Crop Science, University of Ruhuna, Kamburupitiya.
2Department of Soil Science, University of Ruhuna, Kamburupitiya.

In vitro propagation of Kaempferia galanga (L)

Kaempferia galanga (L) is an aromatic perennial herb, which is widely used in Ayurvedic medicine. Dry tubers are imported in large scale to Sri Lanka due to lack of mass production in Sri Lanka. Disease susceptibility and higher cost of production have restricted its cultivation. Propagation of Kaempferia galanga is normally by rhizome cuttings but disease susceptibility of tender rhizomes restricts propagation in large scale. Propagation through other vegetative methods is not possible. Rahman et al. (2004) reported the possibility of obtaining plants through somatic embryogenesis but the survival rate was low. Therefore an attempt was made to develop a protocol for mass propagation of Kaempferia galanga through direct organogenesis. Leaf discs and axillary buds were used as explants. Axillary buds isolated from rhizomes of Kaempferia galanga mother plants were sprayed with 0.2% Captanä 2-3 days before collection. After that, they were placed on a wet paper lined tray and covered again with another wet paper. Five to six days later young axillary buds were emerged from nodes and they were used as explants. For leaf disc explants leaves were washed with soap and soaked in a solution of Teepolä for 15 minutes, and washed with running tap water for 45 minutes.

Both leaf discs and axillury buds were dipped in 5% Chloroxä (5.25% Sodium hyperclorite v/v) for 10-15 minutes under sterile conditions. Then they were washed 10% Cloroxä for 3 minutes and 70% ethanol for one minute each followed by two successive washings in sterile distilled water. Explants were cultured on MS basal medium (Murashige and Skoog, 1962) supplemented with different concentrations of Benzyl amino purine (BAP) and Indole acetic acid (IAA) (2.00 mgl-1 – 2.25 mgl-1 and 0.30 mgl-1 – 0.70 mgl-1 respectively). Sucrose 3% (w/v) and 0.8% agar were added to the media. pH was adjusted to 5.8.

Cultures were incubated under 16 hr light /8 hr dark at 26 + 1 0C temperature for 21 days. Callusing was not observed from both tested explants in any of the media tested. After 15 –18 days of incubation axillary buds were elongated in all combinations tested. MS supplemented with 2.25 mgl-1 BAP and 0.5 mgl-1 IAA showed the highest elongation (490 ± 10 mm). After 25-30 days of incubation in vitro grown shoots were cut and separated from the explant. Then they were subcultured on the same medium and incubated in 16 hr light at 26 + 1 0C temperature for shoot multiplication. MS medium was used as basal medium with above combinations of growth regulators.

The highest multiplication was observed in 2.25 mgl-1 BAP and 0.5 mg-1 IAA (7.0 + 0.02) shoots per explant. Further sub culturing on to the same medium induced roots. Seven-weeks old plantlets were removed from culture vessels, washed well to remove all agar and transferred to small plastic pots containing sand, soil and compost in 1:1:1 proportion by volume and kept in shade house covered with polythene bags, for acclimatization. 100% survival was observed when acclimatized plants were transferred to the field.

 K M R T Wijekoon and W T P S K Senarath
Department of Botany, University of Sri Jayewardenepura, Sri Lanka

Collection, conservation, evaluation and use of durian germplasm at Horana

Unavailability of high quality varieties is one of the constraints in commercial durian production. A program to collect durian germplasm was initiated at Horana. Fruits from 26 seedling trees were evaluated for fruit weight, number of arils, number of seeds, weight of husk and seeds using six fruit per tree. Aril size, seed size, % rind, % seed and % aril were calculated. Aril color, flavor and overall acceptability were also recorded with a panel test. Results showed high variability in fruit quality traits exists among trees. Highest variability was found in number of seeds per fruit (CV=36.2%) while % rind showed the lowest variability (CV=7.26%). Number of seeds varied from 3.7 -19.2 per fruit while % rind varied from 60.5-79.5%. Nine accessions selected were planted with five replicates for further evaluation. Plant height and stem girth showed significant differences at early stages but became non significant by three years after planting. At 42 months after planting plant height varied from 368-492 cm while stem girth ranged from 42.0 – 52.8 cm. Principal component analysis of selected fruit and leaf characteristics of six accessions showed that first three PCs accounted for 89.35% of the variation in the characteristics used for the analysis indicating that the varieties of the collection are diverse. Collection of germplasm continued with establishment of a field gene bank to conserve accessions with two replicates. Nine selections were also further tested in farmer fields for adaptability.

K H S Peiris
Fruit Crops Research and Development Center, Department of Agriculture, Kananwila, Horana

Collection, conservation, evaluation and use of durian germplasm at Horana

Unavailability of high quality varieties is one of the constraints in commercial durian production. A program to collect durian germplasm was initiated at Horana. Fruits from 26 seedling trees were evaluated for fruit weight, number of arils, number of seeds, weight of husk and seeds using six fruit per tree. Aril size, seed size, % rind, % seed and % aril were calculated. Aril color, flavor and overall acceptability were also recorded with a panel test. Results showed high variability in fruit quality traits exists among trees. Highest variability was found in number of seeds per fruit (CV=36.2%) while % rind showed the lowest variability (CV=7.26%). Number of seeds varied from 3.7 -19.2 per fruit while % rind varied from 60.5-79.5%. Nine accessions selected were planted with five replicates for further evaluation. Plant height and stem girth showed significant differences at early stages but became non significant by three years after planting. At 42 months after planting plant height varied from 368-492 cm while stem girth ranged from 42.0 – 52.8 cm. Principal component analysis of selected fruit and leaf characteristics of six accessions showed that first three PCs accounted for 89.35% of the variation in the characteristics used for the analysis indicating that the varieties of the collection are diverse. Collection of germplasm continued with establishment of a field gene bank to conserve accessions with two replicates. Nine selections were also further tested in farmer fields for adaptability.

K H S Peiris
Fruit Crops Research and Development Center, Department of Agriculture, Kananwila, Horana

Comparison of litter decomposition rate constant for Yagirala and Horton

An estimation of rates of litter decomposition was carried out in two forest types; Yagirala Forest Reserve (FR) in the Low Country Wet Zone and Horton Plains natural forest (NF) in montane zone of Sri Lanka. Yagirala forest reserve was located between 6°21′ to 6°26′ north altitude and 80°6’to 80011’east longitude in the lowland wet climatic zone in Sri Lanka. Horton Plains natural forest was located between 6° 47′- 6° 50′ north latitude and 80° 46′- 80′ 51′ east longitude in mid country of Sri Lanka.

Three 300m line transects with three plots (100 m distance between 2 plots) were established in each forest. Litter decomposition rates were determined using the mixed species litter bags method. A total of 54 bags were placed in the both Forests (9 replicates for one plot). The experiment was conducted for a period of 8 months. The rates of decomposition of litter recorded during this were fitted to the exponential decay model proposed by Olson (1963).

x/x=e-kt

Where, x is the weight of litter remaining after time ‘t’, x0 is initial weight of litter and k is decomposition rate constant. Results revealed that the mean annual litter decomposition rate constant for moderately exploited Yagirala forest reserve was 2.19 year-1 while the value for Horton Plains natural forest was 1.35 year-1.Litter accumulation rates for Yagirala Forest is 668.86 tons ha-1 year-1, and this value for Horton Plains natural forest is equal to 226.54 ha-1 year-1. According to the results, it was clear that Yagirala forest reserve situated in the low country wet zone recorded higher litter decomposition rates compared with Horton Plains natural forest situated in the Montane zone of the country.

L A M C Amarasekara and D M S H K Ranasinghe
Department of Forestry and Environmental Science, University of Sri Jayewardenepura, Sri Lanka.

Assessment of soil erosion hazard of Victoria catchment area using GIS as a

Victoria reservoir is located at an elevation of 340 m to 440 m with a geographical position of 7° 15¢ to 7° 19¢ N and 80° 39¢ to 80° 48¢ E which has been constructed by damming the River Mahaweli at Victoria fall, in Sri Lanka in 1983. The reservoir storage capacity is 721.2 MCM and upstream dam site comprises 1338 km2 in the districts of Kandy, Nuwara-Eliya and Matale. The elevation of the catchment ranges from 340 m to 2100 m.

Soil erosion is a major water quality issue in the upland reservoirs. The objective of this paper is to analyze catchment issues contributing to soil erosion in the Victoria reservoir and to evaluate the soil erosion risk areas in the catchment. Study was carried out from 2002 to 2004.

Soil erosion occurs due to natural causes such as rain fall, rainfall runoff and wind, and due to human activities. Universal Soil Loss Equation ( RKLSCA = P ) introduced by Wischmeier and Smith in 1965 is a most widely used method for estimating soil erosion. This encounters detachment of soil particles and its transport by raindrops and surface runoff, which depends on the rainfall erosivity (R), erodibility of soil (K), slope length factor (LS), cover and management factor (C) and the support practice factor of the equation.

The data on erosivity points were interpolated with 50 m resolution grid cells. The erodibility value relevant to each soil group was entered into the attribute table, which was converted into grid cells with 50 m resolution, containing soil erodibility values. The Triangulated Irregular Network (TIN) was created by contour interpolating with 20 m interval, and grid cell was 50 m. Using TIN, slope percentages map was derived, which was used to obtain LS factor. The C factor values relevant to each landuse type were entered into the attribute table. The map was converted into grid cells with 50 m resolution, containing C factor values.

The soil erosion of the Victoria catchment was categorized into five erosion categories, namely; low, moderate, high, very high and extremely high, which extend within Kandy, Nuwara-Eliya and Matale districts. All categories were spread both in the left and right banks.

Extremely high erosion areas extend over 24.34km2 with a percentage of 1.82, Very high erosion areas extend over 121.24 km2 with a percentage of 9.06, High erosion areas extend over 302.91km2with a percentage of 22.63, Moderate erosion areas extend over 434.01km2 with a percentage of32.43 and low erosion area extends over 454.97 km2 with a percentage of 33.99. The results of mapanalysis were confirmed through field verifications. The soil erosion is high in the high slope regionsand in the areas where soil conservation methods are inadequate or poor.

In vitro callus induction of Spilanthes calva DC [Spilanthes acmella auct. nonL,.] (Maha Akmella)

Spilanthes calva DC. (Maha Akmella) is a valuable medicinal plant belongs to Family Asteraceae. It is widely used in indigenous medicine to treat toothache in most of the Asian countries. Not only it has anesthetic properties, but also contain secondary metabolites, with the insecticidal properties, which could be used as potential bio insecticide. This is an annual plant, which grows to a height about 30 cm. After flowering mother plant is dried off. Four to six weeks later seeds are germinated and new seedlings are produced. Viability of seeds loses within short period of time. Even though seeds are germinated percentage of germination is low (about 30%). Rooting of cuttings is also not possible. This is a limitation in using this valuable medicinal plant for commercial production. Therefore it is very important to develop a protocol for mass propagation through tissue culture and establishing cell cultures will be useful for large-scale chemical extraction in industrial purposes.

Leaf discs were used as explant for callus initiation. In order to identify the suitable maturity stage for callus initiation, leaves were harvested at different maturity stages i.e first, second and third fully opened leaf.

Leaves were washed with Dettolä soap and soaked in a solution of Teepolä for 5 minutes. After that leaves were washed with running tap water for 45 minutes. In order to surface sterilize. Leaves were washed with 10% Cloroxä (5.25% Sodium hypochlorite v/v) for 5 minutes and then with 70% alcohol for 30 seconds each followed by three successive washings in sterile distilled water. These operations were carried out inside the laminar airflow cabinet before inoculation. Basal media tested for the study were full strength MS (Murashige and Skoog, 1962) medium and ½ MS (both macro and micronutrients) medium. Media were supplemented with different concentrations (1.0 mgl-1 – 3.0 mgl-1) of BAP and 2,4-D. Cultures were incubated under complete dark at 25±1°C in the growth room.

Study conducted by Haw and Keng (2003) on the same species produced multiple shoots from axillary bud explants without inducing callus in MS medium supplemented with 2.0 mgL-1 BAP. In the present study, callusing was observed within 5 days of incubation in full strength MS medium supplemented with BAP and 2,4D. It took longer period to initiate callus when both macro and micro nutrients in the basal medium was lowered to half and the amount of callus produced was also very low even after 6th week of incubation. In order to observe the time taken to produce maximum amount callus fresh weight was measured after 2nd, 4th and 6th week of incubation. It was observed that maximum amount of callus was produced within 4 weeks in all explant types tested with a maximum of 0.88 g ± 0.23 in leaf discs obtained from first fully opened leaf.

In order to determine the best growth regulator combination for callus initiation, calli fresh weights were measured after fourth week of incubation in different growth regulator combinations tested. Highest amount of calli were in MS medium in the presence of 2.25 mgl-1 BAP and 1.0 mgl-1 2,4-D. Fragile calli, which were transulant and mucilaginous in nature were observed within 15 days of incubation, which could lead to cell suspension cultures.

In vitro callus induction of Spilanthes calva DC [Spilanthes acmella auct. nonL,.] (Maha Akmella)

Spilanthes calva DC. (Maha Akmella) is a valuable medicinal plant belongs to Family Asteraceae. It is widely used in indigenous medicine to treat toothache in most of the Asian countries. Not only it has anesthetic properties, but also contain secondary metabolites, with the insecticidal properties, which could be used as potential bio insecticide. This is an annual plant, which grows to a height about 30 cm. After flowering mother plant is dried off. Four to six weeks later seeds are germinated and new seedlings are produced. Viability of seeds loses within short period of time. Even though seeds are germinated percentage of germination is low (about 30%). Rooting of cuttings is also not possible. This is a limitation in using this valuable medicinal plant for commercial production. Therefore it is very important to develop a protocol for mass propagation through tissue culture and establishing cell cultures will be useful for large-scale chemical extraction in industrial purposes.

Leaf discs were used as explant for callus initiation. In order to identify the suitable maturity stage for callus initiation, leaves were harvested at different maturity stages i.e first, second and third fully opened leaf.

Leaves were washed with Dettolä soap and soaked in a solution of Teepolä for 5 minutes. After that leaves were washed with running tap water for 45 minutes. In order to surface sterilize. Leaves were washed with 10% Cloroxä (5.25% Sodium hypochlorite v/v) for 5 minutes and then with 70% alcohol for 30 seconds each followed by three successive washings in sterile distilled water. These operations were carried out inside the laminar airflow cabinet before inoculation. Basal media tested for the study were full strength MS (Murashige and Skoog, 1962) medium and ½ MS (both macro and micronutrients) medium. Media were supplemented with different concentrations (1.0 mgl-1 – 3.0 mgl-1) of BAP and 2,4-D. Cultures were incubated under complete dark at 25±1°C in the growth room.

Study conducted by Haw and Keng (2003) on the same species produced multiple shoots from axillary bud explants without inducing callus in MS medium supplemented with 2.0 mgL-1 BAP. In the present study, callusing was observed within 5 days of incubation in full strength MS medium supplemented with BAP and 2,4D. It took longer period to initiate callus when both macro and micro nutrients in the basal medium was lowered to half and the amount of callus produced was also very low even after 6th week of incubation. In order to observe the time taken to produce maximum amount callus fresh weight was measured after 2nd, 4th and 6th week of incubation. It was observed that maximum amount of callus was produced within 4 weeks in all explant types tested with a maximum of 0.88 g ± 0.23 in leaf discs obtained from first fully opened leaf.

In order to determine the best growth regulator combination for callus initiation, calli fresh weights were measured after fourth week of incubation in different growth regulator combinations tested. Highest amount of calli were in MS medium in the presence of 2.25 mgl-1 BAP and 1.0 mgl-1 2,4-D. Fragile calli, which were transulant and mucilaginous in nature were observed within 15 days of incubation, which could lead to cell suspension cultures.

 S Hewage and W T P S K Senarath
Department of Botany, University of Sri Jayewardenepura, Sri Lanka.

Effect of bio control agent Trichoderma (T. viride and T. konnigii) on basal rot of Cloropytum comosum ‘laxum’ caused by Sclerotium rolfsii

At present, the biological control of soil borne fungal diseases is becoming popular in foliage industry of Sri Lanka, which is a nature-friendly ecological approach to overcome the problems caused by standard chemical methods of plant protection. With a suitable bio control agent pathogen can be suppressed and reduced the disease incidence could be reduced effectively. This experiment was conducted over a period of six months in polytunnel to identify a potential bio control agent for basal rot of Cloropytum comosum ‘laxum’ caused by Sclerotium rolfsii with five treatments of Trichoderma viride, Trichoderma konnigii and combination of Trichoderma viride and Trichoderma konnigii, Pormarsol forte 80% wp and control. The mean disease incidences of above treatments were 1.75, 2.75, 1.5, 1.75 and 10.75 respectively. It was revealed that Trichoderma viride and combination of Tricoderma spp. are suitable for the highly effective control of plant diseases caused by Sclerotium rolfsii.

K A L Priyadarshani and D B Kelaniyangoda
Department of Horticulture and Landscape Gardening, Wayamba University of Sri Lanka, Sri Lanka