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. and then washed with water. The seeds were then treated with sodium hypochlorite/chlorine solution (0.1 %) for 15 min and then were washed with D.W. three to four times. They were then treated with 70 % alcohol for 5 mins. The seeds were thoroughly washed with sterile D.W. and blotted. Seed coats were removed and the embryos were inoculated on MS basal medium (Murashige and Skoog, 1962). Nodal sectors from germinated seedlings were used as explants for shoot growth. iii) Media used for growth of nodal sectors 1) For growth of nodal sectors from established plant material in all, 44 combinations of growth regulators in MS medium (Murashige and Skoog, 1962) and 16 combinations of growth regulators In WPM i.e. woody plants medium (Lloyed and MaCown, 1981) respectively were used. Shoot induction and growth was mainly studied. MS basal medium and WPM were supplemented with different growth regulators like BAP, Kinetin, TDZ, lAA, NAA either singly or in combinations at different concentrations. Combinations of growth regulators used in the media have been given in appendix 1. 2) For growth of nodal segments from in vitro and in vivo seedlings, about 25 combinations of growth regulators in MS medium were used. MS Medium was supplemented with different growth regulators like BAP, Kinetin, TDZ, lAA and NAA. The combinations tried have been given in appendix 2. Combinations which gave the best results were used for further studies. MS medium and WPM were prepared with 3 % sucrose and agar 0.8 %.
24 pH for the medium was set at 5.8 with 1 N NaOH. Sterilization of medium was carried out at 15 lb/ inch ^ pressure for 20 minutes at 121° C. Incubation conditions used were as follows- Light- white fluorescent light, 40 W, day light tubes with 25 piE /m^ /s intensity. Photoperiod - 12 hours, Temperature- 24+ 2° C. For every treatment 20 replicates were used. iv)Subculturing Subculturing was carried out after every 4 weeks on fresh medium, responses were observed and recorded upto 8 subcultures. v) Observations and analysis of data Observations were recorded after every week for percentage of bud induction/ break, days taken for bud induction and shoot length. To confirm the results, a set of experiments was repeated for the media combinations which gave the best results. 20 replicates were used for these sets. Two way ANOVA was carried out for determining locality dependent and subculture dependent variations. Paired t test was carried out for comparison of responses on different combinations of growth regulators. D) iMicropropagation of C. fragrans - Root Induction I) Medium used- Root induction was carried out for one month old in vitro shoots, raised on MS medium supplemented with 8.8 |iM BAP. MS medium was supplemented with different auxins like lAA, IBA and NAA singly at different concentrations. 10 combinations of MS medium were tried for root induction. MS medium was supplemented with 2.46 pM, 4.96 pM, 9.8 pM and 13.7 pM IBA respectively, 2.8 pM, 5.7 pM 11.4 pM lAA and 2.68 pM, 5.37 pM, 11.7 pM, NAA respectively. MSB was used as control. il) Observations and analysis of data Observations were recorded after every week, for days taken for root induction, number of roots produced, percentage of root induction and root length. Entire set of experiments was repeated twice to confirm the results. Observations of 20 cultures were considered for analysis. Two way ANOVA was carried out for
25 determining locality dependent and subculture dependent variations. Paired t test was carried out for comparison of responses on different combinations of media. E) Hardening of in vitro plantlets— In vitro plantlets (from both localities) were successfully grown and maintained in vermiculite and soil (1:1) as well on red soil and sand mixture (1:1). In vitro plantlets were thoroughly washed under tap water to remove traces of medium. Then the plantlets were the transferred to vermiculite and soil (1:1) as well on red soil and sand mixture (1:1) in small bottles to begin with. (Plate 2 G). The plantlets were maintained in the bottles for about 1 year. Then the plantlets were transferred to the pots containing sand and red soil mixture (1:1).and maintained.
F) Induction and estabiishment of calius in Clionemorplia fragrans. Callus was raised and established on different combinations of media like MS medium and WPM using various explants. i) Expiants used- leaves, internodes, in vitro roots (LI and L2) and cotyledonary leaves from in vitro raised seedlings from L1. ii) Preparation of explants- For leaf explants, pieces of 1 cm^ were cut. They were surface sterilized with the same procedure as was used for nodal explants. In vitro roots and cotyledons and internodes were cut aseptically in pieces of 1 cm length and were inoculated on selective media. iii) Combinations of growth regulators used for callus induction - MS medium and WPM were supplemented with different auxins like 2, 4- D., lAA, and NAA signally at different concentrations. Suitable combinations using auxins like lAA, NAA and cytokinin like BAP were prepared. In all, 30 combinations of media were prepared which are given in appendix 3. Iv) Observations and analysis of data- Observations were recorded for percentage callus induction for different explants on different combinations of media after a week. 20 replicates were kept for each treatment and the entire set of experiment was repeated twice to verify and confirm the results. Fresh weight and dry weight of callus was recorded after 4 weeks for selective media which showed better callus growth. Two way ANOVA was carried out for determining
26 locality dependent and subculture dependent variations. Paired t test was carried out for comparison of responses on different combinations of media. Growth index for callus was calculated by using the formula given below: G. I.= Final fresh weight in (a V- initial fresh weioht in (a^ Initial fresh weight in (g) Results a) Micropropagation In C. fragrans. The plant was studied mainly for its in vitro responses to different growth regulators or their combinations in the medium so as to find out best suitable combinations of media for bud induction, shoot growth and root induction. The parameters studied were bud induction percentage, days taken for bud induction on different combinations of media and effect of seasons on bud induction. Bud induction response in C. fragrans was obsen/ed and the results obtained were as given in table no 6. Table 6 - Bud induction* response in C. fragrans
No. Medium combination Locality Bud break Days required (%) for bud break 1. MS Basal (control) L1** 100 8 1 p*** 100 4 2. MS+4.4 |iM BAP L1 100 4 L2 100 4 3. MS+8.8UM BAP L1 100 4 L2 100 4 4. MS+13.2^MBAP L1 100 4 L2 100 4 5. MS+ 4.4 uM BAP +5.7uM lAA LI 60 15 L2 80 10 6. MS+ 8.8 \M BAP +5.7U M lAA L1 50 10 L2 70 7 *Allt he results are mean of 20 replicates ,**L1-Kam ataka ***L2 - Kerala As seen from table no. 6, bud induction was found to be 100 % in presence of BAP. Auxins added along with BAP led to callus formation at the base of explants thus reducing % of bud induction and delaying bud break. Bud induction was found to be 100 % in presence of BAP for seedling nodal sectors
27 also. Kinetin and TDZ added to MS medium showed lower % of bud break in mature nodal sectors and in seedling nodal sectors also .Such combinations of media have not been included in the table. Effect of seasons on bud induction was also recorded. The results were as given in table no 7. A significant reduction in % bud induction was observed in winter season in plants from L1 (Karnataka) as compared to 12 (Kerala). This may be because L2 is near equator and closer to the seashore as compared to Ll.Thus distinction between the seasons is not very sharp in L2 and climatic conditions are more or less uniform throughout the year. A slight delay in bud break and slow shoot growth in winter was observed for L1. The observations recorded were as given below in table no 7. Table 7- Effect of seasons on percentage bud induction* response in C. fragrans No Medium locality %bud %bud %bud Combination break break break Summer rainy winter 1. MS Basal L1 100 80 60 L2 100 80 70 2. MS+4.4U M BAP L1 100 80 70 L2 100 90 80 3. MS+8.8 uM BAP L1 100 80 70 L2 100 90 90 4 MS+13.2UMBAP L1 100 80 70 L2 100 80 80 5 MS+ 4.4 uM BAP + 5.7u M lAA L1 60 60 40 L2 80 70 50 6 MS+8.8 uM BAP+ 5.7 uM lAA L1 50 50 40 L2 70 50 40 *AII the results are mean of 20 replicates b) In vitro shoot growth in C. fragrans i) Shoots raised from nodal sectors from established parents - BAP, Kinetin and TDZ were used in MS medium for encouraging shoot growth. The most suitable growth regulator for shoot elongation was found to be BAP. MS medium supplemented with 8.8 tiM BAP was found to be the best for shoot growth (Graph 1).Thus subculturing was carried out on this medium combination
28 after every 4 weeks. The results obtained on various combinations of growth regulators have been presented in graph 1. Graphi- Effect of cytokinins on in vitro shoot growth* in C. fragrans (L2)
Effect of cytokinins on shoot growth(L2) From graph 1, it was clear that addition of BAP to MS medium
E7 has a positive influence on shoot O growth. Addition of TDZ to MS
?4 medium also stimulated shoot
o2 growth though the effect was not as pronounced as BAP. Kinetin
0,2.2,4,4,8.8,13.2 0,2.32,4.64,9.29, 0,2.25,4.5,9 13.5 was observed to be the least MMBAP 13.9IJMKIN (iMTDZ effective amongst 3 cytokinins. Concentration of cytoklnin in MS medium
*Mean shioot lengtti of 20 replicates +S. E
MS+8.8 |iM BAP was the most beneficial combination for shoot growth for locality L2. At higher concentration of BAP i.e. 13.2 [iM shoot growth was retarded. Nodal explants from locality L1 also showed similar results (Graph 2). Graph 2- Effect of cytokinins on in vitro shoot growth * in L1.
Effect of cytokinins in in vitro shoot growth in C. The graph 2 showed that fragransLI MS+BAP was the most E 6 .= 5^ beneficial combination for £ 4 shoot growth for nodal = 3 - 2 explants from locality L1 also. MS+8.8 liM BAP was the
iM0,2.2,4.4,8.8,13.2 0,2.32,4.64,9.29 , 0,2.25,4.5,9,13.5 best combination for increase UMBAP 13.9 MMKN HMTDZ in shoot length. Concentration of cytokinins in MS medium
* Mean shoot lengtfi of 20 replicates+ S.E.
29 TDZ added to the medium encouraged increase in slioot length but was less effective as compared to BAP. Kinetin was found be the least effective cytokinin. MS medium and WPM were used to select a better medium for shoot growth in C. fragrans. In both these media, BAP was added at specific concentrations. The results of comparison of increase in the shoot length on MS and WPM containing the same concentrations of BAP have been presented in graph 3 and 4. Graph 3- Comparison of shoot length* in MS and WP medium with different concentrations of BAP(L2).
Effect of medium on shoot growth From graph 3, it was clear that, addition of BAP to both the media showed a • Basal " 6 • 2.2MMBAP positive influence on shoot D 4,4 (JMBAP growth upto the DS.SpMBAP
• 13,2MMBAP concentration 8.8 |iM BAP. Shoot growth on WPM was
MS+BAP WP+BAP 60 % of shoot growth Medium combinations obtained on MS medium. * Mean shoot length of 20 replicatesj^S.E Graph 4- Comparison of shoot length* in MS and WP medium with different concentrations of BAP(L1). Graph 4, showed that, Effect of in«diuiii on shoot growtli (LI) addition of BAP to both the • basal media showed a positive • 2 2uMBAP influence on shoot growth in • -1 /I |JM BAP L • 8 8 MM BAP L1 explants upto the 1 ^^^^^KV^^ —zrjfc • 12 3|JMBAP concentration 8.8 |iM BAP
MS+BAP WPM+BAP WPM did not encourage Medium combinations shoot growth in nodal
* Mean shoot length of 20 replicates4^S.E sectors of L1 and L2.
30 WPM did not encourage shoot growth probably due to less concentration of major nutrients as compared to MS medium, which could be an important limiting factor. In another set of experiments, MS medium and WPM were supplemented with different concentrations of Kinetin. The effect of kinetin on shoot growth was observed. The results have been shown in graph 5. Graphs- Comparison of shoot length in MS and WP medium with different concentrations of kinetin (L1)
Effect of medium on shoot growth Graph 5, it was clear that the
8n shoot length obtained on MS as • Basal well as WPM medium " 6 D 2.32 ^M KIN c supplemented with Kinetin was • 4.64 jiM KIN 014 D 9.2nM KIN lesser as compared to shoot «; 3 -mTl I2 — • 13.8nMKIN length obtained on MS medium fa supplemented BAP. WPM+ 0 fl 1 MS+KIN WFMfKIN combination kinetin was not Medium combinations found suitable for shoot growth *Mean shoot length of 20 replicates ± S. E as compared to MS+ Kinetin combination.
Similar results were obtained for explants from locality L2 hence are not separately shown. To enhance shoot growth, combinations of different cytokinins were tried In MS medium. BAP was added along with Kinetin at different concentrations. In graph 6, effects of combinations of cytokinins on shoot length have been shown. From graph 6, it was seen that the shoot length obtained on MS medium supplemented with 4.4 [iM BAP and 4.64 ^M kinetin was lesser as compared to shoot length obtained on MS medium supplemented with 8.8 \iM BAP (Graph 1 and 2). Thus combination of BAP and kinetin did not seem to give an additive effect and encourage shoot growth. BAP signally added to MS medium had a better effect in terms of shoot growth. 31 Graph 6 -Effect of combination of cytokinins in [\/IS medium on in vitro shoot length*(locality L1 and L2)
Effect of cytokinin combination • 2.22 ^M BAP +2.32 |aM Kinetin on shoot growth • 2.22 |iM BAP + 4.64 \M Kinetin n 4.44|a M BAP+ 2.32 |iM Kinetin D 4.44^ M BAP+ 4.64 [M Kinetin • 4.44|j M BAP+9.28 \M Kinetin • 8.88 nM BAP +2.32^ M Kinetin • 8.88 JJM BAP +4.64 pM L1 L2 Kinetin Shoot growth in L1 and L2 D 8.88 nM BAP + 9.28 ^M Kinetin
* Mean shoot iengtli of 20 replicates+ S.E. Statistical analysis- Statistical analysis was carried out to decide whether the given treatment was beneficial for shoot growth in vitro. ANOVA was carried out for important combinations of medium which showed promising results. The results have been represented below in table no 8. Table-8- ANOVA analysis for effect of cytokinins on growth of nodal sectors of C. fragrans (L2). Medium Mean shoot Medium Mean shoot Medium Mean combination length in cm combination length in cm combination shoot +S.E. +S.E length in cm +S. E. MSB(L2) 1.95+0.06 MSB(L2) 1.95 + 0.06 MSB(L2) 1.95+0.06 MS+ 2.22 2.45±0.03 MS+ 2.32 2.3 ± 0.06 MS+ 2.25 2.5 ±0.03 UMBAP [xM kinetin ^MTDZ
MS+ 4.44 2.9 ± 0.03 MS+4.64 \iM 2.6 J: 0.04 MS+ 4.5 2.9 ±0.03 uMBAP kinetin [iMJDZ MS+ 8.88 7.0 ± 0.06 MS+ 9.28 2.9 + 0.06 MS+9 |JM 4.0± 0.03 ^M BAP UM kinetin UMTDZ MS+ 13.3 3.9 ±0.04 MS+13.9nM 3.4 + 0.02 MS+13.5 2.5 j^0.03 uMBAP kinetin UMTDZ F value for F=11.3* F=7.5* F=10.6* treatments *F values significant at P=0.01
32 The ANOVA analysis results showed that addition of BAP, kinetin and TDZ to MS medium had a positive influence on shoot growth. There was insignificant variation between the replicates. Similar results of ANOVA analysis were obtained for L1 also and therefore are not presented in a separate table. Seed germination in C. fragrans Seed germination in vivo- Percentage of seed germination in vivo was observed for seeds from L1. It was 100 % for mature embryos (0.5 cm to 1 cm length), 90 % for highly mature embryos (length 1.2 cm or more) and 80 % for very young embryos (length upto 0.5 mm). Young embryos showed slower growth as compared to mature embryos. Highly mature embryos showed slight delay in germination, showed slow growth initially, but after about 2 weeks they showed equivalent growth rate to those of mature embryos. Seeds with the seed coats failed to show germination in vivo. Thus the hard seed coats may also be responsible for this. The seedlings, 15 days old were transferred to glass bottles with soil. 70 % of seedlings survived and developed into plantlets (Plate 1E). The seeds from locality L2 could not be obtained, therefore germination studies for seeds from L2 were not carried out. Seed germination in vitro - For seed germination in vitro, different combinations of growth regulators were used. In all, 25 combinations of growth regulators were used (Appendix 2). 100 % germination of mature embryos was observed on MS Basal medium (Plate 2A). Very young embryos (length 5 mm) and highly mature embryos (length more than 1 cm) showed 90 % germination on MS basal. Thus age of the seed also seemed to affect germination process in C. fragrans. The results of seedling growth on different combinations of growth regulators in MS medium have been shown in graph 7. MS medium supplemented with 2.2 pM BAP and 5.37 \iM NAA proved to be the best combination for seedling growth. Addition of lAA along with BAP to MS medium did not give promising results for seedling growth and therefore these combinations have not been shown in the graph. Seeds with the seed coats failed to show germination in vitro. Germination of seeds and seedling in vitro have been shown in plate 2 (A and B).
33 Graph 7- Seedling growth in vitro *m C. fragrans (LI) MS basal, MS+ 4.4 \M In vitro growth of seedlings
• MS Basal BAP MS+ 8.8 uM BAP and the combination MS+ • MS+2.2 \M BAP 2.2 ^^/l BAP+ 5.37 jilVI D MS+4.4 nM BAP NAA encouraged seedling DMS+8,8nMBAP growth. NAA added to the • MS+2.2 ^M medium seemed to have BAP+5.37 ^M NAA Combinations of IVIS medium enhancing effect on
* Mean length of seedling of 20 replicates + S.E. seedling growth.
Growth of seedling nodal sectors in vitro- Nodal sectors from seedlings in vitro were further grown on various combinations of media containing growth regulators like BAP and NAA. . The response of seedling nodal sectors was compared with the response of nodal sectors raised from parents. The results have been presented in graph no 8. Graph 8- Growth response* of seedling nodal sectors and nodal sectors MSB did not prove to be Response of nodal sectors and seedling nodal sectors good for growth of both types of nodal sectors • MS Basal (L1). Nodal sectors from • MS'-2 2 JMEAP seedlings grown on MS+ OMSM 4 JM EAR 4.4 [iM BAP and MS+
nMS«-8 8 JM EAP 8.8 laM BAP showed increase in shoot length. nodal sectors seedling nodal • MS*2.2JMEAP+5 37 sectors MM NAA explant
*Mean shoot length of 20 replicates + S.E. Addition of NAA along with BAP encouraged growth of seedling nodal sectors. The most suitable combination seemed to be MS+ 2.2 |iM BAP+ 5.37
34 MM NAA for seedling nodal sectors. Subculturing was carried out on this combination of medium. The effect of BAP on shoot length was more pronounced for nodal sectors from the established parents as compared with the seedling nodal sectors Nodal sectors from the parent plants showed the best response in MS+ 8.8 |iM BAP combination (Graph 8) but reduction in shoot length was observed with addition of NAA along with BAP to the medium. Callus Induction was also observed with addition of NAA, hence affecting length of shoot. Both the types of nodal sectors showed a good response on MS+ 8.8 pM BAP, therefore the same medium was used for subculturing. The subculture response was noted upto 8 subcultures. The same growth rate was maintained upto 8 subcultures. The growth rate of nodal sectors established from parents on MS+ 8.8 pM BAP was more as compared to seedling nodal sectors. In vitro shoots raised from seedling nodal sectors on MS+ 8.8 pM BAP were delicate, with thin slender stem and thin pale leaves, thus probably showed slower growth rate. Statistical analysis Results of ANOVA for shoot growth of seedling nodal sectors on MS medium have been presented in table no 9. Table 9- ANOVA analysis for effect of growth regulators on the growth of seedling nodal sectors(LI) Mean shoot Mean shoot Medium combination length* in cm + Medium combination length in cm + S.E. S.E. MSB 2.0iL 0.05 MSB 2.0 ±_ 0.05
2.2 + 0.05 MS+2.22HM BAP 2.5 + 0.04 MS+ 2.22nM BAP +2.68 liM NAA 3.3 + 0.03 MS+2.22 pM BAP 6.2 +0.06 MS+ 4.44U M BAP +5.37U M NAA 4.5 + 0.05 MS+4.4 \iM BAP 5 +0.03 MS+8.88UM BAP +5.37MM NAA F value for different 11.2" F value for different 16** treatments treatments * All results are mean of 20 replicates. ** F values significant at P=0.01
35 ANOVA analysis showed that addition of BAP and NAA + BAP to MS medium had a favorable effect on growth of seedling nodal sectors. Variation between the replicates was insignificant. Root induction in C. fragrans i) Root induction in one month old in vitro shoots (obtained by using nodal sectors from the established parents from LI) was obtained by using IBA, lAA and NAA in MS medium. Addition of 4.96 [iM IBA to MS medium could induce 100 % rooting in in vitro shoots whereas addition of 5.37 |JM NAA and 5.7 [iM lAA to MS medium could induce only 70 % and 80 % root induction (Graph 9). Similar results were obtained for shoots raised from the plants of L2. (Graph 10) Graph 9 -Effect of auxins on % root induction* in C. fragrans (LI)
Effect of auxin on root induction(LI) As seen from graph 9, IBA 100 was more effective for root 1 80 U induction as compared to 3 60 •D NAA and lAA. On MS ! 40 o 2 20 medium containing 2.68 |JM NAA or 2.8 nM IAAor2.45 2.68,5.37 MM 2.8,5.7 ^JM IAA 2.45.4.£6pM mNAA IBA [iM IBA, root induction % Concentration of auxin in IVtS medium obtained was low.
*Mean of 20 replicates+ S.E. Graph 10 -Effect of auxins on % root induction* in C. fragrans (L2) The best suitable combination Effect of auxins on root induction (L2) 100 was MS+ 4.96 |iM IBA. The o 80 combinations of growth regulators i.e. 2.68 |iM NAA or 2.8 nM lAA or 2.45 [iM IBA, were not used further as % root 2.68,S.37|aM 2.8.5.7nMIAA 2.45.4.96 ^M IBA induction was low on these NAA Concentration of auxin in fiM combinations.
*Mean of 20 repiicates+ S.E.
36 ii) Difference in root induction response was observed in in vitro shoots raised from nodal sector and seedling nodal sectors (L1). The observations were recorded on MS medium supplemented with IBA, lAA and NAA. Root induction was not obsen/ed on MS medium containing 2.68 |iM NAA or 2.8 \i M lAA or 2.45 |iM IBA. IBA was more effective for root induction as compared to NAA and lAA. Like in vitro shoots, shoots raised from nodal sectors also showed 100% root induction on MS medium supplemented with 4.96 JJM IBA. Thus root inducing ability was observed to be lower in in vitro shoots raised from seedling nodal sectors. The results obtained were as presented in graph no. 11. Graph 11- Root induction* response of seedling raised shoots
Root induction in seedling raised shoots and In vitro shoots raised from nodal sector raised shoots g 100 seedling nodal sectors I seedling 80 nodal showed lower percentage I 60 sectors 40 modal (80 %) of root induction on 20 sectors 1 MS+ 4.96 |iM IBA as 0 I I I 5.37 [JM 5.7MM IAA 4.96 ^M compared to plantlets NAA IBA Auxin concentration in MS medium raised from nodal sectors (100%). Mean of 20 replicates + S.E. Number of roots produced per shoot, days taken for root induction and length of roots were also recorded for in vitro shoots (L1) on MS medium containing different concentrations of auxins. The results have been given in table no 10. TablelO—Effect of auxins on root growth* in C. fragrans (Locality LI, after 4 weeks) Medium Medium Medium Medium Medium combination combination combination combination combination MSB MS+ 4.96 MS+9.8 MM MS+5.7 (iM MS+5.37 MM UMIBA IBA IAA NAA Days taken — 7 7 10 10 (root induction) No. of roots. 0 6j^0.01** 3+0.01 4+0.04" 4+0.01" Root length in — 3+0.1 0.5+0.1 4+0.05 4.5 +0.07 cm 'Results are mean of 20 replicates+ S.E., "statistically significant at P=0.01 37 From the table it was clear that the most effective treatment was 4.96 [iM IBA which resulted in maximum number of roots in a short duration. Thus for next set of experiments MS medium supplemented with 4.96 jiM IBA was used. ANOVA analysis carried out for testing the effect of auxins on number of roots produced in vitro showed that that all 3 the auxins used IBA, lAA and NAA had a favorable effect on number of roots produced/shoot at P=0.01 level. The effect of auxins on root growth was studied for in vitro shoots for locality L2 also. Similar results were obtained. Hence the results have not been included in the table. Hardening of plantlets-The % of survival of plantlets in sand and red soil mixture (1:1) was 80 % for plantlets raised from plants from both localities (L1 and 12) and 70 % in vermiculite and soil mixture (1:1). Seedling raised plantlets (LI) showed 70 % survival on both the combinations of mixtures. Hardening of plants has been shown in the photograph (Plate 2G). The morphology of hardened plantlets was similar to those of parents and no changes were observed. e)Callus induction and establishment in C. fragrans Callus induction response of different explants on different combinations of growth regulators was studied mainly to select the explant showing maximum ability of callus induction. The results of callus induction were as shown in table no. 11. It was clear from the table, that leaf explants showed the highest capacity (100 %) of callus induction on most of the combinations of growth regulators and the cotyledons showed minimum (80 %). Addition of auxins at a higher concentration to MS medium also gave 100 % callus induction in different explants like internodes and in vitro roots. Leaf explants showed the highest % of callus induction on most of the media combinations, therefore used for further experiments on callus establishment and biomass production. The results obtained were as given table no.11.
38 Table no.11- Callus induction* response in C. fragrans Combination of Locality % Callus % callus % callus % callus growth hormone used induction induction induction induction (Leaf) Internode In vitro cotyledon roots
MS+ 2.26 uM 2,4 -D L1** 80 70 50 30 1 p*** 80 80 60 ~ MS+4.52 uM 2, 4- D L1 100 90 90 80 12 100 90 90 ~ l\/IS+ 9 ulVI 2, 4 -D LI 100 100 100 80 l\/IS+ 9 uM 2, 4 -D L2 100 100 100 — IVIS+2.8 uM lAA L1 70 50 60 30 L2 80 60 60 — MS+5.7 \iM lAA L1 100 90 90 50 L2 100 90 80 — MS+11.4uMIAA L1 100 100 80 80 L2 100 100 100 — MS+2.68 uM NAA L1 70 50 70 30 L2 70 60 70 — IVIS+5.37 \iM NAA L1 100 90 90 60 L2 100 80 80 — MS+10.7|iMNAA L1 100 100 100 70 L2 100 100 100 ~ IVIS+2.22 |iM BAP L1 100 90 90 40 +5.37 uM NAA L2 100 90 90 l\/IS+ 2.22 ^M BAP L1 90 70 70 60 + 5.7 \xM lAA L2 100 70 70 WPM+ 2.26 ^M 40 2,4-D L1 80 60 60 — L2 80 70 70 WPM+4.52 uM 2, 4- D L1 100 70 70 60
L2 100 80 70 Results are mean of 20 replicates. —Cotyledons not used for study *L1-Karnataka locality, ***L2-Kerala locality
39 As the leaf explants showed the best response for callus Induction, growth (increase in fresh weight) of leaf callus was studied at the end of 4 weeks to decide the best combination of medium for callus biomass production. The combinations of growth regulators which showed promising results were as given in table no 12. Table 12-Effect of growth regulators on callus biomass production* in C. fragrans (after 4 weeks)
Medium combination Medium combination F value MSB MS+4.52UM 2,4-D increase in fresh 0.12+0.03 1.72+0.03 8.1 wt. in g LI increase in fresh 0.13+0.04 1.85+0.03 8.5 wt. in g L2
MS+5.7 uM lAA MS+11.4UMIAA Increase in fresh 0.87+0.04 1.4+0.04 7.1 wt. in g L1 increase in fresh 0.90+0.03 1.52+0.05 7.3 wt. in g L2 MS+5.37 uM NAA MS+10.7UMNAA increase in fresh 0.85±0.05 1.55+0.05 6.6 wt. in g L1 increase in fresh 0.88+0.04 1.64+ 0.03 6.7 wt. in g l_2 WPM basal WPM+ 4.52 |iM 2, 4-D
increase in fresh 0.10±0.04 1.30 ±0.04 6.01 wt. in g L1 increase in fresh 0.12+0.05 1.34+0.06 6.1 wt. in g L2 MS + 2.22 nM BAP + MS+ 2.22 nM BAP+ Paired t 5.37 uM NAA 5.7uM lAA test increase in fresh 1.52+0.05 **t=8.2.7 wt. in g L1 1.34+0.04 resp. increase in fresh 1.48±0.03 **t=8, wt. in g L2 1.46+0.05 7.5 resp. *mean of 20 replic ates + S. E ** statistica ly significant at P=0.01
Maximum increase in callus biomass was obtained on MS medium supplemented with 4.52 \iM 2,4,D. NAA and lAA were not as effective as 2, 4- D
40 for biomass production and were effective to some extent at higher concentrations only. Combination of cytol 41 Shekhawat (2005).The ratio of BAP to NAA used was higher i.e. ranging from 2 to 10. Hirata et al. (1987) used seedling nodal sectors of Catharanthus roseus as explants and cultured on MS medium containing 0.1 mg BAP/I and obtained multiple shoot induction. Multiple shoot induction in Vinca minor was obtained on MS medium supplemented with 13.2 |iM BAP by Tanaka et al (1995). In Holarrhena antidysenterica Raha and Roy, (2001) used MS medium supplemented with 15 |iM BAP for multiple shoot induction. Agrawal etal. (2005) used 15 |iM BAP in MS medium for multiple shoot induction in Holarrhena antidysenterica. Gerald etal. (2005) also could get multiple shoot induction in the H. pubescens by using BAP alone in MS medium at 6.5 mg/l concentration. It was thus observed that for multiple shoot induction in Holarrhena antidysenterica BAP concentration needed in MS medium ranged from 2 mg/l to 6.5 mg/l. In Wrightia tomentosa multiple shoot induction was obtained on MS medium containing 5 mg BAP/I (Purohit etal. 1996). Patil and Jayanti (1997) obtained proliferation of axillary buds in Rauvolfia tetraphylla and Rauvolfia micrantha on MS medium supplemented with 2 mg BAP/I. For multiple shoot induction in Carissa carandus, Rai and Mishra, (2004) used MS medium supplemented with 3 mg BAP/I. In Mandevilla velutina, for multiple shoot induction. Biondo etal. (2007) used 1/3 MS medium containing 0.44 [iM BAP. In our experiments we used BAP at different concentrations ranging from 2.22 \iM -22.2 \iM and obtained the best shoot growth by using 8.8 [AM BAP in MS medium. We used different concentrations of BAP (0.5 - 13.2 |iM) in MS medium but could not get multiple shoot induction in C. fragrans.Though we did not get multiple shoot induction on this combination, sprouting of 2 or more axillary buds was observed in C. fragrans. (Plate 2). Addition of NAA along with BAP was found to be beneficial for multiple shoot induction in different genera of family Apocynaceae. Yuan et al. (1994) induced multiple shoots in Catharanthus roseus on MS medium containing 7 mg BAP/I and 1 mg NAA/I. 42 Roja et al. (1989) used MS medium with 0.5 mg NAA/I and 1 mg BAP/I to obtain multiple shoot induction in Rauvolfia serpentina. Roy etal. (1994) used axillary buds as explants and induced multiple shoots in R. serpentina on MS medium with 1.5 mg BAP/I and 0.5 mg NAA/I. Sarkar et al. (1996) obtained multiple shoots through callus mediated organogenesis in R. serpentina using MS medium supplemented with 0.5 mg BAP/I and 0.1 mg NAA/I. nodal segment explants of Holarrliena antidysenterica were cultured on MS medium containing BAP (1.0-3.0 mg/l) with NAA (0.2-1.0 mg/l)by Ahmad et al. (2001). Shahrear et al. (2002) grew axillary buds of R. serpentina on MS medium containing 0.1 mg BAP/I and 0.1 mg NAA/I and induced multiple shoots in R. serpentina. Tiwari et al., (2003) used MS+2.5 mg BAP/1 along with 0.1 mg NAA/I for the same. Kataria and Shekhawat, (2005) also used BAP along with NAA in MS medium. The combination of growth regulator used for multiple shoot was 3 mg BAP/I + 0.5 mg NAA/I in MS. In vitro clonal propagation of Rauvolfia serpentina through direct regeneration from shoot tip explants was carried out by Baksha et al. (2007) Multiple shoots (eight shoots per explant) induction were obtained on MS medium supplemented with 4.0 mg/l BAP and 0.5 mg/l NAA. The elongated shoots rooted well in half strength MS medium with 0.5 mg/l N/VA. The ratio of BAP to NAA used was higher i.e. ranging from 2 to 10. In our experiments, we observed that addition of BAP along with NfiJK was beneficial for growth of seedlings and seedling nodal sectors. Callus formation at the base of nodal explants was observed due to addition NAA along with BAP. Ghosh and Banerjee, (2003) obtained multiple shoot induction in Rauvolfia tetraphylla. In vitro culture of shoot apices of Rauvolfia tetraphylla on MS medium, supplemented with NAA and BAP or lAA and BAP resulted in the formation of 7-15 multiple shoot buds. The use of NAA and BAP stimulated callus formation at the base of the multiple shoots. Addition of lAA and BAP in MS medium stimulated only multiple shoot buds. Higher levels of BAP with NAA suppressed shoot elongation but increased the shoot multiplication rate. 43 Handro et al. (1988) cultured Leaf, stem and root explants of Mandevilla velutina and obtained vigorous callus on LS basal medium containing 2, 4-D or NAA along with BAP. Subculture of calli on MS medium with NAA (1.0 mg/l) and BAP (5.0 mg/l) caused profuse regeneration of shoots. We tried different combinations of NAA + BAP with ratio 10- 1 but did not get multiple shoot induction In C. fragrans. For shoot induction in Mandevilla velutina, Biondo et al. (2007) used different cytokinins like BAP, Zeatin and TDZ. The authors found 0.44 pM BAP the most effective for shoot Induction and growth. We have also obsen/ed shoot elongation in vitro due to addition of BAP / TDZ in MS medium and obtained similar results. MS medium supplemented with 0.5-10 pM TDZ was used by Faisal and Ahmad (2005) for multiple shoot induction in Rauvolfia tetraphylla. In our experiments we observed shoot elongation due to addition of TDZ in MS medium but BAP was the most effective cytokinin for shoot growth in C. fragrans. Multiple shoot induction was obtained on MS medium with 0.5 mg kinetln/l and 2 mg BAP/I for Holarrhena antidysenterica by Ahmad et al. (2001). Similarly Kinetin along with BAP was used for multiple shoot induction in Tabernaemontana fuchsiaefolia (Oliviera et al. 2003), and in Tabernaemontana divaricata (Sahu et al. 2006).. 2 mg BAP/I along with 5 mg KInetin/l in MS medium was used by these workers. In our experiments we got a positive effect on shoot growth by addition of kinetin /BAP to MS medium but the effect was as significant as BAP. Combination of cytokinins (BAP + kinetin) was not found to be effective as BAP. Nishitha et al. (2006) have used MS+13.3 pM BAP along with 2.45 pM IBA for micropropagation of C. fragrans. In our experiments we did not try addition of IBA to the medium. To get multiple shoot Induction though a large number of combinations of auxins and cytokinins were tried. But none of these could give multiple shoot Induction. Media other than MS medium have been used by different workers for micropropagation. WPM was used by Hatzilazarous et al. (2003) for shoot and 44 root induction in Nerium oleander and for Aspidosperma ramiflorum by Hubner et al. (2007). The authors used BAP along with NAA in WPM for micropropagation. B5 and White's medium was used for Rauvolfia serpentina by Tiwari et al. (2003). In our experiments we did not find WPM medium suitable for in vitro growth of C. fragrans. For root induction, ISA , lAA and NAA were used by us and ISA was found to be the best auxin for root induction. Many workers have found ISA more suitable for root induction. (Purohit et al. 1996, Raha and Roy, 2001; Shahrear et al., 2002, Oliviera et al. 2003, Rai and Mishra, 2004; Kataria and Shekhawat, 2005; Faisal and Ahmad , 2005 and Nishitha etal. 2006). Purohit ef a/. (1996) found that pulse treatment with ISA was beneficial for root induction in Wrightia tomentosa .Patil and Jayanti (1997) used 2 mg IBA/I for root induction In plantlets of Rauvolfia serpentina and Rauvolfia micrantha. Raha and Roy (2001) used 35 |iM IBA for root induction in Holarrhena antidysenterica and obtained 80 % of root induction . Half strength MS medium supplemented with 0.5 mg/l each of IBA, lAA and NAA was used by Ahmad et al., (2001) for Holarrhena antidysenterica and 95 % rooting was achieved. Shahrear et al. (2002) used IBA for root induction in Rauvolfia serpentina. Oliviera et al.,{ 2003) obtained 100 % root induction by giving short pulses of IBA (5.0 mg/l) in Tabernaemontana fuchsiaefolia. Rai and Mishra (2004) used Vi MS medium supplemented with 10.5 |iM IBA and 0.5 tiM NAA for root induction in Carissa carandus. Similar treatment was also used in Tabernaemontana fuchsiaefolia by Oliviera et al. (2003). Faisal and Ahmad (2005) used 0.5 nM IBA for root induction in Rauvolfia tetraphylla. We obtained 100% root induction in C. fragrans using 4.96 |iM IBA . NAA was used for root induction in Mandevilla velutina by Biondo et al. (2007) and the authors used 26.85 piM NAA. Agrawal et al. (2005) induced rooting in Holarrhena antidysenterica by using 10 nM lAA in MS medium. We obtained entire in vitro plantlets on MS+8.8 |iM BAP and MS+ 4.96 |iM IBA combination. We successfully obtained root induction using lAA and NAA also. Thus some of our results for studies on micropropagation of C. fragrans are 45 in accordance with the results obtained by the earlier workers for different genera. Studies on callus induction has been carried out by Handro et al. (1988). They cultured leaf, stem and root explants of Mandevilla velutina and obtained vigorous callus on LS basal medium containing 2, 4-D or NAA along with BAP. Heijden et al. (1989), used MS medium , B5 and SH medium and these media were supplemented with lAA, NAA and 2, 4- D as well as BAP/Zeatin/ kinetin for callus induction in various species like Tabernaemontana divaricata, T. Africana , T. chippi and T. psorocarpa etc. Callus induction % obtained was 80 % -100 %. MS medium was found to be the most suiable medium by the authors. We also found MS medium as more suitable than WPM. For callus induction of Rauvolfia sellowii, Rech et al. (1998) used Gamborg B5 medium supplemented with 2, 4- D and kinetin. B5 medium was used for getting friable callus. For callus induction in Rauvolfia tetraphylla, Anitha and Ranjitakumarl, (2006) used 2, 4- D. They recorded 95 % callus induction for leaf explants on MS medium supplemented with 9 nM 2, 4-D for Rauvolfia tetrapfiylla. Growth value for callus biomass in Rauvolfia tetraphylla recorded by the authors was 46%. In our experiments we obtained 100 % callus induction on MS medium supplemented with 4.5 [M 2,4, D. Gl as 5.6 (56 %) indicating vigorous growth of callus. Callus was also established by us in media containing lAA, NAA and BAP+ lAA and BAP+ NAA combinations. Thus most of our results are in accordance with earlier results. In vitro plantlets have been successfully acclimatized by different workers for different genera. In Holanfiena antldysenterica, regenerated plantlets were successfully acclimatized and established in soil. About 90% of plantlets survived was achieved by Ahmad et al. (2001). Shahrear et al. (2002) acclimated plantlets of Rauvolfia serpentina using autoclaved coco-peat as potting mixture then that of garden soil, compost and sand in a ratio 2:2:1. Biondo et al., (2007) achieved 100 % survival in the plantlets of H/landevilla velutina. For soil acclimatization, rooted plantlets were directly transferred to 46 plastic bags containing soil/sand mixture (1:3). Each plantlet was covered with a glass test tube (40 ' 80 mm) and kept inside a greenhouse (50 % light) with daily irrigation. In Rauvolfia serpentina, Baksha et al. (2007) acclimatized in vitro raised plantlets in glass house and successfully transplanted to field condition with 80 % survival. In T. divaricata plantlets 100 % survival was obtained by Sahu etal. (2006). In our experiments we obtained 80 % survival for plantlets of C. fragrans in soil and sand mixture(1:1). We also obtained 70% survival in the plantlets raised from seedling nodal sectors. In conclusion it can be said that most of our results are in accordance with earlier reports on different genera from family Apocynaceae. Difference in response to different cytokinins and auxins was observed in C. fragrans belonging to two different localities. Locality dependent variations and genotype variations could be responsible for this. Therefore further investigations on this plant are necessary. 47 PLATE -2 G H Chonemorpha fragrans A - EMBRYOS ON MSB (1.5X) B - SEEDLING (0.5X) C - ROOTED PLANTLET(SEEDLING NODAL SECTOR) (0.3X) D -AXILLARY BUDS SPROUTED (1X) E - ROOTED PLANTLET (0.5X) F &H - ENTIRE PLANTLETS (0.5X) G - HARDENING I - CALLUS(UNTREATED AND TREATED 100 PPM TRYPTOPHAN)(0.7X) CHAPTER II (B) Production of secondary metabolite in Chonemorpha fragrans Introduction and review of literature: Plants produce many chemical compounds than are necessary for their basic functions, i.e. survival and propagation. Primary metabolism refers to all biochemical processes for the normal anabolic and catabolic pathways which result in assimilation, respiration, transport, and differentiation. By and large, basic, or "primary" metabolism Is shared by all cells, while secondary metabolisms generate diverse and seemingly less essential or non-essential byproducts called secondary products. The secondary products give plants the colors, flavors, and smells. These products are sources of fine chemicals, such as drugs, insecticides, dyes, flavors and fragrances. While primary metabolism consists of biochemical pathways that are common to all cells, secondary metabolisms consist of a large number of diverse processes that are specific to certain cell types. Plant pigments, alkaloids, isoprenoids, terpenes, and waxes are some examples of secondary products. The role of many of the secondary products has been rather ambiguous, and initially they were thought to be just waste materials. However, considering their sequesterationary localization in a place and the lack of sophisticated immune system unlike animals, plants had to develop their own defense system against pathogens and predators. The products of plant secondary metabolisms, despite their enormous diversity, are grouped into the following categories, alkaloids, glycosides, tannins, phenols and terpenoids etc. Plants as sources of drugs have been investigated for secondary metabolites since a long time. For isolation of compounds, suitable solvents like acetone, ethyl acetate, ethanol or methanol are used. TLC, preparative TLC, HPTLC, column chromatography, HPLC are routinely used for isolation, separation and purification of these compounds. 48 TLC is the simplest technique and has been widely used for preliminary phytochemical investigations and identification of alkaloids. In Catharanthus roseus. Renaudin et al. (1984) have used TLC for identification of alkaloids. Preparative TLC along with RPLC also has been carried out for indole alkaloids in C. roseus by Hirata et al. (1987). An exhaustive account on TLC of alkaloids in genus Tabernaemontana, has been given by Van Beek etal. (1984). Different alkaloids occurring in genus Tabernaemontana, their Rf and chromogenic reactions with different reagents like FCPA, (ferric chloride perchloric acid) CSSA (eerie sulphate in sulfuric acid) and TCNQ (7, 7, 8, 8 tetra cynoquinodimethane) and the color of alkaloid spots under UV have been given by these authors. Pereira etal., (1983, 1984) also have used TLC for isolation and identification of alkaloids In Tabernaemontana dichotoma. Ferrari and Verotta (1988) have used Centrifugal TLC for separation of alkaloids from plants. Batista et al. (1996) had used TLC for separation of alkaloids from crude alkaloid extract from Rauvolfia sellowii. TLC may not prove the best method for routine analysis when numerous samples are involved. Moreover, crude plant extracts often contain many alkaloids and TLC is not always able to separate them adequately. HPTLC is another technique which enables identification and quantification of compounds by comparing the profile of a standard or pure compound and the extracts and by densitometric scanning. Conessine in Holarrhena antidysenterica was quantified by using HPTLC by Patel and Prajapati (2008) and camptothecin from the stem powder of Nothapodytes foetida by Dighe etal. (2007). HPLC is another technique commonly used for detection and identification of metabolites. A review of recent developments in HPLC analysis of alkaloids from C. roseus has been given by Hisiger and Jolicoeur (2007) in which extraction phase, HPLC separation and analysis protocols and identification methods at the analytical and semi-preparative scales were reviewed in details. Tikhomiroff and Jolicoeur (2002) used two direct HPLC analytical methods for the screening of the major indole alkaloids of Catharanthus roseus hairy roots and 49 their iridoid precursors. Separation was achieved on a reversed-phase column allowing the separation of catharanthine, serpentine, tabersonine, vindoline, vinblastine, and vincristine etc. Quantitative HPLC assay of the culture extracts of multiple shoot cultures of Catharanthus roseus carried out by Hirata et al. (1987) showed that vindoline and catharanthine were mostly found in the shoot, especially in the leaf tissue, whereas ajmalicine was localized in the unorganized tissue. Heljden et al. (1987) have used HPLC for determination of indole alkaloids in a suspension cell culture of Tabernaemontana divaricata. Heijden et al. (1989) have also used RPLC and ion pair CCC for isolation of different alkaloids In Tabernaemontana species. Standard or pure compound was loaded along with the extracts, allowing identification of compounds. HPLC was used for Rauvolfia sellowii by Rech et al. (1998) for identification of indole alkaloids from suspension cultures. For structural elucidation GC-MS, IR, N.M.R. and UV spectroscopy have been used by several authors (Joshi et al. 1984 ; Kam et al. 1999; Gunasekera et al. 1989) and have compared the spectra with the spectra of authentic samples or the values given in the literature. Family Apocynaceae is known to possess alkaloids, mainly steroidal alkaloids and indole alkaloids. Indole alkaloids have been isolated from C. roseus, Tabernaemontana species and Ochrosia elliptica whereas steroidal alkaloids have been isolated from Chonemorpha fragrans, Holarrhena antidysenterica and Alstonia scholaris. There are a few reports on steroidal alkaloid production, in Chonemorpha fragrans (Chatterji and Banerji, 1972; Banerji and Chatterji, 1973) Steroidal alkaloids like chonemorphine and funtumafrine have been isolated from root bark by these authors. The % of funtumafrine obtained from root bark was 0.002 %. There are no reports of the metabolic profiling and secondary metabolite production in vitro of this plant. Thus secondary metabolite production in C. fragrans was taken up for the present work. 50 Material and methods: OProcurement of plant material used for analysis, la; in vivo or field material lb) in vitro plant material were used for plant analysis. la) in vivo plant material- i) The plant material of C. fragrans was procured from 2 localities, L1 (Karnataka) and L2(Kerala). The material, collected from Kamataka state (L1) was mature (stem diameter 2 cm) whereas that from Kerala state {L2) was young (stem diameter 0.5 cm). The stem with stem bark and leaves were separated, washed and were shade dried at room temperature to constant moisture content. The dried plant material was powdered and stored in plastic bottles till further use. ii)Seedlings, six months old, established in thermocol pots were also dried and used to prepare ethanol extract for HPLC analysis, ill) A few hardened plantlets were surrendered and used to prepare ethanol extracts for HPLC analysis. lb) In vitro plant material - In vitro shoots grown on MS+8.8 nM BAP and leaf callus grown on MS+ 4.52 nM 2,4- D were dried, powdered and used for preparation of extracts. 11) Preparation of extracts- I) Cold extraction was carried out using 50 g of powdered material was added to 500 ml ethanol, methanol, acetone and ethyl acetate respectively, it was kept at room temperature, for forty-eight hours. The extracts were filtered and centrifuged at 9000 g for five min. The clear supernatant obtained was passed through the membrane filter (cellulose-nitrate, 0.20 |im, Pall Gelman). The extracts were evaporated to dryness to get the residue. The weight of the residue was recorded and the % extractive values were calculated. Hot extraction was carried out by adding 150 ml of solvent to 20 g powder of stem with bark (L1). The solvent was refluxed for 18 hrs. Solvent was evaporated to get the residue. The weight of residue was recorded. To the residue, 1 ml of ethanol was added. The alcohol extract was used for isolation of alkaloids. Weight of alkaloid fraction was recorded. A part of ethanol extract was used for TLC, HPTLC, HPLC and GC-MS analysis. Total numbers of extracts prepared are listed in the appendix 4. 51 Ill) Pharmacognostic studies of C. fragrans i)Pharmacognostic studies of C. fragrans were carried out for different fractions like acid, base, phenol and neutral fractions . They were separated by using following scheme (Katade, 2005) and % of these fractions was recorded. Ethanol Extract Add 10% Saturated NH40H Solution Triturate & extract with Ether I i 1 Aqueous Layer Ether Layer -1 Neutralize with acid Add10%NaOH Extract with Ether ^ 1 i 1 Ether Layer Aqueous layer Ether Layer-ll Aqueous layer Organic acids Extract with dil. HCI Neutralize with HCI * Extract With Ether ^ Ether. Layer Aqueoi^s Layer Phenolics Neutralise Neutral compounds .rWithDil.NH40H Extract with Ether Ether Layer (organic base) il) Extraction of alkaloids- It was carried out by the method described by Harborne (1998). % of alkaloids was calculated for stem with bark and leaves and in vitro plant material. ill) TLC of different extracts a) Selection of TLC plates- Ethanolic, methanolic, acetone and ethyl acetate extracts were loaded on glass TLC plates coated with silica for preliminary investigations. Numbers of spots developed in Iodine chamber were recorded. FCPA reagent (3 % FeCb in 35 % HCIO4) was sprayed on the plates for detection of alkaloid spots. The separation of spots was compared for different 52 solvent systems, prepared by using AR grade solvent. Precoated plates (6OF254 pre-coated (20x 20 cm Merck Darmstadt) were used in the last phase of analysis, for analysis of ethanolic extracts for isolation and Identification of alkaloids. b) Choice of solvent systems- Solvent systems tried for separation of compounds were 1) Ethyl acetate : Toluene (1:9)v/v 2) Ethyl acetate: Toluene (7: 3) v/v* 3) Chloroform: Ethyl acetate (1:1) v/v 4) Cyclohexane: Chloroform: Diethyl amine (6:3:1) v/v*. Solvent systems which gave good separation of compounds* were used for further separation of compounds. The chromatographic chamber was allowed to saturate for 30 min. using about 20 ml of the solvent system. The plates were run in duplicates in the solvent systems upto 10 cm of solvent front. c) Localization of compounds-The plates were observed under UV light at 254 nm and 366 nm wavelength. The fluorescent spots were recorded and Rf was calculated for these spots. For detection of alkaloids Dragendorff's reagent/ FCPA reagent was used. The plates were heated in an oven till colors developed. Rf and colour of the spots were recorded. d) identification of aikaioids-The Rf of spots was compared with the Rf values of alkaloids cited in the literature (Devkota, 2005). Identification of Camptothecin was carried out by comparing Rf with standard compound (Sigma, Bangalore)) using the methodology of Fulzele etal. (2001). Iv) HPTLC- HPTLC facility at ARI was used for this analysis. 500 |ig of extracts were loaded on HPTLC plates. The plates were run in duplicates in solvent systems i) ethyl acetate: toluene (7:3) and ii) Chloroform: ethyl acetate (1:1).The chromatographs were scanned by Camag densitometric scanner and the peaks, peak areas and the Rf of the spots were recorded. Authentic/pure sample of Camptothecin was procured from Sigma Aldrich, Bangalore. A standard sample of Camptothecin was prepared by dissolving 40 ^g of Camptothecin in 1 ml of DMSO:methanol (1:50) and run along with the extracts. Rf of standard Camptothecin was recorded. HPTLC profiles of different extracts were compared 53 with that of standard camptothecin to detect presence of camptothecin in the extracts. Identification and characterization of compounds v) HPLC- HPLC facility was used from IVIicrobiology department, IVIES Garware College, Pune. Isocratic analytical HPLC was performed on instrument Perkin Elmer series 2000 using RP18 column (SPHERI 5, 5 mm, 250 *4.6 mm). The mobile phase for alkaloid elutlon was acetonitrile: water (40:60), at a flow rate 1.6 ml/min with a sample size 20 \iL; & UV detection at 254 nm. A standard curve was obtained using authentic sample of camptothecin (Sigma Aldrich). The standard was prepared using DMSO: Methanol (1:50 v/v). Chromatography and co-chromatography of extracts with camptothecin was performed for confirmation. Validation of quantitative method was performed for sample in 5 replications. The profiles of the standard and the extracts were compared on the basis of retention time. Ethanolic extracts of stem with bark, leaves, entire in vitro plantlets, callus, and hardened plantlets (from both localities, L1 and L2) seeds and seedlings (L1) were analyzed with HPLC technique. The areas of the peaks were recorded and were compared with peak areas of the standard camptothecin. The amount of camptothecin was calculated for different extracts. vi) GC-IVIS- For GC- MS work facility at common facility centre, Shivaji University, Kolhapur and Ganware research centre, Pune was used. GC- MS analysis was carried out for ethanolic extracts of stem with bark, callus. For GC-MS analysis instrument used was Schimadzu QP 2000. Helium was used as a carrier gas at 0.7 mL per min. The injection temperature was 270° C. The column temperature was programmed to rise from 100 to 200° C at 10° C per min (for 10 min.) and from 200 to 260° C at 15 °C per min. (for 20 min.). Sample volume was 20 \i\. The retention time, peak area, % of area and M/z values were recorded for the fragments and the values were compared with the values given in NISHT library. 54 Results Phytochemical investigations on in vivo piant material of C. fragrans The Investigations were carried out for finding out the profile of secondary metabolites for this plant. Results of preliminary investigations are given below. i) Phytochemical studies indicated that there was a quantitative difference(%) in different types of fractions. Acid, base, neutral and phenol fractions of stem bark of C. fragrans as. Acid - 0.07 %, Base- 0.050 % neutral- 0.25 % and Phenol- 0.2 %. ib) Phytochemical tests carried out for in vivo ( stem bark and leaves) showed presence of alkaloids, steroids and tannins and in vitro plant material (in vitro plantlets and callus) showed presence of alkaloids. ii) Percentage extractive values for different plant parts of C. fragrans for different solvents were calculated. The values recorded were as given in table no 13. Primary TLC analysis showed identical spots for ethanol, methanol and ethyl acetate extracts. Thus for plant material of L2 only ethanol and methanol extracts were prepared. Similarly acetone and chloroform extracts showed identical spots on TLC, thus only acetone extracts were prepared for L2. Table13 - Percentage extractive values* for C. fragrans Solvent Stem with bark Leaves In vitro callus plants Ethanol LI 6.89(c)** 12.2(h)*" 4.76 (c) 2.3 (c) 3.4(c) L2 7.2 (C), -(h) 4.12 (c) 2.5 (c) 3.7(c) Methanol L1 9.2(c), 15(h) 5.55 (c) L2 10(c), --(h) 5.07 ( c) Acetone L1 3.2 (C ), 5 (h ) 1.2 (c) L2 4(C), --(h) 1.11 (c) Chloroform L1 0.89(c), (h) Ethyl acetate L1 1.5(c), —(h) n hexane 0.7,(c) -(h) *Mean of 2 samples. — extracts not prepared , **-cold extraction, * ** hot extraction 55 It was observed that methanol gave maximum percentage of extractive value and hexane gave the minimum. This indicated that as compared to non polar constituents, polar constituents were more in the plant material of C. fragrans. Extractive values for in vitro plant material were found to be lower as compared to in vivo material. Ratio of extractive values for stem bark and entire in vitro plantlets was 3 :1 (cold extraction) and that of stem bark and callus was 2:1. The callus showed higher percentage of extractive value as compared to in vitro plantlets. Preliminary investigations showed a wide range of metabolite profile for C. fragrans. As family Apocynaceae is well known for alkaloids, studies were focused on alkaloid extraction, isolation and identification of alkaloids. Percentage of alkaloids/ gram dry weight of plant material was calculated to compare alkaloid production in in vivo and in vitro .The results have been given in table no. 14. Table no. 14-Percentage of alkaloids* in plant material of C. fragrans. Plant material Percentage of alkaloids Percentage of alkaloids for plant material from LI for plant material l_2 stem with bark 0.22 0.24 Leaves 0.147 0.155 Callus 0.065 0.076 In vitro plantlets 0.047 0.057 * Mean of two samples. Alkaloid percentage was maximum for stem with bark. The ratio of alkaloid percentage in stem bark and callus was 3.38:1 and that of stem with bark and in vitro plantlets was 4. 68:1. Ratio of alkaloids in callus and in vitro plantlets was 1:0.75. ill) TLC analysis of in vivo plant material of C. fragrans - Ethanolic extract of stem bark was used to separate alkaloids on TLC plates coated with silica. The solvent system initially used was Ethyl Acetate: Toluene (1:9). Number of spots observed under 366 nm were recorded .The plates were sprayed with FCPA reagent ( 3.25 % of FeCIa In 35 % of perchloric acid) and heated in oven to 56 develop distinct colors to detect alkaloid spots. The spots observed under 366 nm, with their Rf in this solvent system were as given in table no 15. Table no. 15 -TLC analysis of ethanolic extract of stem bark of C. fragrans (LI) Ethyl acetate: Rf of spots observed under 366 nm Rf of alkaloid toluene (1:9) spots Ethanol extract 0.117,0.35,0.55,0.85 0.55 Methanol extract 0.117,0.12,0.35,0.55,0.85 0.55 Acetone 0.12, 0.250, 0.558 0.55 Ethyl acetate 0.12, 0.588 .—* chloroform 0.12, 0.688 — *—Not detected In stem with bark for L2 similar TLC profile was observed for solvent system Ethyl Acetate: Toluene (1:9).The solvent system Ethyl acetate: Toluene (1:9) showed separation of few spots. Thus for better separation of alkaloids from stem with bark, leaves, entire in vitro plantlets and callus (LI and L2), another solvent system was tried i.e. Cyclohexane: Chloroform: Diethylamine (6:3:1). The colour of alkaloid spot and Rf were also recorded. Initially Dragendorff's reagent was also tried for detection of alkaloid spots but in did not give distinct colors with alkaloid spots and thus was not used in further analysis. The plates were sprayed with FCPA reagent (3.25% of FeCb in 35% of perchloric acid) and heated in oven to develop distinct colors to detect alkaloid spots. More number of alkaloid spots were observed for extracts of in vitro plant material (callus) of C. fragrans than in vivo plant material. Nodal callus showed maximum number of alkaloid spots as seen from table no 16. 57 Table no16- TLC analysis of ethanolic extracts of C. fragrans Solvent system Rf of spots observed Rf of alkaloid spots Cyclohexane: chloroform under 366 nm and total and total no. of diethylamlne (6:3:1). no. of spots alkaloid spots Stem with bark L1 0.12,0.25,0.35,0.42, 0.35, 0.42,0.56 (3) 0.56 (5) Stem with bark L2 0.25, 0.35, 0.42, 0.56, 0.35, 0.42, 0.56 0.65,0.7 (6) (3) Leaves L1 0.03, 0.26, 0.35, 0.42, 0.35, 0.42 (2) 0.7, 0.75, 0.86, 0.9 (8) Leaves L2 0.03., 0.2, 0.35, 0.62, 0.35, 0.62 (2) 0.75, 0.86, 0.9 (7) Nodal callus L1 0.03, 0.26, 0.35,0.42, 0.35, 0.42, 0.56, 0.56, 0.62, 0.7 (7) 0.62 (4) Nodal callus L2 0.032,0.27,0.35, 0.42, 0.35, 0.42, 0.55, 0.62 0.55, 0.62, 0.68, 0.72 (4) (8) Leaf callus L1 0.03, 0.26, 0.42,0.62, 0.42,0.62 (2) 0.7,0.75,0.86,0.9 (8) Leaf callus L2 0.21, 0.35, 0.62, 0.86, 0.35, 0.62 (2) 0.9 (5) Entire in vitro plantlets L1 0.20,0.35, 0.55, 0.86 0.35,0.55 (2) 0.92 (5) Entire in vitro plantlets L2 0.21,0.35, ,0.65,0.86 0.35 (1) 0.90 (5) From table no. 16, it was observed that there were more or less similar alkaloid spots in stem bark and leaves. In callus, comparable number of fluorescent spots were observed as compared with in vitro plantlets. Nodal callus and leaf callus showed 3 alkaloid spots, common in both with the Rf, 0.35, 0.42, 0.62 respectively. Leaf callus did not show alkaloid spot with Rf 0.56. Callus extracts (callus raised from L1 and L2 explants) showed 3 or 4 alkaloid spots. Two alkaloid spots with Rf 0.42, 0.62 were present in callus but not in in vitro plantlets. Thus more variation in alkaloids was observed in callus than in entire in vitro plantlets. Alkaloid spot with Rf 0.62 was not recorded in the 58 extracts of stem with bark. Thus alkaloid profiles of in vivo and in vitro plant material of C. fragrans seemed to vary in terms of Rf of alkaloid spots. Identification of alkaloids could not be done due to unavailability of standard alkaloids. TLC was also carried out for a known steroidal alkaloid chonemorphine, as per the method described by Devkota (2005). The solvent system used was Petroleum ether: Acetone: Diethyl amine (40:58:2). The Rf given by the author for chonemorphine was 0.52 in this solvent system and no fluorescence was observed under 366 nm UV. A spot with this Rf was detected but this spot showed fluorescence. Thus presence of chonemorphine could not be confirmed in the extracts. This could be because of very low concentration of this compound which might be below detection level. The fluorescent spot could not be identified by TLC due to unavailability of standard alkaloids. HPTLC analysis of C. fragrans HPTLC was carried out for ethanolic extracts of stem with bark of C. fragrans of L1 and L2 .The extracts were run In solvent system, ethyl acetate : toluene( 7:3). The separation of spots has been shown in the plate. HPTLC of ethanolic extracts of stem with bark of C. fragrans. Laneland 2- ethanolic extract of in vitro plantlets (L1 and L2 respectively) Lane 3- Pure sample of camptothecin Lane 4- ethanolic extract of stem with bark(L2) Lane 5- ethanolic extract of stem with bark(LI) In the HPTLC plate, it was clearly seen that a band with the same Rf as that of pure Camptothecin was shown by the stem bark extract of L2. Thus on the basis of comparison of Rf, we can say that Camptothecin was present in stem bark extract of C. fragrans (L2).The peak areas could not be calculated 59 because the scanner could not detect the band fronn the sample , as the quantity was below detection level. 2-HPTLC profiles of ethanolic extract of leaf of C. fragrans The extracts which showed good metabolic profile on TLC plates were selected for HPTLC analysis. 9 extracts of in vivo and in vitro plant material were screened initially. The solvent system used was ethyl acetate: toluene (7:3). Only 2 extracts, leaf extracts (L1 and L2) showed separation of 5 or 6 spots. The spots recorded under 366 nm had been shown in the profile. The chromatograms were obtained as shown below. HPTLC profile of ethanolic extract leaf of C. fragrans(L2) The spots with Rf 3SB- 1 ' m- 1 0.07,0.11.0.25,0.67, ZSB- 0.82, 0.89, 0.96 were 2M- recorded. The peak i. ise- 1 i I areas and the 1L J 1 12 3 percentage of peak 1 58- 1u i L areas were calculated. B 1 r- 1" • 1 ' ' ' Peak area of compound n n »t n -^ e»"3 a 4 a c 0 C a 7 0- 0 a Q n 7 was maximum and for peaks 6 and 4 respectively was lesser. Identification of these spots could not be done due to unavailability of standard alkaloids. 3. HPTLC Profile of ethanolic extract of leaf (LI) The spots showed Rf 0.07, 0.86 and 0.96 similar to those in leaf extracts of L2. The profile shows that the quantity of these metabolites is higher in extracts of L2 as compared to L1. The peak areas were low and thus are not 8,8 8,1 U 8,3 8.^ 8.5 U V 8.8 3.5 shown. 60 HPTLC for in vitro plant material was carried out. Entire in vitro plantlets and callus extracts were analyzed. Both types of plant materials did not show detectable peaks in HPTLC ( profile no. 4, 5, 6, 8). 4. Summary of HPTLC profiles of all samples (ethanolic extracts) ^^ iwe A 393 z^X: a- £ 0.8 e.i a e 3 B* B.s «.6 a. e e e.« CHT] Fi ]e >Ane. miKXlLS ;r«cA I to 9 a ui.a* s--ti:aiBafi«9s otnAG sarrirfeiE ic» 19% £OH«tER 3: a3IB3B Sample 1-Leaf (L2), Sample2-Leaf callus (L2) precursor fed 100 ppm tryptophan) Sample3-Leaf callus untreated (L2), Sample 4-Leaf callus precursor fed 100 ppm tryptophan) (L1), Sample 5 - Leaf callus untreated (LI),Sample 6- entire in vitro plantlets (L1),Sample7-leaf (L2), Sample 8-Leaf callus elicitor treated (L2), Sample 9-Leaf (L2) 5 - HPTLC for detection of Camptothecin in ethanolic extracts of in vitro Lanel-entire plantlet fed with 100 ppm tryptophan (L1) Lane 2-ln vitro plantletsLI Lane 3 -In vitro plantletsL2 Lane 4 In vitro plantletsLI treated with elicitor 125 \xg glucose equivalent Lane 5 - pure sample of camptothecin Lane 6-stem with bark L2 Lane7- stem with bark LI Lane 8 - Tabernaemontana Lanel 2 3 4 5 6 7 8 alternifolia stem with bark 61 The solvent system was ethyl acetate : chloroform (1:1). The wells, number 2 and 3 show spots of extracts of in vitro shoots of L1 and L2, grown on MS+ 8.8 |iM BAP. The fifth well shows spot is of pure sample of Camptothecin. It is clearly seen that in one month old in vitro plantlets, did not show spot having same Rf as that of pure Camptothecin. Thus on the basis of comparison of Rf , we can say that Camptothecin was absent in the extracts of in vitro plantlets or was below the level of detection. Ill) Results of HPLC analysis of ethanolic extracts of C. fragrans HPLC analysis of different extracts was carried out mainly for detection of Camptothecin in C. fragrans. Retention time of pure sample Camptothecin was compared with the retention time of compounds in the extracts. The spots were detected under UV at 254 nm. The profiles recorded for in vivo and in vitro plant material were as shown in the figures. HPLC profiles for ethanolic extracts of C. fragrans- Peaks having same retention time of camptothecin were observed in ethanolic extracts of 1. Stem bark of L1 and L2 2. Leaves of LI and L2 3. Callus of L2 4. Hardened plantlets (6 months old) L1 and L2 The HPLC profiles obtained were as follows: 62 ) n met a*K • K a t = • » u 1. pure sample Camptothecin at concentration 40 \xg /ml. in solvent system DMSO: Methanol (1:50)Retention time- 3.75 min. peak area- 4448905 ?RS 8 aRWfiSSflsa fl& 8 9 2- Chonemorpha stem with bark. (LI)Retention time-3.81 min. peak area- 22121543 3. Chonemorpha leaf (LI) Retention time- 3.75 min. Peak area- 35,85,992 4- Chonemorpha leaf (LI) Co chromatography (10 \i\ sample+10 |il standard) Retention time- 3.84 min peak area- 3986237 " 58 S? ? A« e s 5-Chonemorpha hardened plantlets (6 months old) Retention time - 3.87 min. peak Area-17669 63 6- camptothecin standard Retention time- 3.96 min. Peak area-4527486 7- HPLC profile of stem with bark (L2) Retention time- 3.95 min. Peak area- 882926 8-HPLC profile of Chonemorpha stem ethanolic extract (L2) Co chromatography (10 [x\ sample+10|il standard) retention time3.92 min peak area- 2335405 n s s f f J I 1 "Tf^^^T^ 1 9-HPLC profile leaf extract (L2) retention time-3.86 min. Peak area- 886093 10-HPLC profile of Chonemorpha callus (L2) Retention time-3.89 min. peak area-34628 64 Peaks of camptothecin were not detected for ethanolic extracts of 1) Seeds and one month and six month old seedlings, 2) Entire in vitro plantlets ( L1 and L2), one month old and six months old respectively. 3) Hardened plantlets , one months old (L1 and L2).4) Callus raised from L1 leaf explants. Quantification of camptotliecin in the sampies The pure sample of Camptothecin 40 jig/ml in Methanol: DMSO (1:50) was analyzed by HPLC for five times. The concentrations of standard used for plotting SDR were from 5ng/ml to 35|ig/ml. The peak areas obtained Standard graph of camptothecin were: 4448502.9, 4514971.6, 3SOO00O, 4472644.5, 4622038.6, and 3000000 4683424.3. (Mean 2S00000 4548316.4, SE = 44994.407, 2000000 ISOOOOO CV = 2.2%) lOOOOOO 500000 4 0 0 10 20 30 40 5C Concentration of camptothecin in ng/ml For calculation of amount of camptothecin in the samples, following values were considered 1) Retention time of pure camptothecin 2) Retention time of compounds in the samples. The peak in the extracts showing same retention time as that of standard camptothecin. 3) Peak area shown by the standard 4) Peak area of peak showing the same retention time as that of standard camptothecin 5) Total volume of the extract prepared 6) Dry weight of the plant material used to prepare the extract. Percentage of camptothecin was calculated for the samples. The values calculated on dry weight basis of plant material were as shown in 65 table no17. Table17-Percentage of camptothecin in plant material of C. fragrans (Dry weight basis) Plant material % of camptothecin % of camptothecin Locality l(Karnataka) Locality L2(Kerala) Stem with bark 0.0013 0.0012 Leaves 0.0009 0.0007 Hardened plantlets 0.0003 0.0004 (6 months old) Callus * 0.0003 *— Not detected Camptothecin peaks were observed in mature plant material like stem with bark and leaves. These were observed in plant materials from both localities. Though both the plant materials showed comparable values of camptothecin, the plant material from L2 was younger as compared with the plant material of LI which was highly mature. This observation suggests higher synthetic ability of plants from locality L2. GO-MS analysis GC-MS analysis carried out for ethanolic extracts yielded following chromatograms. 66 GC-MS Chromatogram 1- Ethanolic extracts of Chonemorpha bark(LI) "T ' r- 20.0 30.0 The retention times for the fragments were as given below. GC-IVIS 2 Ethanolic extracts of Chonemorpha callus (LI) I ^iM^iM^V^*^^ -T T ~T —iT-i • ..—1-" 20.0 30.0 GC-MS- 2 Ethanolic extracts of Chonemorpha stem with bark (L2) . v.- .—..^U—^...^—_-^^^^ .^J^^-.^.. iVat»if^*W*' .afca**'^ ^tVWt^'>>m"^*^"'»»>'*^»l^i ••.pf^^^mi—»•'> ».J^'^»' 67 Fragment analysis- GC-MS-3 -Ethanolic extracts of Chonemorpha stem with bark (L2) 43 0 456 97.0 439 960 396 « sleo 67.0 348 33« 1 * 25%- M.O 111 0 2P 82. C 224 i ^3 ; * * ': 123 0 264 0 172 163 29 0 'I 1 '"i » t 136 "1 :' ji * 295 0 125 0 222 0 180 0 122 • : li ^0 ,1 • 1! 112 114 39. D ^ Ira J i 103 • 152 0 « ii 1 » fa 0 71 A 50 I. ii i llf. •' ! jj'';' i ;, iteS „*i1i, !i'li;!lM!«i: iui ii till lli,ii ii i.iL. ii. i, 1. i' . : i • 54< 6 J 50V- 41,0 419 4 143 0 247 25%-i • 75 0 55 0 189 29 0 174 158 • « 69 0 133i • 129 0 42.0 101.0 106 227 0 270 C 91 83 0 91 « 78 83 27.0 72 t 171 0 • 1 53 » 51 4 , ( iji. i:., iii'i. 2} ,:„, il, . •! ! : i :J i. 1 07o-f i 68 The fragment analysis and M/z values revealed the presence of fatty acids like hexadecanoic acid and octadecanoic acid esters and Hexadecanolc methyl ester. These are fatty acid precursors of steroids/ steroidal alkaloids. GC'MS analysis could not be used in identification of secondary metabolites, as no standard compounds from this plant were available. Phytochemical investigations revealed some of the important points about metabolic profile of in vivo and in vitro plant material of C. fragrans, which are as follows: 1. Callus showed a better alkaloid profile as compared with the alkaloid profiles of in vivo and in vitro plant materials. 2. One of the alkaloids in stem bark and callus was identified as camptothecin on basis of TLC, HPTLC and HPLC analysis. This is the first report of detection of camptothecin from this plant. 3. The studies revealed age dependent synthesis of camptothecin in vivo and in vitro plant material. 4. GC-MS analysis revealed presence of fatty acid esters which are primary compounds giving rise to steroidal alkaloids. Discussion Secondary metabolite production has been studied in many plants from family Apocynaceae, Most of them show presence of alkaloids, steroids, phenolic compounds and tannins. In Chonemorpha fragrans funtumafrine from root bark was isolated (Chatterji and Banerjii, 1972). Shah etal., (1987, 1989) isolated chonemorphine, rapanone, stigmasterol and ecdysterone from root bark of this plant. It was obtained by fractionation, purification of ethanol fraction of root bark. In other plants of Apocynaceae, alkaloid production has been studied. Total percentage of alkaloids has been calculated by different authors tor different plants. For Tabernaemontana heyneana. Meyer et al. (1973) obtained 0.34 % of alkaloids in the dried roots. Knox and Siobbe (1975) isolated alkaloids from Ervatamia orientalis stem bark and alkaloid % ranged from 0.13 % -0.20 %. 69 Locality dependent variation was observed in production of alkaloids by these authors. Gunasekera et al. (1979) isolated Camptothecin and 9 methoxy camptothecin from the wood and stem bark of this plant. Methanolic extract was used and from neutral fraction of this extract, the alkaloids were isolated .The percentage of Camptothecin was 0.00013 % whereas 9 methoxy camptothecin was 0.00004 %. We have recorded comparable levels of camptothecin in the stem bark in C. fragrans. Srivastava et al., (2001) obtained 0.03 % of alkaloids from fruits of Tabemaemontana heyneana. Thirumalapad et al. (1982) isolated alkaloids from Ervatamia dichotoma and obtained 3 % of alkaloids (34 g/1.1 kg) from the root bark. Tertiary indole alkaloids were extracted from Tabemaemontana pachysiphon by Van Beek et al. (1984) and have recorded 0.58% of alkaloids in stem bark and 0.73 % of alkaloids from the root bark. Aspidosperma ramiflorum, was studied by Marquis et al. (1996) for alkaloid production and 0.77 % alkaloids were obtained from ethanolic extracts of stem of this plant. In our studies we obtained 0.22 % total alkaloids for stem bark of C. fragrans plant material. It was thus clear that C. fragrans is a plant with moderate alkaloid content as compared with other genera of family Apocynaceae. Investigations on total alkaloid production in C. fragrans showed that the plants from L2 seemed to have higher ability of alkaloid production. We tried to find out the reason for the difference in the ability to synthesize alkaloids in C. fragrans belonging to two different localities. Soil analysis was carried out for soil samples from both localities (table no 5). Analysis of soil showed that the soil from L2 contained a low amount of exchangeable K^ and a very high amount of Iron and chlorides. Probably the plant must be facing nutritional stress and therefore producing more amount of secondary metabolites. Environmental conditions, microbial associations in soils also should be taken into account in this context. As standard compounds were unavailable, identification of alkaloids from this plant was not possible. 70 Secondary metabolites in in vitro piant material -Indole alkaloid production in suspension culture of C. roseus was studied by Lounasma and Galambos, (1989) and Zhao et al. (2000). It was obsen/ed that in vitro production of alkaloids was low as compared to intact plants. In our experiments, the alkaloid content was observed to be low in in vitro plantlets as compared to the parent plants. Production of specific alkaloids in vitro has been studied. Vinblastine production in callus and multiple shoot cultures in C. roseus was low (Miura et al. 1988). Moreno et al. (1993) did not get production of vincrystine in suspension culture of C. roseus. In Rauvolfia serpentina Roja etal. (1987) detected ajmalicine, reserpine and serpentine in multiple shoot cultures and 0.15 % ajmalicine production was obtained in shoot cultures. These values were comparable with the roots and the intact plants. Roja et al. (2000) studied callus cultures of Ochrosia elliptica and obtained camparable values of ellipticine production with that of parent plant. Panda et al. (/9^32) isolated and quantified connesine from suspension culture from Holarrhena antidysenterica.AnHa and Ranjitakimari (2006) have extracted 0.9 mg/g reserpine from callus culture of Rauvolfia tetraphylla. Oliviera et al. (2001) reported that Aspidosperma ramiflorum callus cultures produced 1/4 and 1/6 of alkaloid content of intact plant. Ramiflorine A and B were detected in the callus. % of alkaloid recorded was 0.294 for callus and 1.16 % for the intact plants. Thus the ratio of alkaloid production in intact plant and in callus cultures was 4:1. In our experiments, percentage of alkaloids calculated for callus was far lower as compared to the intact plants. Callus showed more percentage of alkaloids as compared to in vitro plantlets. This was probably due to cell to cell contact, age of the plant and limited differentiation in callus. Heijden et al. (1989) showed that percentage of alkaloids in fresh callus varied from 0.003 % to 0.086 % for different species of Tabemaemontana. % of alkaloids in callus of C. fragrans obtained by us was 0.065 % and 0.076 %. Thus the values seemed to be comparable in both these plants. 71 We observed that callus cultures of C. fragrans produced one or two different spots of alkaloids than the ones produced In in vitro plants. This observation was similar to the observations recorded by Heljden et al. (1987). A different alkaloid pattern in vitro cultures, mainly callus and suspension cultures had been observed In Tabernaemontana species (Heljden et al. 1987) and In T. divaricata (Pawelka and Stocklgt, 1983). The plant showed a complex pattern of alkaloid synthesis in vitro. An Important achievement was detection of camptothecin In C. fragrans on the basis of HPTLC and HPLC analysis. The amount of camptothecin calculated in stem bark showed that the amount was 10 times higher than the amount reported in Tabernaemontana tieyneana by Gunasekera et al. (19CW.) .Whereas it was 1/10 the amount reported in Camptotheca acuminata by Sakato et al. (1974). There are only a few plant sources of camptothecin and Tabernaemontana heyneana has been the only one from family Apocynaceae. Thus Chonemorpha fragrans could be a potential source of Camptothecin. Further fractionation, purification of extracts, GC-MS analysis and NMR analysis can be carried out for confimriatlon of the compound. C. fragrans can be further studied for production camptothecin under different ecological conditions for finding the best genotype/elite plant. It had been shown in Camptotheca acuminata (Sakato et al. 1974) and Nottiapodytes nimmoniana (Padmanabha etal. 2006) that production of camptothecin is age dependant. Similarly in our studies camptothecin was detected in mature plant parts but it was not detected in seeds or seedlings one to six months old. It was also detected in a small amount in six months old hardened plantlets. Thus, our studies confirmed age dependent synthesis of camptothecin in C. fragrans. In callus cultures of C. fragrans from locality L2 it was detected in a very small amount. This also pointed towards better potential of secondary metabolite production of plants from L2. The plant can be further studied for production of camptothecin under different cultural conditions having different growth regulators , different biotic and abiotic elicitatiors. 72 CHAPTER II (C) Enhancement of Secondary metabolite production in vitro In Chonemorpfia fragrans Introduction and review of literature: Pharmaceutically significant secondary metabolites include alkaloids, glycosides, flavonoids, tannins, resins etc. Some of the plant derived natural products are morphine, codeine, cocaine, quinine, Catharanthus alkaloids, belladonna alkaloids, reserpine and steroids like diosgenin, digoxin and digitoxin. Plant-derived drugs in western countries also represent a huge market value. Higher plants are rich source of bioactive constituents or valued at more than US$30 billion in 2002 in the USA alone. Vincrystine and vinblastine were isolated from Catharanthus roseus in 1960's .Similarly camptothecin isolated from Camptotheca acuminata and derivatives of camptothecin (CRT) are also used for treatment of various types of cancers (Takeuchi et at. 1991). Currently, most of these secondary metabolites are Isolated from wild or cultivated plants because their chemical synthesis is economically infeasible. Many of these pharmaceuticals are still in use today and often do not have useful synthetic substitutes that possess the same efficacy and pharmacological specificity. In view of world's growing population and exploitation of plants for medicines, a large number of herbs and trees are facing extinction (Houghton, 1997). Biotechnology is a boon and has offered attractive altematives like cell and tissue culture techniques, recombinant DNA technology and bioprocess technology. It has offered the great opportunities of exploiting totipotency, biosynthetic and biotransformation capabilities of plant cells in vitro (Stockigt et al. 1985). To date, this has only limited commercial success because of a lack of understanding of how these metabolites are synthesized. Biosynthesis in cultured plant cells- In vivo biosynthetic capacities of plant cells are maintained in vitro. Almost all groups of natural products like triterpines, furanocoumarines, indole alkaloids and carotenoids are produced by plant cultures (Stockigt et al. 1985). There are reports that some products are 73 produced in cell or suspension cultures are equal or higher in quantities than the whole plants. Ajmalicine production in in vitro cultures of Catharanthus roseus was higher in quantities than the whole plants (Drapeau etal., 1987). In most of the cultures, production of secondary metabolites has been low. This has been a major hurdle in commercial production of plant products and attempts are needed for enhancement in secondary metabolite production in vitro. Production of secondary metabolites in vitro can be enhanced by screening of high yielding cell lines, media modifications, precursor feeding, elicitation, large scale cultivation in bioreactor system, hairy root culture, biotransformation and others. Elicitation, is a process in which the treatment of cultures with a substance which, when introduced in small concentrations, initiates or improves the biosynthesis of specific compounds or metabolites. Elicitors may be abiotic or biotic. Abiotic factors predominantly are inorganic salts, like Cu and Cd, ions like Ca ^*' high pH, UV light. Heavy metals. Biotic elicitors are substances with biological origin and include polysaccharides derived from plant cell walls (pectin or cellulose) and micro-organism extracts or filtrates containing chitin or glucans. Though the molecular and biochemical mechanisms related to elicitation are not universal, enhanced production of end products is observed. Biotic elicitors- In plant tissue cultures, stress induced by inactivated fungi or fungal enzymes has been used to enhance production of biologically active secondary metabolites. In several instances it has been reported that fungal elicitation led to overproduction of pentacyclic triterpenes instead of some other expected metabolites. Heijden et al. (1988), reported overproduction of pentacyclic triterpenes in tissue cultures of Tabernaemontana species., normally producing indole alkaloids. These cultures were subjected to stress induced by fungi, bacteria, or enzyme like cellulase or pectinase. The cell cultures produced ursane-type pentacyclic triterpines, 3.3 times the normal rate (2 % of dry mass). No increase in the production of indole alkaloids occurred. Heijden et al. (1989) used Candida albicans as an elicitor for treatment of suspension cultures of Tabernaemontana divaricata. Accumulation of pentacyclic triterpines was 74 observed accompanied with growtfi inhibition and inhibition of indole alkaloid accumulation in suspension cultures. Several parameters in relation to elicitors are important in metabolite production. They are i) elicitor concentration ii) duration of elicitor exposure ill) age of culture, iv) nutrient composition. Elicitor concentration plays a very important role in elicitation process. Namdeo et at. (2002) reported higher accumulation of ajmaliclne in C. roseus cultures when treated with different concentrations of elicitor extracts of T. viride, A. niger and F. moniliforme. Ajmalicine accumulation was higher in cells elicited with higher concentration (5.0 %) of elicitor extracts as compared to the cells treated with lower concentration of the elicitor (0.5 %).However, increasing the concentration further upto 10.0 % adversely affected the accumulation of ajmalicine. Similar results were obtained by Rijhwani and Shanks (1998), Zhao et al. (2001). They tested several fungal elicitors including Aspergillus niger, to enhance alkaloid production in suspension cultures Of C. roseus. Different fungal mycelium homogenates stimulated different kinds of indole alkaloid (ajmalicine, serpentine and catharanthine) accumulation, which ranged from 2- to 5-fold higher than the control. Some fungal culture filtrates also efficiently elicited the biosynthesis of different indole alkaloids. The optimal elicitor addition and exposure time for the maximal alkaloid production has been given by the authors. Duration of elicitor exposure is an important factor. In cells of C. roseus exposed with elicitor extracts of T. viride, A. niger for 24h, 48h, 72h and 96h showed about 3-fold increase in ajmalicine production by C. roseus cells elicited with extracts of Trichodenva viride for 48 h , higher concentrations were inhibitory for alkaloid production.Two-fold increase was observed in cells elicited with Aspergillus niger and Fusarium . moniliforme ( Namdeo et al. 2002). However, further increasing exposure time resulted in decrease in ajmalicine content. Inhibition of growth and alkaloid production was observed in Tabernaemontana divaricata due to treatment of Candida albicans leading to accumulation of triterpenoids (Heijden etal. 1988). 75 Age of subculture plays is an important role in production of bioactlve compounds by ellcitation. C. roseus cells of 20-day-old cultures showed higher yields of ajmalicine on ellcitation. Highest amount ajmallcine (166 pig/g Dry Wt.) accumulated in 20-day-old cells elicited with extracts of T. viride followed by 90 and 88 |ig/g Dry Wt. ajmalicine in cells elicited with A. niger and F. moniliforme respectively (Namdeo et at. 2002). Similar observations were reported from various workers Rijhwani and Shanks (1998), Ganapathi and Kargi. (1990). Media modifications have been tried by several workers. Effect of addition of organic and inorganic salts for catharanthine production was studied by Smith etal. (1987). They studied the effect of different concentrations of NaCI, KCI and sorbitol on catharanthine production. Diluting the mineral salts of the culture medium decreased the alkaloid production. Addition of high levels of Ca^^, Mg^^ or Sr^"^ to B5 media in which the mineral salts were diluted to 5-40 %, increased the alkaloid production. Decendit et al. (1992) confirmed enhanced accumulation of indole alkaloids in Catharanthus roseus cell cultures due to addition of cytokinin. Similar results were shown by Roja et al. (1996) in Rauvolfia serpentina. Kaudio et al. (1985) observed effect of growth regulators on alkaloid production in Ochrosia elliptica. They observed that reduced levels of 2, 4- D in the nutrient medium increased alkaloid content. Effect of ethylene and cytokinin on alkaloid production in C. roseus was investigated by Papon et al. ( 2005) and more production was observed due to addition of cytokinins. Sherawat et al. (2002) have reported that 2, 4- D in medium leads to decrease in production of alkaloids in C. roseus.. Other plants studied are Holarrhena antidysenterica, Tabemaemontanadivaricata(Heijden etal. 1989). Nutrient composition of medium or selection of medium also plays a vital role for elicitation process. Ajmalicine accumulation in C. roseus was observed more in Zenk's medium as compared to Murashige and Skoog's medium Namdeo et. al. (2002). Instability in alkaloid production was observed by Deus- Neumann and Zenk (1984) in C. roseus suspension cultures after a long period of cultivation. 76 Abiotic ellcitor used in C. roseus was Vanadium for increase in production of indole all 77 secondary metabolites, combined efforts of experts in pharmacognosy, microbiology, phytochemistry, biochemistry, molecular biology, and fermentation technology are needed. There are no reports on enhancement in secondary metabolite production in C. fragrans and Tabernaemontana altemifolia. Therefore studies in secondary metabolite production and their enhancement in secondary metabolite production in vitro in these two plants were taken up. MATERIAL AND METHODS: I) Enhancement in secondary metabolite production using precursor. For enhancement in alkaloid production, precursor selected was tryptophan as a primary precursor of alkaloids biosynthesis. i) Expiants used- la) One month old in vitro shoots( from both localities) were grown on MS medium +8.8 pM BAP and used for precursor treatment. ib) Leaf callus (from both localities) was grown on MS medium+ 4.5 pM 2, 4- D and used for treatment with a precursor, tryptophan. ii) Precursor addition- ii a) For treatment of in vitro shoots, MS medium + 8.8 pM BAP was supplemented with tryptophan at 25 mg/l, 50 mg/l*, 75 mg/1 and 100 mg /I* of tryptophan respectively. The selection of concentrations was made on the basis of in vitro responses of the expiants. The best suitable concentrations * (50 mg/l and 100 mg/l) were selected for further studies, lib) For treatment of callus, MS medium + 4.5 pM 2, 4- D was supplemented with, tryptophan at 25 mg/l, 50 mg/l 75 mg/1 and 100 mg /I of tryptophan respectively. The selection of concentrations was made on the basis of culture responses. The best suitable concentrations i.e. 50 mg/l and 100 mg/l of tryptophan respectively were used for further studies. II) Enhancement in secondary metabolite production using a biotic elicitor OSelection of an elicitor- For studies on elicitation, Aspergillus niger was used as a precursor, as it is a broad range pathogen. ii) Preparation of Aspergillus n/ger extract Pure culture of Aspergillus niger was obtained from culture collection of Department of Botany, University of Pune, Pune, India. It was grown on Curie's 78 medium for 15 days. The mycelium and spores were collected and added to D.W. and autoclaved. This was filtered and carbohydrate (glucose equivalent) estimation of the filterate was carried by anthrone method (Sadasivam and Manikkam, 1996).The selection of concentrations of elicitor was made on the basis of initial studies on culture responses. Five concentrations respectively, 60, 125, 175, 250 and 300 |ig glucose equivalent were added to MS medium. The best suitable concentrations of the elicitor, i.e. 125 |ig glucose equivalent and 250 |ig glucose equivalent were selected for further studies. iii) Elicitor treatment- ilia) For treatment of in vitro shoots, MS medium + 8.8 \iM BAP was supplemented with fungal extract, 125 |ig glucose equivalent and 250|ig glucose equivalent respectively, iiib). For studies on effect of elicitor on callus, 125 \ig glucose equivalent and 250 |ig glucose equivalent of elicitor respectively was added to MS medium + 4.5 |iM 2, 4- D. 20 replicates kept for each treatment and the entire set of experiment was repeated twice to verify and confimi the results. For in vitro shoots effect of precursor and elicitor on growth and other morphological characters were recorded. They were further used for phytochemical analysis mainly for alkaloid production. The callus was observed after every week and recorded as + ( meagre), ++( moderate) and +++ ( heavy callus). Fresh weight and dry weight of callus was recorded after 4 weeks for to find out the effect of precursor and elicitor on biomass production. Callus was further used for phytochemical analysis mainly for alkaloid production. Results Enhancement in secondary metabolite production in C. fragrans Effect of precursor on production of callus biomass was noted. The results recorded were as given in table no. 18. Biomass production in callus cultures was observed for both localities (table 18). Biomass production was seen to be reduced by 30 % and 25 % for LI and L2 respectively, with addition of 50 ppm tryptophan feeding, whereas it was reduced by 40 % for LI as well as L2 at 100 ppm concentration of tryptophan feeding. 79 Table 18- Effect of precursor feeding and elicitor treatment on biomass production* in cailus of C. fragrans (after 4 weelcs) Medium combination *lncrease in fresh Increase in fresh wt. of callus in g+ wt. of callus in g+ S.E(LI) S.E( L2) MS medium + 4.5 |iM 2, 4, D 1.72+0.05 1.85+0.05 (control) MS medium + 4.5 |iM 2, 4, D 1.23+0.06 1.27+0.07 + 50 mg tryptophan /I MS medium + 4.5 |iM 2, 4, D 1.02+0.04 1.1t0.02 + 100 mg tryptophan /I MS medium + 4.5 |iM 2, 4, D 1.35+0.03 1.45±0.07 +elicitor 125 ug glucose equivalent MS medium + 4.5 |iM 2, 4, D 1.11+0.06 1.23+0.05 +elicitor 250 ug glucose equivalent Mean of 20 replicates A similar observation was made with elicitor treatment also. 20 % reduction in biomass production was observed for LI and L2 respectively due to the elicitor concentration at 125 ug glucose equivalent whereas at elicitor concentration 250 ug glucose equivalent it was by 35 % for L1 and L2 Treated callus material was collected and dried and powdered and used to prepare ethanolic extracts. This was further used for phytochemical analysis. The extractive values for ethanol for treated callus and plant material were recorded as given in table no 19. Table no19-Percentage extractive values* for callus and in vitro plant material of C. fragrans fed with tryptophan Ethanol extract Percentage Percentage extractive values extractive values LI L2 Entire plantlet (control) 2.3 2.52 Entire plantlet ( 50 ppm tryptophan) 2.5 2.7 Entire plantlet (100 ppm tryptophan ) 2.13 2.23 Callus (control) 3.4 3.71 Callus (50 ppm tryptophan) 3.75 3.97 callus (100 ppm tryptophan) 3.25 3.56 *Mean of 2 samples 80 There was an increase in percentage extractive values of in vitro plantlets as well as callus at 50 ppm tryptophan concentration in MS medium (for in vitro plantlets 8 % for L1 and L2 both and for callus, 10 % for L1 and 7 % for L2 respectively). At 100 ppm concentration of tryptophan in the medium, there was decrease in percentage extractive values of in vitro plantlets (4.3 % decrease for LI and 11.5% for L2) as well as callus (4.4 % for L1 and 4% for 1-2). 100 ppm concentration of tryptophan supplement showed a slight in inhibitory effect on metabolite production. Table no 20- Percentage extractive values* for ellcltor treated callus and in vitro plant material in C. fragrans. Ethanol extract Percentage Percentage extractive value LI extractive value L2 Entire plantlet untreated 2.3 2.52 Entire plantlet treated with elicitor 125 |ig glucose equivalent 2.7 2.7 Entire plantlet treated with elicitor 250 |ig glucose equivalent 1.93 2.00 Callus untreated 3.4 3.71 callus treated with elicitor 125 \1Q glucose equivalent 3.85 4.07 callus treated with elicitor 250 kig glucose equivalent 3.00 3.06 * mean of 2 samples. Increase in percentage extractive values were observed for in vitro plants and callus cultures due to treatment at 125 ng glucose equivalent elicitor concentration. It was 17 % and 8 % for in vitro plantlets and for callus they were 13 % and 8 % for LI and L2 respectively. Decrease in percentage extractive values for in vitro plantlets was observed . The decrease was 16% and 20% for LI and L2 respectively. For callus it was 11.7 % for LI and 17% for LI and L2 respectively. This indicated that higher concentrations of the elicitor had more inhibitory effect on metabolite production as compared to higher concentrations of precursor. Effect of precursor and elicitor treatment on alkaloid production, (percentage of alkaloids) was evaluated for both in vitro material of C. fragrans. 81 As seen from table no. 21, callus cultures(L1 and L2) showed more amounts of alkaloids as compared to in vitro plantlets when fed with 50 ppm tryptophan. Table no 21-Percentage of total alkaloids* In precursor treated in vitro material of C. fragrans Ethanol extract %of total % of total alkaloids LI alkaloids L2 Entire plantlet (control) 0.047 0.057 Entire plantlets fed with 50 ppm typtophan 0.049 0.062 Entire plantlets fed with 100 ppm tryptophan 0.035 0.041 Callus (control) 0.065 0.076 Callus fed with 50 ppm tryptophan 0.075 0.091 Callus fed with 100 ppm tryptophan 0.051 0.060 *Mean of 2 samples In callus and in vitro plantlets, decrease in percentage of alkaloids ( LI and L2) was observed at 100 ppm concentration of tryptophan in the medium. It was noted that there was increase in alkaloid % in in vitro plantlets (25.5 % for LI and 26 % for L2) and callus (15 % for LI explants and 10 % for L2 explants) due treatment of elicitor 125 [ig glucose equivalent. There was decrease in alkaloid production In in vitro plantlets as well as callus at higher concentrations of elicitor,( 21 % for LI as well as L2 explants) and thus these concentrations seemed to be Inhibitory for metabolite synthesis (table 22). Table 22 -Percentage of total alkaloids *in elicitor treated in vitro material %of total alkaloids % of total Ethanol extract LI alkaloids L2 Entire plantlet untreated 0.047 0.057 Entire plantlet (125 pg glucose 0.059 0.072 equivalent) Entire plantlet (250 ng glucose 0.036 0.040 equivalent Callus untreated 0.065 0.076 callus (125 ng glucose 0.075 0.081 equivalent) callus (250 |ig glucose 0.051 0.061 equivalent) *Mean of 2 samples Results indicated that, elicitor treatment seemed to be more effective for increase in alkaloid production in C. fragrans as compared to precursor 82 treatment. Higher concentrations of precursor as well as ellcitor seemed to have an inhibitory effect on alkaloid production in both i.e. in callus as well as in vitro plantlets. TLC analysis for plant material treated with precursor and ellcitor. Identification of alkaloid spots- Rf of fluorescent spots recorded under UV 366 nm for the plantlets for L1 and L2 for solvent system was Cyclohexane :Chloroform: Diethyl amine (6:3:1). The plates were sprayed with FCPA (3.25% FeCIa in 35% perchloric acid) reagent were studied for locating the spots. The results were as given in table no. 23. Table- 23 -TLC of precursor treated in vitro plantlets of C. fragrans (Fluorescence under 366 nm UV) untreated untreated 50mg 50mg 100mg/l 100 mg (L1)(control) (L2)control) tryptophan/1 tryptophan/1 tryptophan tryptophan/1 Rf of spots Rf of spot (LI) (L2) (LI)Rfof (12) Rfof Rf of spots Rf of spots spots spots 0.20 0.21 0.19 0.23 0.2 0.20 0.35* 0.35* 0.23 0.56* 0.35* 0.31 0.55* 0.65 0.42* 0.67 0.96 0.96 0.86 0.86 0.56* 0.96 0.92 0.90 0.96 *alkaloid spot The extracts of untreated plantlets showed 2 spots of alkaloids. Extracts of plantlets grown on medium with 50 ppm tryptophan also showed similar Rf for alkaloid spots. Plantlets grown on medium with 100 ppm tryptophan showed less number of alkaloid spots whereas plantlets from L2 did not show spot of alkaloids. The spot of alkaloid having Rf 0.56 did not appear in the extracts of plantlets grown on medium with 100 ppm concentration of tryptophan. The spot of alkaloid having Rf 0.35 did not appear in the extracts of plantlets treated with 50 ppm concentration of tryptophan. Thus there was change in the alkaloid profile of in vitro plantlets due to tryptophan treatment. 83 TLC for plant material treated with precursor and elicitor. TLC was carried out for elicitor treated plant material also. The alkaloid profile obtained was as given below in table in 24. Table 24- TLC of ethanolic extracts of precursor treated callus of C. fragrans (Fluorescence under 366 nm UV) Cyclohexane :Chloroform: Diethyl amine (6:3:1). Callus L1 Callus L2 Callus(L1) callus(l_2) callus (L1) callus (L2) untreated untreated 50 mg 50 mg 100 mg lOOmg (control) (control) tryptophan/1 tryptophan/1 tryptophan/1 tryptophan/1 Rf of spots Rf of spot Rf of spots Rf of spots Rf of spots Rf of spots 0.03 0.26 0.19, 0.23, 0.2, 0.2, 0.26 0.42* 0.23, 0.35* 0.35* 0.35* 0.42* 0.61* 0.42,* 0.56*, 0.96 0.96 0.62* 0.7 0.56*, 0.96 0.7 0.75 0.96 0.75 0.86 0.86 0.95 0.90 * alkaloid spot In extracts of untreated callus , 2 spots of alkaloids were observed . In callus treated with 50 mg tryptophan/1 also, 2 spots of alkaloids were observed. The spot having Rf 0.61 did not appear in the extracts of treated callus. Spot with Rf 0.35 was observed in callus grown on medium with lOOmg tryptophan/1 which was observed to be present in the extracts of in vitro plantlets, stem and leaf. Thus a change in the alkaloid profile of callus in terms of Rf due to tryptophan treatment was observed. To study the effect of elicitor on alkaloid profile, TLC was carried out. The Rf values recorded were as presented in table no 25. 84 Table 25- TLC of ethanolic extracts of elicitor treated in vitro plantlets of C. fragrans (Fluorescence under 366 nm UV) (Cyclohexane :Chloroform: Diethyl amine (6:3:1). (control) (Control) (LI) (L2) (L1) (L2) L1 L2 Elicitor 125 Elicitor 125 Elicitor ,250 Elicitor, 250 untreated untreated |ig glucose ^g glucose |ig glucose |ig glucose equivalent equivalent equivalent equivalent Rf of spots Rf of spots Rf of spots Rf of spots Rf of spots Rf of spots 0.20 0.21 0.19 0.23 0.2 0.2 0.35*, 0.35* 0.23 0.56* 0.35* 0.35* 0.55* 0.65 0.42* 0.96 0.96 0.96 0.86 0.86 0.90 0.56* 0.92 0.96 *alka oid spot It was clear that alkaloid spot with Rf 0.35, did not appear in the extracts of in vitro plantlets treated with elicitor 125 [ig glucose equivalent whereas it was present in the extracts of in vitro plantlet treated with elicitor 250 |ig glucose equivalent . The spot of alkaloid with Rf 0.35 was observed in extracts of callus and in vitro plantlets grown on medium with 100 mg tryptophan/1, as well as in the extracts stem and leaves. Thus change in the alkaloid profile in terms of Rf due to elicitor treatment was observed. Callus extracts were also analyzed for alkaloid spots. The Rf of spots recorded as given in table no. 26. 85 Table 26 - TLC of ethanolic extracts of elicitor treated callus of C. fragrans (Fluorescence under 366 nm UV) (Cyclohexane :Chloroform: Diethyl amine (6:3:1). (L1) (L2) (L1) Elicitor (L2) (L1) (L2) untreated untreated 125 ^g Elicitor 125 Elicitor 250 Elicitor 250 (control) (control) glucose (ig glucose (ig glucose ^g glucose equivalent equivalent equivalent equivalent Rf of spots Rf of spot Rf of spots Rf of spots Rf of spots Rf of spots 0.03 0.26 0.19, 0.23, 0.2, 0.2, 0.26 0.42* 0.23, 0.35* 0.35* 0.35* 0.42* 0.61* 0.42,* 0.56* 0.96 0.96 0.62* 0.7 0.56* 0.96 0.7 0.75 0.96 0.75 0.86 0.86 0.95 0.90 'Alkaloid spot The alkaloid spot with Rf 0.35 was seen in the extracts of treated callus at both the concentrations of precursor treatment. The spot did not occur in the extracts of untreated callus. The spot with this Rf is present in stem bark and leaves as well as in vitro plantlets. Thus due to elicitor treatment, changes in the alkaloid profile was observed. Thus TLC analysis carried out revealed that tryptophan used as a precursor and Aspergillus niger as an elicitor were effective in enhancement in production of total amount of alkaloids. The alkaloids could not be identified due to unavailability of standard alkaloids. HPTLC analysis- HPTLC analysis was carried out for precursor fed and elicitor treated material gave following results. 86 HPTLC of precursor treated entire plantlets of C. fragrans. The HPTLC profile shown below shows 7 wells. The first well on the left shows spot of extract in vitro plantlet grown on MS medium supplemented with 100 mg tryptophan. The fifth well shows band of pure camptothecin (standard). There is no spot of same Rf as that of pure Camptothecin. Thus on the basis of comparison of Rf we can say that Camptothecin is absent in in vitro plantlets supplemented with 100 mg tryptophan. Extracts of plantlets treated with precursor (50 ppm) as well as elicitor (250 |jg glucose equivalent) did not show presence of camptothecin, thus these profiles are not produced over here. Though precursor treatment (at 50 ppm concentration) and elicitor treatment (at 125 |ig glucose equivalent) led to increase in total alkaloid production, they affected production of camptothecin. Camptothecin probably is not synthesized, or alterations in the pathway might have taken place. The steps in synthesis might be blocked or impaired, or converted to some other intermediates. HPLC analysis Treated in vitro plantlets, (LI and L2), as well as callus failed to show peak of Camptothecin. Thus HPLC chromatograms for treated in vitro plants has been shown . 87 12-HPLC profile of ethanolic extracts Elicitor treated(125 |jg glucose equivalent) entire in vitro plantlet (L2). No peak of camptothecin detected As elicitor treated plant material (250 |ig glucose equivalent) and precursor fed (50 as wll as 100 ppm tryptophan) material did not show peak on camptothecin, the profiles have not been produced over here. Alkaloid production in C. fragrans was found to be moderate in quantity as compared to other plants belonging to family Apocynaceae. In vitro plant material showed less alkaloid production as compared with the parent plants. Camptothecin was detected in the plant extracts of C. fragrans by HPLC and HPTLC. Age dependent synthesis of camptothecin was confirmed in in vivo plant material of C. fragrans. Treatment of in vitro plant material with lower concentration of tryptophan as well as Aspergillus niger elicitor enhanced alkaloid production in vitro whereas higher concentrations of these showed reduction in in vitro alkaloid production. Alkaloid profile of in vivo and in vitro plant material differed with respect to Rf of alkaloid spots. Changes in alkaloid profile were observed due to tryptophan as well as elicitor treatment. Production of camptothecin seemed to be altered due to these treatments. DISCUSSION The reduction in the alkaloid production at higher concentration of precursor was obtained by Lucumi et al. (2002) in suspension cultures of Tabernaemontana elegans which had lost the capacity of biosynthesis were fed with tryptamine and loganin. It was probably due to blocking various steps in alkaloid synthesis or production of different intermediates. It was observed that 88 biosynthesis of alkaloids was impaired at several steps. We have obtained similar results. In callus of Rauvolfia tetraphylla increased reserpine production was obtained by Anita and Ranjitakumari, (2006) by adding lower levels of tryptophan (50 mg/l). Reserpine production was obsen/ed to reduced at higher levels 75 mg/l) of tryptophan in the medium. The reduced levels of alkaloids may be due to inability of cultures to utilize precursor, or toxic levels of precursor, or diverting the precursor molecule to other pathways other than alkaloid pathways. We could not detect presence of camptothecin in tryptophan treated and elicitor treated material. Probably these concentrations are higher affecting utilization of precursor or toxic to the plant. Response to lower concentrations of both these should be studied. The values of % of alkaloids in treated plantlets and in callus showed an increase in alkaloid production in in vitro plantlets as well as in callus, due to elicitor treatment. 125 pg glucose equivalent elicitor concentration in the medium seemed to beneficial for alkaloid production but higher concentration i.e 250 ng glucose equivalent elicitor concentration seemed to be inhibitory for alkaloid production for entire plantlets as well as callus. Reduction in production of alkaloids in vitro due to treatment of an elicitor at a higher concentration had been obsen/ed in Tabemaemontana species, Cattiaranttius roseus etc. In Ctionemorptia fragrans also similar trend was observed. It would be interesting to study effect of abiotic elicitors, changes in composition of nutrient media on camptothecin production in C. fragrans. C. fragrans might be useful in future as a new plant source of camptothecin with further fractionation, purification and documentation. 89 CHAPTER II D Studies on antimicrobial activities of Chonemorpha fragrans Introduction and review of literature Finding iiealing powers in plants is an ancient idea. The plants are still widely used in ethno medicine around tine world. New plant sources are being investigated. The use of plant extracts, as well as other alternative forms of medical treatments, is enjoying great popularity since 1990's. Pharmacological industries have produced a number of new antibiotics in the last three decades. Resistance to these drugs by microorganisms has increased. In general, bacteria have the genetic ability to transmit and acquire resistance to drugs, which are utilized as therapeutic agents. The continued emergence or persistence of drug resistant organisms and the increasing evolutionary adaptations by pathogenic organisms to the antimicrobials have reduced the efficacy of antimicrobial agents currently in use. Therefore, to continue studies to develop new drugs, either synthetic or plant sources, becomes necessary (Fransworth and Morris, 1976). The use of plant compounds for pharmaceutical purposes has been in practice since a long time. In developed as well as developing countries, use traditional medicine, has tremendously increased. Therefore, medicinal plants should be investigated to better understand their properties, safety and efficiency. Approximately 20 % of the plants found in the world have been submitted to phamnacological or biological tests (Suffredini et al. 2004). Plants possess antimicrobial properties due to secondary metabolites synthesized in them. These metabolites may be alkaloids, glycosides, steroids, triterpenoids or phenolic compounds which are part of the essential oils and tannins . Different plants from family Apocynaceae have been studied for their antimicrobial activity. They are Wrightia spp. (Hadi and Bremner, 2001), Alstonia scholahs (Goyal and Varshney, 1995; Versha etal. 2003 and Khan etal. 2003), Rauvolfia tetraphylla (Shariff etal. 2006), Nerium o/eanofer(Hussain and Gorsi, 2004), Picralima nitida (Nkere and Iroegbu, 2005), Baissea axillaries Hua 90 Abere and Agoreyo, (2006), Plumeria acutifolia (Rasool et al. 2008), Holarrhena pubescens (Chakraborty and Brantner, 1999) and Tabemaemontana chippi (Beekefa/. 1985) Antimicrobial activity of Alstonia scholaris was studied by Goyal and Varshney (1995) and reported the antimicrobial properties of the plant constituents of A. scholaris (alkanes, alkanols and sterols). Chattopadyay et al. (2001) tested various extracts for antibacterial activity of Alstonia macrophylla leaves. Butanol extract showed antimicrobial activity against various strains of Staphylococcus aureus, Staphylococcus saprophyticus, Streptococcus faecalis, Escherichia coll, Proteus mirabilis. The minimum inhibitory concentration (MIC) values ranges from 64 to 1000 |jg /ml for bacteria. The strains of Pseudomonas aeruginosa, Klebsiella sp. and Vibrio cholerae showed resistance against in vitro treatment of the extracts up to 2000 pg/ml concentration, while the two yeast species were resistant. The stem bark extract prepared similarly was found to be less active compared to the leaves. Phytochemical study of leaves and the bark revealed the presence of tannins, flavonoids, saponins, sterols, triterpene and reducing sugars. Similar antibacterial activity of leaves of Alstonia scholaris was demonstrated by Versha et al. (2003). Khan et al. (2003) evaluated the antibacterial activity of the petrol, dichloromethane, ethyl acetate, butanol fractions of crude ethanolic extracts of the leaves, stem and root barks of Alstonia scholaris and reported antibacterial activity of butanol fraction. Antimicrobial activity of Rauvolfia tetraphylla and Physalis minima leaf and callus extracts were studied by Shariff et al. (2006). Leaves and call! were extracted using absolute alcohol, benzene, chloroform, methanol and petroleum ether. Leaf and callus extracted In chloroform of were found to be more effective against pathogenic bacteria and fungi. R. tetraphylla leaf extract showed MIC of 2.0 and 4.0 mg/ml against all the tested fungi for absolute alcohol and chloroform extracts. MIC ranged between 1.0 to 6.0 mg/ml for callus extracts in absolute alcohol, chloroform and methanol. Aqueous extracts of Apocynaceae and Solanaceae members from native of Amazon rain forest and Atlantic forest were tested for their antimicrobial 91 activity against sXaphylococcus aureus, Enterococcus faecalis following broth microdilution method, and they showed some degree of inhibition of bacterial growth at concentrations of 100 |ig/ ml (Suffredini etal. 2004). For Aspidosperma ramiflorum alkaloid extract of bark showed antimicrobial activity against gram-negative bacteria (Oliveira etal., 1999). Nerium oleander was investigated by Hussain and Gorsi, (2004). The ethanolic, methanolic and chloroform extracts of Nerium olearider leaf and roots were tested. The extracts showed considerable antimicrobial activity against Bacillus pumillus. Bacillus subtilis, Staphylococcus aureus and Escherichia coli. Antimicroibial activities were studied for Picralima nitida by Nkere and Iroegbu (2005). Ethanol, benzene, chloroform and aqueous (cold and hot) extracts of seed, stem bark and roots were tested against five bacterial strains using the agar-well diffusion method. The ethanol extracts of the root and stem bark were active against the test organisms. The benzene and chloroform extracts exhibited no activity. The MIC for the bacteria was also calculated. Abere and Agoreyo, (2006), tested Inhibitory activities of aqueous and ethanolic extracts of leaves of Baissea axillaries Hua against clinical strains of Escherichia coli, Pseudomonas aeruginosa. Staphylococcus aureus and Streptococcus faecalis and inhibitory activities were compared with Togamycin (Spectinomycin). Minimum inhibitory concentration (MIC) against the test organisms was calculated. The extracts inhibited the growth of Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus to varying extents, but only the ethanolic extract inhibited growth in Streptococcus faecalis. Chakraborty and Brantner (1999) studied Holarrhena pubescens for its antibacterial activity. Stem bark extracts were tested for antibacterial efficacy against Staphylococcus aureus, Staphylococcus epidermidis. Streptococcus faecalis, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa using the microdilution broth method as well as the disc diffusion method. The crude methanolic extract was shown to be active against all tested bacteria. Further chemical fractionation indicated that the antibacterial activity was mainly associated with the alkaloids. 92 Raman et al. (2004) tested the alkaloid holarrlfine-24 ol isolated from the stem bark of Holarrhena antidysenterica for its antibacterial and antifungal activity against ten pathogenic bacteria and plant pathogens. The alkaloid showed good antibacterial and antifungal activity against almost all the test organisms. The activity was compared with the antibiotic ampiciilin. The MIC values of the alkaloid were also calculated by the authors. Thus alkaloid holarrifine-24ol was shown to have antibacterial properties against almost all the test bacteria. Similar antibacterial activity of extracts of stem bark of Holarrhena antidysenterica has been reported by Ballal ef a/.(2001). Kavitha et al. (2004) also evaluated the alkaloids from the ethanolic extract of H. antidysenterica seeds for their antibacterial activity against clinical isolates of enteropathogenic Escherichia coli (EPEC) in vitro. The plasmid DNA, whole cell lysate and outer membrane protein profile of a clinical isolate of EPEC was determined in presence of alkaloids of H. antidysenterica. The disc diffusion and agar well diffusion methods were used to evaluate the antibacterial efficacy. The alkaloids showed strong antibacterial activity against EPEC strains. Lindsay et al. (2000), tested dichloromethane extract of the wood of Carissa lanceolata for antibacterial activity against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. The extract showed activity having a minimum inhibitory concentration less than 0.5 mg/ml against S. aureus and E coli. Rasool et al. (2008) tested ethanolic extract of Plumeria acutifolia Poir. Stem bark was tested for antimicrobial activity against Gram-positive bacteria {Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus). Gram-negative bacteria {Escherichia coli, Klebsiella Pnumoniae, Psuedomonas aeruginosa, Salmonella typhimurium) by disc diffusion method. Minimal inhibitory concentration (MIC) and acute toxicity were also assayed. The ethanol extract showed the strong in vitro antimicrobial activity against £. faecalis, B. subtilis, S. aureus, P. aeruginosa, S. typhimurium. Gentamycin was the standard antibiotic kept as positive control. Inhibition was observed at 125-250 ng /ml. 93 Thus the literature survey made it clear that a number of plants from family Apocynaceae have been shown to exhibit a broad spectrum of antibacterial activity. Some plants have not been investigated for their antimicrobial activity though they have been used as medicinal plants since a very long time. C. fragrans is used in medicinal preparations in Kerala Ayurvedic system since a very long time. Therefore the antibacterial activities of this plant were taken up for our studies. Material and methods: i) Selection of micro-organisms Selection of micro-organisms- For testing activity, Gram positive and Gram negative organisms were selected. Following organism were procured from NCIM, NCL, Pune and Department of Biotechnology, Sinhgad College, Pune. Organism Accession number Medium for growth Type Bacillus subtilis ATCC 6633 Nutrient agar Gram positive Salmonella typhae — Nutrient agar Gram negative Klebsiella pneumonae — Nutrient agar Gram positive ii)Preparation of extracts for testing antimicrobial activity Ethanolic extracts of stem bark from L1 and leaf callus (L1) was used to test the activity. Ethanol was added to the extracts, to get the concentration 250, 500 ppm. Callus powder 25 g was added to ethanol and extracted for 24 hours. The solvent was evaporated to obtain the residue. Known volume of ethanol was added to the extracts, so as from the stock, required concentration could be obtained (250 and 500 ppm). Antimicrobial activity of ethanolic extracts of leaves, stem bark from L2 and leaf callus (L2) was not studied. iii)Preparation of medium- Nutrient agar medium was prepared, autoclaved at 15 lb pressure/sq. inch for 20 min. about 20 ml of medium was dispensed in each petridish. Bacterial cultures were inoculated into nutrient broth and incubated at 35±2° C .After 24 hours, the bacterial suspension was centrifuged at 6000 rpm for 15 min. The pellet was suspended in sterile distilled water and the 94 transmittance of suspension was corresponding to 70-80 % at 530 nm and a constant number 10 ^cells/ml. iv)Preparation of microbial suspension- 0.1 ml of suspension was spread with a spreader uniformly. The plates were allowed to dry for 10 min. The wells were prepared by using 0.6 mm borer and 5 wells were prepared in each petridish. 75 |il of extract was loaded in each well. The solvent ethanol was kept as a negative control and the standard antibiotic Cephotaxime (250 and 500 ppm respectively) was used as a positive control. 3 replicates were kept for each set of experiment and each set was repeated twice. Inhibition zone was recorded in mm after 24 hours. Inhibition zones recorded were as given in table no-27. Table no. 27-Antibacterial activity of Chonemorpha fragrans (LI) Plant Size of inhibition zone (mm)* Extract Bacillus subtilis Klebsiella pneumoniae Salmonella typhae Stand ard Extract Stand ard Extract Stand ard Extract 500 250 500 250 500 250 500 250 500 250 500 250 ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm Stem 23 20 13 12 20 18 13 11 26 19 11 10 Callus 23 20 12 10 20 18 11 10 26 19 *«^ *- Mean of 3 replicates The results are also presented in graph 12. Graph 12--Antibacterial activity of etiianolic extracts of Chonemorpha fragrans (LA) From the table and the graph, it is • Bacillus • Klebsiella clear that the stem D Salmonella extracts showed moderate activity against Klebsiella pneumonae and Standarad S Plant extract Bacillus subtilis and no activity against Salmonella typhae. It is important to note that callus also shows comparable antibacterial activity to that of stem extracts against all these 95 organisms. Callus extracts showed maximum inhibition of Klebsiella pneumonae and minimum Inhibition of Salmonella typhae. In plate 3, Inhibition zones produced by ethanollc extracts of C. fragrans plant material in vivo and in vitro have been shown. The activity of bark extracts may be assigned to the secondary metabolites I.e. alkaloids , steroids and tannins present in the bark. The results obtained also are supported by our phytochemlcal investigations which have shown higher percentage of alkaloids in the bark as compared to callus. The antibacterial activity of stem with bark extracts In comparison with the standard antibiotic showed that the extracts possessed activity equivalent 282 ppm of standard antibiotic for stem extracts and equivalent to 260 mg of standard cefotaxime for callus extracts for B. subtilis as well as Klebsiella pneumonae. For salmonella typhae only the stem bark extracts showed activity whereas the callus extracts did not show inhibition of Salmonella typhae. Salmonella typhae is known to be an antibiotic resistant pathogen not inhibited by many of plant extracts. Thus important observation was antibacterial activity exhibited by callus extracts of C. fragrans (L1). For Holarrhena antidysenterica methanolic extracts inhibition zones of 6mm, 3 mm, 2 mm and 2 mm and 0 mm respectively for B. cereus and S. epidemiidis E. aerogenes, P. vulgaris and Salmonella typhemurium (Parekh and Chanda, 2007). Holarrhena floribunda methanol extract was tested for antibacterial activities against B. subtilis and an inhibition zone of 18 mm was recorded. Chloramphenicol 10 ng gave 26 mm of inhibition zone against B. subtilis.{Pa\r\ce etal., 2007). Tabernaemontana catharinensis methanol extracts were shown to have weak activity against Gram negative bacteria (Guida et al. 2003). The inhibition zones recorded were 10 mm, 13 mm, 24 mm, 25.5 mm for methanol extracts (500,1000,2000, and 4000 \ig respectively) against B. subtilis. Rasool et al. (2008) tested ethanollc extract of Plumeria acutifolia Poir. Stem bark was tested for antimicrobial activity. The ethanol extract showed the 96 strong in vitro antimicrobial activity against E. faecalis, B. subtilis, S. aureus, P. aeruginosa.S. typhimurium. Gentamycin was tlie standard antibiotic l 97 Bacillus suibtllis Plate L1 (left) 1,1'- inhibition zones produced by C.fragrans ethanolic extract stem with bark 1' -extract 250 ppm 1- extract 500 ppm , 2'- callus extract-250 ppm 2- callus extract 500 ppm. Plate R 2-(Right) 2'- callus extract-250 ppm 2- callus extract 500 ppm. 3, 3' inhibition zones produced by standard antibiotic Cephotaxime 3- 500 mg and 3' 250 mg Klebsiella pneumoniae Plate LI (left) 1,1' inhibition zones produced by C.fragrans ethanolic extract stem with bark r -extract 250 ppm 1- extract 500 ppm , 2'- callus extract-250 ppm 2- callus extract 500 ppm. Plate R 2-(Right) 3,3' inhibition zones produced by standard antibiotic Cephotaxime 3- 500 mg and 3' -250 mg 2, 2' inhibition zones produced by C. fragrans callus ethanolic extract 2'- callus extract-250 ppm 2- callus extract 500 ppm Salmonella typhae Plate LI (left) 3, 3 both inhibition zones produced by standard antibiotic Cephotaxime 500 mg 1,1' inhibition zones produced by C.fragrans ethanolic extract stem with bark 1-500 mg extract 1' -250 mg extract Plate R 2-(Right) 3', 3' both inhibition zones produced by standard antibiotic Cephotaxime 250 2, 2' inhibition zones produced by C.fragrans callus ethanolic extract PLATE -3 Bacillus iubtUh KiebsMta pneumoniat' SalmoneUa typbae Sivm vxtmci CRUOS citrsct Chonemorpha fragrans antibacterial activity