II. MATERIAL AND METHODS

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1 Effect of Vermicompost on the growth of Strawberry (Fragaria ananassa Duch) plant and standardization of micro propagation of elite germplasm Manikuntala Bhandari and Subhash Kanti Roy Post Graduate Department of Biotechnology, Oriental Institute of Science and Technology, Dewandighi, Katwa Road, Burdwan, West Bengal, India Abstract-- Effect of Vermicompost on Strawberry plant growth and yield was studied in field condition in the district of Burdwan, West Bengal, India. Significant morphological growth of the plant and increase in fruit yield were observed. However, fruit yield significantly decreased with the increase in diameter of bushes and total fruit yield increased with the decrease in diameter of lamina and length of leaf petiole. In vitro micro propagation of Strawberry plant was studied using nodal segment, shoot tip, runner tip, leaf base and rolled leaf as explant. MS basal media supplemented with high concentration of NAA (5mg/l) and BAP (5mg/l) played an important role for its proliferation and multiplication. Index Terms-- Growth regulators, micro propagation, Strawberry, vermicompost. I. INTRODUCTION The Strawberry ( Fragaria ananassa Duch ), is a stoloniferous herb and hybrid species cultivated throughout the world. It is popular as a nutritious soft fruit[11],[9] and has a wide range of medicinal values, such as, lowcalories carbohydrate, high fiber content and natural source of antioxidants[21],[12],[5][8],[13]. Various uses of Strawberry plant and its fruit in commercial food product industries and others for human welfare have been established [6]. In India, Maharashtra is the leading state for strawberry production and it is also grown in Dehradun, Nainital, Srinagar and in parts of West Bengal [13]. Recently, Strawberry is being cultivated in some areas of Bankura and Nadia district, West Bengal, India [17], during winter season (November to March). Non-availability of saplings compels the growers to import mother plants and it is very expensive for commercial cultivation. The use of organic matters, such as, animal manures, human waste, food waste and composts has long been recognized as beneficial for plant growth and yield in agriculture and maintenance of soil fertility [1]. Vermicompost applications are also known to increase Strawberry plant growth and yield [4],[14],[1],[15]. Micro propagation of elite germplasm is the only means to supplement the burning demand of Strawberry growers. Although in vitro methods for micro propagation of Strawberry have been successfully introduced from different parts of the world [7],[13],[9],[16],[18], the information in regard to the heterogeneous agro ecological conditions in Indian context, particularly in West Bengal, is meagre.in the above context, an attempt has been made to find out the effect of vermicompost on Strawberry plant growth and yield followed by the standardization of its in vitro micro propagation in West Bengal. II. MATERIAL AND METHODS Strawberry (Fragaria ananassa Duch ) plants were collected from California and cultivated in the Institute s field (Fig: 1). Plants were allowed to grow in the field for 60 days (Fig: 2). All the plants were treated with normal fertilizer that is cow dung, oilcake and NPK ( 10:26:26). Four rows containing 10 to 14 plants in each were maintained. One row was kept as control and three other rows (R1, R2 & R3) of plants were treated with vermicompost with doses of 15 gm per plant at every fifteen days interval. Comparative study was done with the control plants where no vermicompost was used. A. Morphological Parameters Morphological parameters were studied by following ways- plant height was measured by cm scale at three different positions and average height was calculated. Diameter of bushes (study of canopy) were measured by cm scale in three different directions diagonally. Runners of each plant were counted and length of each runner was measured by cm scale. Leaf petiole length was measured by taking three different sizes (small, medium, 442

2 large) of leaf of a plant by using cm scale. Lamina diameter was measured by taking three different sizes (small, medium, large) of a plant using cm graph paper. Then the average diameter expressed as square cm was recorded. Flowering (opened) of each plant was counted. Fruits (Fig: 2) were picked up at afternoon and weighed individually of each plant by digital weight machine. Fig 1: Cultivation of Strawberry plant in field Fig 2: Strawberry plant with fruits and runners B. In vitro Culture After using vermicompost healthy plants were used for in vitro culture. Shoot tip, runner tip, nodal segment, leaf base, rolled leaf etc of Strawberry ( Fragaria ananassa Duch ) mother plants were taken as explants. Then plant materials were excised in short pieces containing meristematic portion. The materials were then washed under running tap water for 30 mins. After washing the plant materials were surface sterilized with liquid detergent (Tween 20, Teepol about 2-3%) for 15 minutes on magnetic stirrer. Plant materials then washed thoroughly with double distilled water for 3-4 times. After that the plant materials were treated with 0.1% HgCl 2 solution for 5-7 minutes followed by washing with sterile double distilled water for 3-4 times. Then the sterilized plant material was cut into small pieces to form explants and inoculated in MS basal media[19] with different combinations & concentrations of growth regulators viz., Auxin ( NAA) and Cytokinine (BAP). The cultures were then kept in standard tissue culture laboratory with a photoperiod of 16h (white fluorescent light, Philips India Limited; µmol m -2 s -1 ) at 25±2ºC and 78% relative humidity [10]. C. Statistical analysis Standard Error of Mean (SE) & Coefficient of Variation (CV) of all the morphological parameters and tissue culture data have been analyzed by standard statistical methods. III. RESULTS A. Morphology It has been observed that vermicompost has played a key role in plant growth and yield (Table I, II, III & IV). The major findings (Fig: 1, 2 & 3) have been summarized as follows: Plant height ranged from 10 cm to 23 cm (Table II) in vermicompost treated soil as compared to control plant (without vermicompost) where height ranges between 10 cm to 17 cm (Table I). The bush diameter in vermicompost treated soil showed maximum cm (Table II) and minimum cm (Table III). After using vermicompost the number of runners were increased significantly. The maximum number seven (07) was observed (Table II, III, & IV). As an effect of vermicompost leaf petiole length was increased in compared to the control plants. The maximum length observed was 14.73cm (Table III). The maximum lamina diameter (33.17sq cm) was observed in vermicompost treated plant (Table IV) where as control plants showed only 28 sq cm (Table I). By using vermicompost number of flowers increased significantly with a maximum of 20 flowers for a plant (Table II). The effect of vermicompost on total yield of Strawberry showed very satisfactory result as compare to control plants. The maximum and minimum fruit weight ranges between gm (Table IV) to gm (Table II) where as the control plants showed gm to gm respectively (Table I). 443

3 percentage of growth ISSN: con R1 R2 R Parameters Parameters: 1= Plant height, 2 = Diameter of Bush,3 = No of runners,4 = Length of runners,5 = Leaf petiole length,6 = Lamina diameter,7 = No. of flowers,8 = No. of fruit,9 = Total yield Fig 3: Graphical representation of morphological parameters of control & vermicompost treated strawberry plant Table I: Study of Morphological Parameters Plant No. Plant Ht Diameter of Bush No. of Runners Length of Runners Leaf Petiole length Lamina diameter (sq cm) Flowering (no. of flower/ plant) Fruit set/plant Total yield/plant (gm) C O N T R O L Pt Pt Pt Pt Pt Pt Pt Pt Pt Pt Mean SE(Mean) CV(%) No. = Number, Pt = Plant, Ht= Height, Wt = Weight

4 R1 ISSN: Table II: Study of Morphological Parameters No. Length of Leaf of Runners Petiole Runner length s Plant Plant Diamete Lamina Floweri Fruit Total No. Ht r of diamete ng (no. set/ yield/ Bush r of plant plant (sq cm) flower/ (gm) plant) Pt Pt Pt Pt Pt Pt Pt Pt Pt Pt Pt Pt Pt Pt Mean SE(Mean) CV(%) R1=Row1, No. =Number, Pt = Plant, Ht= Height, Wt = Weight Table III: Study of Morphological Parameters Plant Plan Diamete No. of Length of Runners Leaf Lamina Floweri Fruit Total No. t Ht r of Runner Petiole diamete ng (no. set yield/ Bush s length r of /plan plant (sq cm) flower/ t (gm) plant) Pt Pt Pt Pt R2 Pt Pt Pt Pt Pt Pt Pt Pt Pt Mean SE(Mea n) CV(%) R2=Row2, No. =Number, Pt = Plant, Ht= Height, Wt = Weight 445

5 Plant No. ISSN: Plant Ht Diameter of Bush Table IV: Study of Morphological Parameters No. of Length of Runners Leaf Runners Petiole length Lamina diameter (sq cm) Flowering (no. of flower/ plant) Fruit set/plant Total yield/plant (gm) R3 Pt Pt Pt Pt Pt Pt Pt Pt Pt Pt Pt Pt Pt Mean SE(Mean) V(%) R3=Row3, No. =Number, Pt = Plant, Ht= Height, Wt = Weight B. In vitro culture In vitro culture of Strawberry plant has been performed by using shoot tip, runner tip, leaf base, rolled leaf and nodal segment as explants with MS basal media supplemented with auxin (NAA) and cytokinine (BAP). Nodal segment showed better response compared to other explants (Fig: 4). Poor response in lower concentration of auxin (0.1 mg/l) and cytokinine (0.5 mg/l) was observed. Increasing trend in both the initiation and establishment of explants up to a certain level was noticed with the increase in hormone concentration. Optimum result was obtained (Table V) with the concentration of NAA (5 mg/l) and BAP (5mg/l). Further increase in concentration beyond 5 mg/l of both NAA and BAP decreased the initiation and establishment of explants. (A) (B) (C) (D) Fig 4: (A) In vitro initiation (B) Establishment (early) (C) Establishment (late) and ( D) Multiplication of Strawberry plants using MS media supplemented with suitable growth regulator 446

6 Table V: Establishment of cultures of Strawberry (Fragaria ananassa Duch) using Nodal segments as explants & Basal Medium : MS Auxin Cytokinine Response (%) Explants showing Multiplication (%) NAA(mg/l) BAP(mg/l) initiation of shoot Bud(%) SE( Mean) : CV(%) : IV. DISCUSSION The cultivation of Strawberry (Fragaria ananassa Duch) in parts of West Bengal, India has been relatively a recent addition to the already existing traditional routine crop cultivation practices. Although it is a crop of winter climate [17], the prevailing heterogeneous agro ecological condition is not congenial for Strawberry plant growth and yield. In West Bengal this crop is grown during the short duration winter season. Some records of commercial cultivation of Strawberry in Bankura and Nadia district, West Bengal, India are available [17]. In the present study, satisfactory plant growth was not observed with the normal manure application practices. Vermicompost is known to increase growth of various plants [4],[14],[1]. All the findings on the present investigation revealed that vermicompost played an effective role on the morphological growth of Strawberry plant and increase in fruit yield (Fig: 3). Similar result in respect of the growth has been recorded in Strawberry plant [ 3],[4],[14],[1]. However, it was observed that increase in diameter of bushes significantly decreased the fruit yield. On the contrary total yield increased with the decreased diameter of lamina and length of petiole. This observation reflected that extensive growth of the plant adversely affected the yield. In vitro studies, nodal segment showed better response as compared to other explants. This study is consistent with the earlier findings of some researchers [20]. A good number of literatures are available on the micro propagation of Strawberry from different parts of the world [13],[9],[20],[16],[6],[2]. In this investigation MS [19] as basal media supplemented with high concentration of NAA (5mg/l) and BAP (5mg/l) played an important role for its proliferation and multiplication (Fig: 4). Optimum initiation and establishment (proliferation of shoot bud & plantlet) has been shown with NAA (5 mg/l) and BAP (5 mg/l) concentration (Table V). It is found that increase in concentration beyond 5 mg/l of both said hormone decreases explants initiation and establishment. Similar result was obtained in in vitro investigation of Pineapple [10] where BAP (10mg/l) and NAA (10mg/l) played an important role in highest number of shoot bud proliferation. This investigation will therefore be of immense importance for understanding the basic and applied research as well as educational purpose of this plant. ACKNOWLEDGMENT Authors are very much thankful to Secretary, Oriental Institute of Science and Technology for providing research facilities and constant inspiration to conduct this work. Thanks have also been given to Mr. Achintya 447

7 Mondal, Secretary, and OAER for giving the field facilities for cultivation of Strawberry plant unless this work has not been completed. REFERENCES [1] M.A. Abduli, L. Amiri, E. Madadian, S. Gitipour and S. Sedighian, Efficiency of Vermicompost on Quantitative and Qualitative Growth of Tomato plants, Int. J. Environ. Res., vol. 7, issue 2, pp , [2] N. Adak, Studies on determining the appropriate hormone concentrations on Meristem culture of some strawberry (Fragaria spp.) cultivars, Journal of food, Agriculture Environment. vol. 9, issue l2, pp , [3] N.Q. Arancon, C.A. Edwards, P. Bierman, C. Welch and J.D. Metzger, Influences of vermicomposts on field strawberries, Bioresource Technology,vol. 93, pp ,2004. [4] N.Q. Arancon and C. A. Edwards, Effect of vermicompost on plant growth, ISWVT, Los Banos, Philippines, pp. 1-25, November, [5] P.C. Bickford, T. Gould, L. Briederick, K. Chadman, A. Pollock, D, Young, B. Shukitt-Hale and J. Joseph, Antioxidant-rich diets improve cerebella physiology and motor learning in aged rats, Brain Research 866, pp , [6] M.K.Biswas, U.K.Roy, R. Islam and M. Hossain, Callus culture from leaf blade, nodal, and runner segments of three strawberry (Fragaria sp.) clones, Turk J. Biol.,vol. 34, pp.75-80, [7] P.Boxus, The production of strawberry plants by in vitro micro propagation, Journal of Horticulture Sciences, vol.49, pp , [8] M.J. Chung, S.H. Lee and N.J. Sung, Inhibitory effects of whole strawberries, garlic juice or kale juice on endogenous formations of N-nitrosodimethylenamine in humans, Cancer Letter, vol.182, pp.1-10, [9] S.C. Debnath and J.A.T. da Silva, Strawberry culture in vitro: Application in Genetic Transformation and biotechnology, Fruit Vegetable and Cereal Science and Biotechnology, vol.1, issue 1, pp.1-12, [10] G. Gangopadhyay, S.K. Roy, S.B. Gangopadhyay and K.K. Mukherjee, Agro bacterium mediated genetic transformation of Pineapple Var. Queen using a novel encapsulation-based antibiotic selection technique, Plant cell Tiss Organ cult, vol.97, pp , [11] J.F. Hancock, J.L. Mass, C.H. Shanks, P.J. Breen and J.J. Luby, Strawberries (Fragaria), Acta Horticulturae, vol.290, pp , [12] M.I. Heinonen, A.S.Meyer, E.N. Frankel, Antioxidant activity of berry phenolics on human low- density lipoprotein and liposome oxidation, Journal of Agricultural and Food Chemistry, vol.46, pp , [13] P. Joshi, R. Kaur and D.Singh, Improvement of Strawberry using tissue culture techniques-a review, Agric.Rev. vol. 24, issue 4, pp , [14] R. Joshi and A. Pal Vig, Effect of vermicompost on Growth, Yield and Quality of Tomato (Lycopersicum esculentum L ), African J. of Basic & Apl.Sci., Vol. 2, issue (3-4), pp , [15] C. Lazcano and J. Dominguez, The use of vermicompost in Sustainable Agriculture: Impact on Plant Growth and Soil Fertility, In: Soil nutrients, chapter- 10, pp [16] W. Litwinczuk, E. Okolotkiewicz and I. Matyaszek, Development of in vitro shoot cultures of strawberry (Fragaria x ananassa Duch.) Senga Sengana and Elsanta under the influence of high doses of gibberellic acid, Folia Horticulturae, Ann.21/2, pp.43-52, [17] H. Mondal, Strawberry fields are forever for Nadia farmers, Hindustan Times (Kolkata), 21 Jun, [18] A.A.Mozafari and M.Gerdakneh, Influence of media and growth regulators on regeneration and morphological characteristics of strawberry cvs Kurdistan and Merck (Fragaria X ananassa Duch.), Int.J. Plant physiol.biochem. vol.4, issue 5, pp , April [19] T.Murashige and F. Skoog, A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol. Plant., vol. 52, pp , [20] S.Sakila, M.B.Ahmed, U.K. Roy, M.K. Biswas,R. Karim, M.A.Razvy,M. Hossain, R.Islam and A.Hoque, Micro propagation of Strawberry( Fragaria x ananassa Duch)- A newly introduced crop in Bangladesh, Am- Euras. J. Sci. Res.,vol 2, issue 2,pp , [21] H. Wang, G. Caw and R.L. Prior, Total antioxidant capacity of fruits. Journal of Agricultural and Food Chemistry, vol.44, pp ,

8 AUTHOR BIOGRAPHY Biography: Manikuntala Bhandari Graduated in 2007 from Burdwan University in Biotechnology (H). Obtained M.Sc. in Biotechnology from Vidyasagar University in Presently, doing PhD. in the field of Plant Biotechnology (Plant tissue culture) under Vidyasagar University. Biography of Corresponding author: (Dr. Subhash Kanti Roy) Born in fifth day of December in Graduated in 1993 from Calcutta University with Botany (H), Masters in 1995 from Calcutta University in Botany. Obtained PhD in 2003 from Kalyani University in the field of Photochemistry and Plant Tissue Culture in Botany. Pursued Post Doctoral Research at Bose Institute, Kolkata in the field of Plant Molecular Biology and Biotechnology during 2004 to Published 08 research paper in International and 11 research paper in National Journal of repute, and have Genbank Accession of 33 EST sequence. Participated several National and International symposia. Having teaching experience of more than 15 years in Graduate and Post Graduate level. Major research areas are Plant Molecular Biology and Biotechnology, Identification and Isolation of medicinally active compounds from plant and microbes. Having 04 PhD students of Department of Biotechnology and Biochemistry, Vidyasagar University under my supervision. 449