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July-Sept 2015 Pdf.Cdr CHAPTER II (A) Micropropagation of Chonemorpha fragrans Introduction and review of literature: Medicinal and neutraceutical herbs have received attention due to their health promising properties. Even today 75 % of world's population depends upon herbal drugs. With increasing population, demand for herbal drugs has tremendously increased. Thus for commercial production of herbal drugs, mass propagation is an essential prerequisite. Tissue culture techniques offer a rapid means of mass propagation of plants to generate uniform plant organs for production of active compounds. They are also useful in conservation of elite germplasm as well as rare and endangered plants. Tissue culture techniques help in creation and dissemination of a large number of plants across the continents without spreading the pathogens. Seed derived clones accelerate screening and selection to obtain the best clones. Micropropagation also helps in creation of genetically engineered plants for suitable traits like disease resistance. Micropropagation has several advantages over conventional or traditional methods of clonal propagation which suffer severe limitations. Tissue culture holds a tremendous potential for production of high quality plants for production of important plant products. Micropropagation can be achieved through i) multiplication of pre-existing meristems ii) callus mediated organogenesis and iii) somatic embryogenesis. i) Multiplication of pre-existing meristems - It is the easiest method in which growth regulators play an important role in stimulating the development of axillary or apical buds. Different auxins and cytokinins are added to the medium either signally or in combinations at different concentrations. ii) Callus mediated organogenesis- In this method callus induction, growth of callus, differentiation and organogenesis is accomplished by different growth regulators in the medium. With the stimulus of endogenous growth 19 substances or addition of growth regulators in the medium, cell division cell growth and tissue differentiation is achieved. iii)Somatic embryogenesis- It is a process where somatic cells lead to fomriation of somatic embryos which resemble zygotic embryos. They can be grown on suitable media to get the seedlings. A large number of somatic embryos are known to be produced in the cultures but efficient development and germination of embryos is an essential prerequisite for commercial production of plantlets. Plants from family Apocynaceae are studied for their in vitro responses. A summary of in vitro reports has been given in the introduction (Table-2 page 4). Scanty reports are available on in vitro studies on Clionemorptia fragrans and Tabemaemontana alternifolia. Thus these two plants were selected for the present work. Chonemorha fragrans (Moon), Alston. Syn. C. grandiflora, M.R. & S. M. Almeida syn. Chonemorpiia macroptiylla G. Don. is a woody climber distributed in tropical and subtropical Asia (Li et a!., 1995). In India, three species of Chonemorpha viz. C. pediciliata, C. assamensis and C. fragrans are known to occur. C. fragrans is distributed in different states in India like Maharashtra (Kulkarni, 1988), Karnataka (Saldanha, 1996), Kerala (Gamble, 1986). C. fragrans has been included in the list of threatened medicinal plants and is assigned endangered status (A1C) in Karnataka and vulnerable (A1C) in Kerala state (Khan et al., 2005). C. assamensis and C. pediciliata are also included in the list of threatened medicinal plants (BSI, 1995, http://envfor.nic.in/bsi/research.htm). C. fragrans is a medicinal plant (Nair and Mohanan, 1998; Sivaraman and Balachandran, 1996), used in several medicinal preparations used in Indian medicinal systems. It is used in preparations like kumariasavam and sudarshanasavum used as tonic. It is used for fever and stomach disorders. Entire plant, roots and root bark are used. The trade is mainly confined to Kerala state under the name Perumkurumba and the dried roots are sold at a price of 2.40 Rs / Kg (http://envis://frlht.org.in.cfragrans.htm, 1993). C. fragrans is latex 20 bearing shrubby climber, occurring in evergreen or semi deciduous forests (Plate 1 A and B). The stem barks is grey and shows lenticels. Roots are adventitious. The leaves are opposite. Flowering occurs from April to September and fruiting during November to February. The fruits are follicle, upto 1 foot In length (Plate 1 D). Seeds are comose with about coma 1 inch in length. Mature seeds are about 1 cm in length, bears 2 seed coats, outer thin and inner white and tough. Materials and methods: A) Collection of plant material Plant material was collected from two localities from India (shown by arrow). Plants were collected from locality 1(L1) Karnataka, once in every season (3 times/ year) for 2 successive years, i.e.2005, 2006 and once in 2007. The parent plant selected from Karnataka locality was a plus shrub with a height 30 feet, diameter of stem (D. B. H) - 8 inches. Age of the plant was approximately 20 years (Plate 1A). In April and September, collection was carried for young and mature stem, leaves as well as flowers. In January, fruits were collected. The plant material from locality 2(L2) i.e. Kerala was obtained as a gift from a research colleague .It was obtained from a plant nursery from Thrissur district, Kerala state. South India. Fruits of the plants from this locality could not be obtained. Specimens from both the localities were dried and the hertaarium sheets were submitted to Botanical Survey of India (Western circle), Pune, for identification. Both the plants were identified as C. fragrans (syn C. macrophylla, syn C. grandiflora). 21 Plant material was collected from two localities from India (shown by arrow). Forest Map of Karnataka KERALA Forest Map MAHARAaHTIU AMOHRA rMOi»H # Slaw e««*ui ® SUtiupiUl ^B DtftH Pertti H DtntiFofwt I I OptH Fertii I I Optn FBfitI TAWLNAOU C^pfngM • 3004 Corner* iMoeM* Pvi . LI (Karnataka) L2 (Kerala) Table 5- Geographic features of localities L1 and L2 Locality 1 (LI) Locality 2(L 2) State Karnataka District- N. Kannada State Kerala, District- Thrissur Village- Joida Plant nursery 14°52'to 15° 12'N latitude 10 "-10-46' North latitudes and 75 " and 74°16' to 74°44'E longitude 55' East, Altitude is 970 m Altitude 500 m temperature ranges -16° to 36°C temperature ranges - 26° to 36°C average annual rainfall is 2500 mm. average annual rainfall is 3500 mm Soil type- red laterite. pH -6.8 , Soil type- red laterite. pH -6.2 , *Total N- 426.2 mg/kg, P-282.8 mg/kg *Totai N-466.81 mg/kg, P-271 mg/kg K-171.1 mg/kg, S04-66.84 mg/kg K-251.1 mg/kg, S04-13.61 mg/kg, CI-171.06 mg/kg, Fe-1197.5 mg/kg CI**- 86235 mg/kg, Fe**-27780 mg/kg Mn-less than 0.547 Mn- less than 0.613 mg/kg C/N ratio-42.6:1 C/N ratio-37.4:1 Tropical evergreen+ deciduous forest Tropical evergreen forest 'Soil analysis as per APHA Ed -21'' ed. ** very high content 22 b) Establishment of plants i) Branches of 1 cm thickness were cut and used for establishment of plants. Every year plant material was collected from one and the same parent plant. Cuttings were raised (for the plants from both the localities) in pots without application of keradix. 100% survival was observed in cuttings. The cuttings were maintained in the Botanical garden, Department of Botany, University of Pune. Nodal sectors and leaves from these plants were used for in vitro studies. ii) Fruits were collected from Karnataka locality (L1) in January every year. The fruits were mature brown, 9 inches to one foot in length (Plate 1D). The fruits contained about 30 -40 seeds in each follicle. The mature seeds were about 5- 10 mm in length, with two seed coats. The seed coats were removed and embryos were grown on sterile filter paper in petridishes. 15 days old seedlings were transferred to soil in glass bottles. 70 % of the embryos developed into plantlets. The plantlets were maintained for about 8 months. The seedlings were very weak and showed very slow growth. ill) Seeds were also germinated in vitro on MS Basal medium (Murashige and Skoog, 1962) and nodal sectors from in vivo and in vitro seedlings were used for further in vitro studies. C^ Micropropagatlon of C. fragrans -In vitro shoot growth i) Explants used-1) nodal sectors from established parent plants (both localities LI and L2). 2) nodal sectors from in v/Vo germinated seedlings (L1) and 3) nodal sectors from in vitro seedlings (LI). Ii) Preparation of explants 1) nodal sectors from established parent plants -Nodal sectors 1 cm in length were cut from the parent plants, washed under running tap water for 20 min. and then with 10% teepol for 10 min. These were then treated with 70 % alcohol for 2 min., washed thoroughly with distilled water (D.W.) and then treated with 0.1 % HgCbfor 5 min. The explants were thoroughly washed with sterile D.W. 3 to 4 times and blotted well. A small part nodal sector at both the ends was cut before inoculation. 23 PLATE -1 Chonemorpha fragrans A-STEM (0.15X) B-HABIT C - FLOWERS D - FRUITS (0.2X) E - SEEDLINGS (0.33X) F - EMBRYOS (1X) 2) Nodal sectors from in vivo germinated seedlings Seeds were soaked in water for two hours. The seed coats were removed and the embryos were kept for germination in petridishes on sterile filter papers. Nodal sectors from 15 days old seedlings were cut. The nodal sectors were washed with water, treated with 70 % alcohol for 2 minutes and with 0.1 % HgClg for 2 minutes. The nodal sectors were then washed with D.W. and blotted thoroughly before inoculation. 3) Nodal sectors from in vitro germinated seedlings Seeds were soaked in water for 2 hours, washed with detergent for 10 min.
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