4.1. Germplasm collection The state of Tamil Nadu is located in the southern eastern part of Indian

Transcription

1 Results

2 4.1. Germplasm collection The state of Tamil Nadu is located in the southern eastern part of Indian peninsula 8 o 5` and 13 o 35` North latitude and 76 o 15` and 80 o 20` East longitude. The climate is tropical with little variation in temperature during winter and summer. Summer season is felt between April and June when temperature reaches 40 o C. November to February is the months when Tamil Nadu experiences winter but the temperature doesn t drops below 20 o C. Fifteen accessions of W. somnifera were collected from different locations of Tamil Nadu (Fig. 1). It was found to be distributed in wild lands and as a weed in cultivated land. No particular association of this species with other plants was noticed during study Analysis of variations among accessions Whole plant traits All the accessions revealed significant difference for all the characters studied. This indicates the considerable variability among the 15 accessions (Table 2 & Fig. 2). The variability ranges observed were as follows: Plant height (14 to 37 cm), number of leaves per plant (4 to 8 leaves), number of roots per plant (4 to 30), root length (6 to 24.5 cm) and root diameter (1.4 to 2.3mm). The plant height was maximum in ACCN 06 (37 cm) followed by ACCN 13 (32 cm) and ACCN 04 (27.5 cm) respectively. The number of leaves per plant was maximum in three accessions ACCN 01, ACCN 06, ACCN 12 (8 leaves) followed by ACCN 02, ACCN 03, ACCN 05, ACCN 07, ACCN 08, ACCN 09 (7 leaves). The number of seeds per berry was maximum (32 seeds) in ACCN 04 and ACCN 06 followed by (28 seeds) in ACCN 01 and ACCN 14. It was minimum in ACCN 11 (18 seeds). The maximum number of roots (27 roots) per plant was in ACCN 06. The minimum number of roots (4) per plant was observed

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4 Table 2. Morphological characterization for various accessions of W. somnifera S. No Plant height (cm) No. leaves per plant No. of seeds per berry No of root per plant Root length (cm) Root diameter (cm) f 8 f 28 i 09 d 15.1 g 1.8 bcd d 7 d 22 e 10 e 09.0 b 1.6 abc c 7 d 26 h 12 f 10.2 c 1.7 abcd g 6 c 32 k 16 h 14.3 f 2.3 e d 7 d 24 g 14 g 12.2 d 1.9 cd i 8 f 32 l 27 k 24.5 j 2.4 e a 7 d 19 b 04 a 06.1 a 1.4 a d 7 d 29 j 12 f 09.2 b 1.7 abcd b 7 d 24 g 08 c 09.5 bc 1.4 a f 5 b 20 c 09 d 13.2 e 1.4 a a 4 a 18 a 05 b 09.5 bc 1.5 ab e 8 f 23 f 25 j 14.3 f 2.0d h 4 a 26 h 30 l 19.1 i 1.4 a g 6 c 28 i 18 i 18.2 h 1.7 abcd e 5 b 21 d 12 f 15.5 g 1.4 a Means with the same alphabet are not significantly different according to Dancan s multiplication range test at 5% level. The values average of four replicates.

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6 Results in ACCN 07. The maximum root length (24.5 cm) was observed in ACCN 06 and minimum (6 cm) in ACCN 07. The root diameter was maximum (2.4 mm) in ACCN 06 and minimum (1.4 mm) in accession ACCN 07. While comparing the overall results in whole plant traits, ACCN 06 was found to be well performing and ACCN 07 was poorly performing Leaf traits The physical leaf traits showed considerable variation among the 15 accessions (Table 3 & Graph 1-6). The maximum leaf thickness (37 µ) was recorded in ACCN 04 and minimum (5 µ) in ACCN 12. The fresh weight was maximum (1.15 g) in ACCN 09 and minimum (0.2 g) in ACCN 08. The leaf dry weight was maximum (216 mg) in ACCN 10 and minimum (0.05 mg) in ACCN 13. The score of leaf dry matter content was maximum (457 mg g -1 ) in ACCN 10 and minimum (137 mg g -1 ) in ACCN 12. The average leaf size was maximum (2805 mm 2 ) in ACCN 09 and minimum (5.31 mm 2 ) in ACCN 09. The specific leaf area was maximum (24196 mm 2 mg -1 ) in ACCN 07 and minimum (8198 mm 2 mg -1 ) in ACCN 04. Among all the leaf traits studied, leaf dry matter content was found to be less effective in revealing variations. The Fitch Margoliash Cladogram based on the class-based stratification matrix of the physical leaf traits revealed four different groups (Fig. 3). The group 1 consisted of accessions ACCN 13, ACCN 02, ACCN 06, ACCN 12, ACCN 10 and ACCN 05. The second group consisted of accessions ACCN 08 and ACCN 03. The third group consisted of ACCN 14, ACCN 15, ACCN 11, ACCN 07 and ACCN 04. The fourth group consisted of accessions ACCN 09 and ACCN 01. K. Mohamed Rafi, Ph.D. Thesis, April

7 Table 3: Physical leaf traits of fifteen accessions ACCN code ACCN01 ACCN02 ACCN03 ACCN04 ACCN05 ACCN06 ACCN07 ACCN08 ACCN09 ACCN10 ACCN11 ACCN12 ACCN13 ACCN14 ACCN15 Average LT µ 8.56 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.61 Average Fwt g 0.53 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.22 Average Dwt mg ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.03 Leaf traits. Average LDMC mg/g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Average leaf size mm ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Average SLA ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Abbreviations LT - Leaf thickness Fwt - Leaf fresh weight Dwt - Leaf dry weight LDMC - Leaf dry matter content SLA - Specific leaf area

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10 Fig. 3 : Cladogram based on physical leaf traits

11 Results Quantification of Withaferin-A content The quantification of Withaferin-A content in leaf tissue was carried out in all the 15 accessions. The solutions of various concentrations of standard Withaferin-A produced peaks of different areas in their respective chromatograms (Fig. 4). The R 2 value for the trend line of the chart (Graph 7) confirmed the positive correlation between the concentrations of the standard compound (1 µg µl -1, 2 µg µl -1 and 3 µg µl -1 ) and their respective peak areas (77 mm 2, 162 mm 2 and 240 mm 2 ). The peaks that showed the retention time of the standard compound solutions seconds were identified as Withaferin-A peaks. Those areas were found to be in comparable range of the known concentrations. The maximum area of the peak was produced by ACCN 06, followed by ACCN 12 and ACCN 13; the lowest area was produced by the chromatogram of sample from ACCN 05 (Fig. 5). The quantification of Withaferin-A in all the accessions revealed, three prominent accessions (ACCN 06, ACCN 12 and ACCN 13) and the Withaferin-A content found to be 5.60 mg, 5.40 mg and 5.26 mg respectively (Table 4 & Graph 8) Seed protein profile SDS-PAGE analysis of total seed proteins of 15 accessions led to the detection of fourteen polypeptide bands whose Rf values ranged from 0.5 to 7.0 of these, 7 out of 14 (50% of bands) were monomorphic with all the accessions. This contributed 56.5% of the total peptides i.e., 186 out of 267 polypeptide bands. Remaining seven bands (50%) varied in their appearance. This fraction of 43.5% i.e. 81 polypeptides, out of 267 were the reason for the genetic variability, expressed by the seed protein profile (Fig. 6). The presence and/or absence of various polypeptides obtained in the seed protein profile is presented in Table 5. K. Mohamed Rafi, Ph.D. Thesis, April

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17 Results The cladogram based on seed protein profile was divergent (Fig. 7). The first group consisted of accessions ACCN 10, ACCN 06 and ACCN 05. The second group consisted of accessions ACCN 12 and ACCN 09. The third group consisted of ACCN 04 and ACCN 03. The accessions ACCN 07 and ACCN 02 were distinct genotypes Isozyme variation and its analysis The esterase zymogram (Fig. 8) provided a total of 62 bands in all the fifteen accessions. Out of these, the band with Rf value 2.3 was universal and all others (47 bands of 75.8% of all bands) were specific to accessions ACCN 05, ACCN 06 and ACCN 08. The esterase zymogram produced a total of 7 alleles producing 67 bands. The polymorphism ranged from production of two to seven allelic subunits. The allelic relative frequency ranged from 0.28 to a maximum of The effective number of alleles produced in esterase zymogram was The overall polymorphism was 28.57% for the esterase subunits detected in the study. The Fitch Margoliash cladogram based on esterase profile revealed a total of three clusters viz., first group consisted of accessions ACCN 13 and ACCN 02 alone. The second group consisted of accessions ACCN 08, ACCN 05, ACCN 06, ACCN 15, ACCN 14, ACCN 07 and ACCN 03. The third group consisted of accessions ACCN 12, ACCN 10 and ACCN 09. The second and third group branched out from the same node. The fourth group consisted of ACCN 11, ACCN 04 and ACCN 01. The accessions ACCN 13 was distinct from all other accessions (Figure 9). K. Mohamed Rafi, Ph.D. Thesis, April

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19 Results In the peroxidase profile, a total of 57 bands were obtained. Their Rf values of alleles were restricted to 0.5, 0.8, 1.2, 2.3, 4.0, 4.5. Only the band of allele with Rf value 2.3 was universal (26.3% - 15 out of 57 bands) and all other bands were (73.6% - 42 out of 57 bands) were specific to accessions. The effective number of alleles produced in peroxidase zymogram was 9.5. The Fitch Margoliash cladogram based on peroxidase profile revealed a total of three groups. The first group consisted of accessions ACCN 15, ACCN 09, ACCN 14, ACCN 04, ACCN 13, ACCN 12, ACCN 05 and ACCN 08. The second group consisted of accessions ACCN 10, ACCN 11, ACCN 06. The third group consisted of accessions ACCN 02 and ACCN 03. The accessions ACCN 01 and ACCN 07 were distinct without falling into any group. The genetic diversity and similarity values and distance values are calculated for 15 accessions. The analysis revealed that the Jaccard s similarity index ranged from 1.0 to The overall allele frequencies at 13 loci (7 from esterase and 6 from peroxidase). The mean observed number of alleles per locus ranged from 6 to 15 (mean 9.15 ± 3.44). The proportion of polymorphic loci (P) (0.99 criterion) varied from 0.20 to 1.00 (mean 0.61 ± 0.23) Estimation of genetic diversity by using RAPD For RAPD assay, 15 random primers were used. Of these, a few failed to produce any amplification while certain others amplified bands. Only four primers were found to be scorable (Fig. 12, 13, 14 & 15; Table 8, 9, 10 & 11). The mean number of bands produced per primer ranged from (OPA03) to K. Mohamed Rafi, Ph.D. Thesis, April

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25 Results (OPA16). The polymorphism was scored for all the 15 accessions. The polymorphic loci ranged from 04 to 09 with a mean of The effective number of loci ranged from 11 to 23 with a mean value of 19. Shannon s information index (I) showed a mean value of 19.00±05.47 For the 15 accessions. Genetic identity measures were estimated the values of which ranged from to The mean value was found to be ± with a mean standard error of 3.0. The overall polymorphism percentage was 35.85% for all the loci detected by four primers. The Fitch Margoliash cladogram based on the RAPD profile yielded three groups (Fig. 16). The group 1 consisted of accessions ACCN 15, ACCN 14, ACCN 08, ACCN 03, ACCN 04 and ACCN 09. The group 2 consisted of accessions ACCN 13, ACCN 06, ACCN 12, ACCN 05, ACCN 11, ACCN 07 and ACCN 02. The group 3 consisted of two distinct accessions ACCN 01 and ACCN Grouping analysis of desired accessions The Fitch Margoliash cladogram using pooled data matrix (Table 12 & Fig. 17) revealed four major groups viz., first group consisted of accessions ACCN 06, ACCN 13 and ACCN 10 in one cluster. The second group consisted of accessions ACCN 12, ACCN 04, ACCN 11 and ACCN 07. The third group consisted of accessions ACCN 15, ACCN 05, ACCN 09 and ACCN 02. The fourth group consisted of ACCN 08, ACCN 14, ACCN 03 and ACCN 01. The accessions which yielded the highest concentrations of Withaferin-A were selected as promising chemotypes. The three accessions ACCN 06, ACCN 12 and ACCN 13 were compared for their traits. When comparing the K. Mohamed Rafi, Ph.D. Thesis, April

26 Table 12 : Presence/absence matrix of pooled data (physical leaf traits and Withaferin A content) PHYSICAL LEAF TRAITS Average leaf thickness (µ) Average fresh weight (g) Average dry weight (mg) Average leaf dry matter content (mg/g) Average leaf size (mm 2 ) Average specific leaf area (mg mm -2 ) Withaferin-A (mg) CLASS ACCESSION CODE (ACCN 01-15) class class class class 4 + class class 1 + class class class 4 + class 5 + class class class 3 class class 5 + class class class 3 class 4 + class 5 class class class class class 5 class class 2 + class class class 5 + class class 3 + class class class class class class 4 class class class class 4 class

27 Fig. 17 Cladogram based on the pooled data

28 Results seed protein profile of the accessions, the three prominent Withaferin-A accessions did not form a group. But the ACCN 06 and ACCN 13 were at the same genetic distance from the root. When comparing the peroxidase polymorphism and the Withaferin content, the cladogram showed ACCN 13 and ACCN 12 forming closely related branches where as ACCN 6 was in a separate unrelated cluster. In esterase zymogram, all the three hyper Withaferin accessions showed in different unrelated groups. The Fitch Margoliash cladogram representing the RAPD data from four recordable primers (OPA03, OPA06, OPA12 and OPA16) showed all the three desired accessions ACCN 06, ACCN 12 and ACCN 13 in the same group. Similarly, the Fitch Margoliash cladogram based on Class Based Stratification Matrix of physical leaf traits produced these three accessions on the same group with equal genetic distance from the root. Similarly, the presence and/ or absence data for all the traits were compiled along with class-based stratification matrix which produced a group where ACCN 12 and ACCN 06 were members. ACCN 12 was found to have the same genetic distance and fall in a closely related group In vitro culture studies Micropropagation Establishment of aseptic culture Nodal segments collected from the field grown plants, required 6 minutes of treatment in 0.1% HgC1 2 for getting 90% contaminant free cultures. Further increase in the exposure time in 0.1% HgC1 2 resulted in the death of explants K. Mohamed Rafi, Ph.D. Thesis, April

29 Results (Table- 13). Surface sterilized nodal segments were cultured on MS medium supplemented with 0.5 mg L -1 BAP and 0.1 mg L -1 NAA. Strong and healthier single shoot was initiated from each explant after 20 days (Fig. 18 a, b & c). Further this shoot initiation medium served to screen uncontaminated sprouting Shoot multiplication Explants viz leaf and nodal segments from in vitro raised aseptic cultures were inoculated on to MS medium supplemented with BAP alone or in combination with NAA/IAA for induction of multiple shoots Direct organogenesis Nodal segment culture On lower concentration of BAP ( mg L -1 ) nodal segments did not respond to multiple shoot formation and only a single shoot from pre-existing axillary bud after 25 days (Table 14). BAP at mg L -1, multiple shoots were produced only from the pre-existing axillary buds. On medium supplemented with mg L -1 BAP, multiple shoots were produced from the pre-exiting meristems of axillary bud as well as from the portions of the nodes below the axillary bud. A gradual increase in the number of shoots was observed when MS medium was augmented with auxins viz NAA / IAA used along with BAP. A maximum of 40 shootlets were initiated from nodal segment of the medium supplemented with 2.0 mg L -1 BAP in combination with 1.0 mg L -1 NAA (Fig. 19 a, b & c). IAA (1.0 mg L -1 ) when supplemented along with 2.0 mg L -1 BAP resulted in 3-4 shoots and leaves of shootlets were broader and with long inter node. K. Mohamed Rafi, Ph.D. Thesis, April

30 Table 13. Effect of HgCl 2 (0.1%) on surface sterilization of nodal explants S. No Exposure time (min) Contamination free plants (%) a b c e d Means with the same alphabet are not significantly different according to Dancan s multiplication range test at 5% level. The values are average of four replicates.

31 Table 14 : Effect of BAP and its combination with auxins (NAA / IAA) on multiple shoot formation from nodal segments (after 30 days) Hormone concentration (mg/l) Percentage of shoot response BAP No of shoots per explant Shoot elongation (cm) b 1 a 1.2 b i 1 a 1.6 c l 12 f 3.4 g n 25 j 3.6 h m 23 h 2.9 de k 24 i 2.8 d BAP + NAA Hormone concentration Percentage of shoot response No of shoots per explant Shoot elongation (in cm) g 06 e 3.8 hi hi 12 f 3.0 def j 22 g 3.2 fg m 40 n 4.0 jk j 32 m 3.8 hi g 30 l 3.4 g f 29 k 3.1 efg e 22 g 3.2 fg BAP + IAA Hormone concentration Shoot elongation (in cm) Percentage of shoot response No of shoots per explant c 2 b 2.8 d d 2 b 3.6 h h 3 c 4.2 lm m 4 d 5.2 o l 3 c 4.6 n k 3 c 4.2 lm g 2 b 3.8 hi g 2 b 4.1 km f 2 b 4.3 m e 2 b 4.1 km d 2 b 4.3 m Means with the same alphabet are not significantly different according to Dancan s multiplication range test at 5% level. The values are average of four replicates.

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33 Results Regeneration of plants from leaf explants When leaf explants were cultured on MS medium supplemented with BAP or NAA or IAA alone, all the cultures formed callus irrespective of the plant growth regulator (Table 15). Leaf segments when cultured on medium containing mg L -1 BAP, callus initiation occurred after 15 days and green compact callus was obtained after 40 days (Fig. 20 a & b). White friable callus (Fig. 20 c & d) mass was formed from the explants cultured on medium supplemented NAA (0.5 mg L -1 to 1.0 mg L -1 ). Yellow friable callus was observed on medium supplemented with IAA (0.5 mg L -1 to 1.0 mg L -1 ). Leaf segments inoculated on medium containing 0.5 mg L -1 IAA in combination with 2.0 mg L -1 BAP produced roots from the veins of the explants after 30 days of culture (Fig. 20 e & f). Concurrent callus formation was also noticed along with the roots. On raising the concentration of IAA to 1.0 mg L -1 to 1.5 mg L -1 along with 2.0 mg L -1 BAP profuse shoot bud development was noticed from the veins as well as from the excised portion of the explant (Table 16). A maximum of 60 shoot buds were initiated from leaf segments on the medium containing 2.0 mg L -1 BAP in combination with 1.5 mg L -1 IAA after 40 days of culture (Fig. 21 a, b & c). The frequencies of explants forming shoot buds decreased at 2.0 mg L -1 IAA in combination with 2.0 mg L -1 BAP. Similarly 2.0 mg L -1 BAP in combination with NAA (0.5 mg L -1 to 2.0 mg L -1 ) formed only with thick white roots and no shoot bud formation Anatomical studies on organogenic process in leaf explants Anatomically, the leaf explant showed several structural changes. Many changes have taken place in the photosynthetic cells. Formation of numerous domes like primordial structure were observed after complete disintegration of K. Mohamed Rafi, Ph.D. Thesis, April

34 Table 15 : Effect of growth regulators on callus formation from leaf explants (40 days of culture) Plant growth regulators BAP IAA NAA Concentration (mg/l) Amount of callus* Nature of callus Green, compact callus Green, compact callus Green, nodular compact callus Green, nodular, compact callus Pale yellow friable callus. Yellow friable callus Yellow friable callus with root formation Yellow friable callus with root formation. White friable callus. White friable callus White friable callus Yellow friable callus. * - Absence of callus *+ Fresh weight of callus ~ g *++ Fresh weight of callus ~ g All the observations were made after 40 days from the inoculation. Means with the same alphabet are not significantly different according to Dancan s multiplication range test at 5% level. The values are average of four replicates.

35 Table 16 : Effect of growth regulators on adventitious shoot formation from leaf explants after (40 days of culture) Growth regulators (mg L -1 ) % of response Number of shoot buds formation BAP + NAA Shoot length (cm) d Callus formation g Callus with root formation 92 e Callus with root formation 94 f Callus with root formation BAP + IAA 90 d Callus with root formation c 55 b 0.80 a b 60 c 1.00 b a 30 a 0.75 a Means with the same alphabet are not significantly different according to Dancan s multiplication range test at 5% level. The values are average of four replicates

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37 Results palisade and spongy parenchyma cells. The emergence of primordia took place at the upper side of the explant. The formation of primordia showed several well distinguished stages. They are as follow, Stage 1: Accumulation of nuclear content with dense cytoplasm (Fig. 22 a ). Stage 2: Next stage is the formation of dome like structure with plenty of nuclei containing cells (Fig. 22 b). Stage 3: Final stage was elongation of apical dome structure into shoot primordia (Fig. 22 c) Indirect organogenesis Regeneration of plants from callus cultures Callus induction In the present study, callus induction was also achieved from internodal segments cultured on MS medium supplemented with 0.5 mg L -1 to 3.0 mg L -1 of auxins ( 2,4-D / NAA / IAA) alone or in combination with 0.25 mg L -1 of BAP. The callus raised in cultures showed variation in morphology, color and quantity based on the growth regulators used (Table 17; Fig. 23 a, b, c & d). Callus formed on medium supplemented with NAA was dry and friable in nature but it never gave rise to roots. Higher concentration of IAA induced moist and friable callus from cut end and low concentration resulted root induction. Maximum callus proliferation was obtained in medium enriched 1.0 mg L -1 2,4-D in combination with 0.25 mg L -1 BAP. The callus was friable, greenish white with small globular structures. K. Mohamed Rafi, Ph.D. Thesis, April

38 Table 17 : Effect of growth regulators on callus induction from internodal segments. Growth regulators (mg/l) % of culture showing response Amount of callus* Nature of callus IAA a - Root induction d e - Root induction b + Moist friable callus c d ++ Moist friable callus d e ++ Moist friable callus a + Moist friable callus NAA b c a + Dry friable callus a + Dry friable callus d e ++ Dry friable callus b ++ Dry friable callus d e + Dry friable callus 2,4-D a ++ Friable, white callus b c +++ Friable, white callus b +++ Friable, white callus b ++ Friable, white callus b c ++ Friable, white callus a ++ Friable, white callus BAP + 2,4-D b +++ Friable, greenish white callus d e ++++ Friable, greenish white callus b c +++ Friable, greenish white callus b ++ Friable, greenish white callus a ++ Friable, greenish white callus * - Absence of callus *+ Fresh weight of callus ~ g *++ Fresh weight of callus ~ g *+++ Fresh weight of callus ~ g *++++ Fresh weight of callus > 3.0g All the observations were made after 40 days from the inoculation. Means with the same alphabet are not significantly different according to Dancan s multiplication range test at 5% level. The values are average of four replicates.

39 Results Regeneration of plants from callus Greenish white callus originated in 2,4-D along with BAP containing medium and was sub cultured on to MS medium containing BAP (3.0 mg L -1 to 4.0 mg L -1 ) produced 25 to 30 shoot buds (Fig. 24 a, b & c). Higher or lower concentration of BAP resulted in a corresponding decrease in the number of shoot buds (Table 18). The calluses derived from NAA and IAA had no impact on shoot development in BAP containing medium Anatomical studies on organogenic process in nodal callus: Transverse section of callus showed two well distinguished cell types i.e. the vacuolated cells and nucleated cells (Fig 25 a & b.). The vacuolated cells act as boundary zone and the nucleated cells act as to produce the primordia (Fig. 25c). The nucleated cells aggregated to produce the nodular region with a primordial segment and they were not consistent through the callus. The formations of vascular elements have also been identified in the basal portion of the primordial segment. Once the vascular strands were well organized the callus produced shoots from the primordia (Fig. 25 d) Shoot elongation The shoot buds derived from nodal, leaf segments and callus cultures were transferred to MS medium containing 0.25 mg L -1 BAP in combination with 0.10 to 1.0 mg L -1 GA 3 (Table 19). Low concentration of GA 3 (0.20 mg L -1 ) in combination with BAP promoted the growth and elongation of shoot buds (Fig. 26 a & b) however increase in concentration of GA 3 resulted in abnormalities in leaves and shoots. K. Mohamed Rafi, Ph.D. Thesis, April

40 Table 18 : Effect of BAP on regeneration of shoot buds from callus cultures. Growth Regulators (mg L -1 ) % of Response BAP Number of shoot buds per explant Shoot length (cm) a 10 a 0.80 a b b 28 c 0.90 b c 30 d 0.80 a b d 25 b 0.70 a All the observations were made 40 days after the subculture of the callus. Means with the same alphabet are not significantly different according to Dancan s multiplication range test at 5% level. The values are average of four replicates.

41 Table 19 : Effect of GA 3 in combination with BAP for shoot elongation Growth regulators (mg L -1 ) % of cultures showing response Average Shoot length (cm) BAP + GA f 4.2 c d h 6.4 g g 5.8 f e 5.2 e d 4.6 d c 4.4 d c 4.1 c b 3.6 b a 3.5 b b 3.6 b Means with the same alphabet are not significantly different according to Dancan s multiplication range test at 5% level. The values are average of four replicates.

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43 Results Rooting and transplantation of microshoots The in vitro shoots derived through various explants were transferred to MS medium containing mg/l NAA / IBA for rooting. When MS medium supplemented with NAA (0.5 mg L -1 to 2.0 mg L -1 ), only callus formation was noticed from the base of the in vitro raised shoots. High frequency of rooting was noticed in MS medium supplemented with 1.0 mg L -1 IBA (Table 20). About 16 roots were formed after 20 days of culture (Fig. 27 a). Lower concentration (0.5 mg L -1 ) of IBA had no impact on root formation and higher concentration (1.75 mg L -1 to 2.0 mg L -1 ) resulted in basal callusing with short roots (Fig. 27 b). Reducing MS concentration to ½ of its strength, increased rooting (18 roots) response compared to full strength concentration (Table 21; Fig. 27 c). Plantlets with 3-4 leaves and roots were transferred to soil using techniques earlier described in materials and methods. After one month, the survival rate was assessed and 90% of the plants were found to grow normally (Fig 28 a & b) In vitro flowering and fruit development The nodal segments collected from in vitro raised seedlings of W. somnifera accessions were used for this study. GA 3 treated seeds required 10 min surface sterilization treatment in 0.1% HgCl 2 for getting 90% germination. Nodal segments excised from 25 days old seedling were cultured in MS medium containing 0.5 mg L -1 to 5.0 mg L -1 BAP / Kn alone or in combination with 0.1 mg L -1 to 1.0 mg L -1 IAA. In the present study Kn alone or in combination with IAA induced in vitro flowering and fruit development (Table 22). Higher frequency of flowering, fruiting and seed setting have been observed on the medium containing 2.0 mg L -1 Kn in combination with 0.2 mg L -1 IAA after two K. Mohamed Rafi, Ph.D. Thesis, April

44 Growth regulators Table 20 : Effect of auxins on root induction % of culture showing response Average number of roots NAA Average root length (cm) Basal callus callus callus callus callus callus callus callus IBA c 12 c 3.4 c d 16 d 3.6 c b 10 b 2.1 b a 4 a 1.5 a Rooting with callus Callus Means with the same alphabet are not significantly different according to Dancan s multiplication range test at 5% level. The values are average of four replicates.

45 Table 21 : Influence of nutrients level with 1.00 mg L -1 IBA on in vitro rooting after 20 days of culture MS medium with IBA ( mg L -1 ) 1 / 4 strength MS medium / 2 strength MS medium / 4 strength MS medium MS full strength % of culture showing response Average no of roots Average root length (cm) 89 a 12 a 3.2 a 95 c 18 c 3.5 b 92 b 16 b 3.4 a b 90 b 16 b 3.6 b Means with the same alphabet are not significantly different according to Dancan s multiplication range test at 5% level. The values are average of four replicates.

46 Table 22 : Effect of growth regulators on in vitro flower formation and fruit development Growth regulators (mg L -1 ) % of culture flowering response Average no of flower No fruits per plant* No of seeds per fruit % of viable seeds per fruit Kn b 18 c 9 b 16 b 92 b g 22 e 14 d 26 e 96 d a 14 a 6 a 12 a 90 a Kn + IAA e 21 d 16 e 22 c 94 c h 26 g 18 f 28 f 94 c f 23 f 16 e 24 d 93 b c d 18 c 14 d 26 e 92 b c 16 b 13 c 24 d 90 a All the observations were made 25 days after the subculture. Means with the same alphabet are not significantly different according to Dancan s multiplication range test at 5% level. The values are average of four replicates.

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48 Results successive subculture (40 days). The flowers produced in vitro appeared morphologically normal, greenish yellow in color, born in axillary clusters and they were bisexual. Fruit formation was observed 6 weeks after flowering. The fruits were berries, small globose, smooth, orange red when ripe and enclosed in the inflated membranous calyx (Fig. 29). Seeds were yellowish in color and reniform shape. K. Mohamed Rafi, Ph.D. Thesis, April

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