IV. RESULTS AND DISCUSSION

Transcription

1 IV. RESULTS A DISCUSSION 4.1. DIRECT ORGANOGENESIS Direct organogenesis has been well established in cucurbit crops. Many explants have been employed successfully in several cucurbit species. Shoot tips (Barnes, 1979; Locy and Wehner, 1982; Lee and Thomson, 1985; Compton et al., 2001; Shiragave and Chavan, 2001), axillary buds (Handley and Chambliss, 1979; Fortunato and Mancini, 1985; Lee et al., 1985; Compton et al., 2001) and cotyledons (Trulson and Shahin, 1986; Gambley and Dodd, 1990; Compton and Gray, 1993; Selvaraj et al., 2007) were most commonly used explants. However no such reports are found in West Indian Gherkin (Cucumis anguria L.). The current study utilized two types of explants (cotyledon and nodes from young shoots) Effect of explant and plant growth regulator The cotyledon and nodal explants were inoculated in the shoot induction medium (MS) containing cytokinins, Kinetin (Kn), BAP and Naphathalene acetic acid (NAA) alone or in combinations. During the first week of culture all the explants get bulged and started producing buds in the second week of culture. The cotyledon explants produced buds and subsequently shoots in the second week whereas the nodal explants differentiated the shoots in the third week of culture. The MS medium containing either BAP or Kinetin alone produced shoots in low concentrations from both explants. Increasing the concentration of BAP from 0.89µM to 4.44 µm and kinetin 0.93 to 4.65 µm increased the percentage of explant response and number of shoots in cotyledon as well as nodal explants. (Table-1and 2). However, increasing the concentration of both hormones beyond this level did not increase the number of shoots but decrease in response was noticed. To study the combination effects of hormones NAA was combined with these 47

2 two cytokinins and the combination of both cytokinins with or without NAA was assessed. NAA either with BAP or Kn produced higher number of shoots and higher percentage of explant response. In all the experiments increasing the NAA beyond 1.62 µm produced callus and stopped the shoot production. The maximum number of shoots (29.4) and the highest percentage of response (91.2) were noted in BAP 4.44 µm with NAA 1.07 µm combinations from cotyledon explants. The same hormonal combination yielded 24.8 shoots and 90.6 percentage of response in nodal explants. Application of other auxins i.e., IBA and IAA did not produce any significant results. Plant growth regulator has played an important role in micropropagation of the cucurbit species. High cytokinin and low auxin medium has been routinely used for micropropagation of many cucurbit species. For the induction of morphogenesis in cucumber, exogenous auxin either alone or in combination is considered essential at the initial culture. Handley and Chambliss (1979), Rajasekaran et al. (1983) and Lou and Kako (1995) reported that auxin free media containing Kn stimulated the production of complete cucumber plants with minimal callus production in axillary bud explants but they suggested that shoot growth could be obtained by a balance of both auxins and cytokinins. In Cucumis sativus L. Vasudevan et al. (2001) reported that BA and NAA is required to produce multiple shoots from shoot tip explants. In the present study BAP with lower concentration of auxin produced higher number of shoots. In contrary to this Gambley and Dodd (1990) obtained shoots from Cucumis sativus L. cotyledons without auxin and callus growth was noticed when auxins were added to the medium. Zhang et al. (2001) also used only Zeatin for the production of shoots from cotyledon explants. 48

3 Among the two explants tested, cotyledon explants showed slightly higher response than the nodal explants. In addition to this the cotyledon explants produced the shoots within two weeks however the nodal explants has taken three weeks. It is suggested that the area adjacent to the shoot apex has been considered as most active site for regeneration (Gaba et al., 1999). Cotyledon based shoot production was successful in other cucurbit crops like Cucumis sativus L. (Gambley and Dodd, 1990; Colijin- Hooymans et al., 1994; Selvaraj, 2002) Cucumis melo L. (Melon) (Leshem, 1989; Gonsalves et al., 1994; Gaba et al., 1999) and water melon (Compton and Gray, 1993). Hence the cotyledon explants have been used as candidate for the genetic transformation studies of Cucumis anguria L Effect of L-glutamine The effect of L-glutamine at different concentrations ( µm) was tested with BAP (4.44 µm), and NAA (1.07 µm) in shoot induction medium to test its role in multiple shoot production from cotyledon and nodal explants. Addition of L-glutamine increased the multiple shoot production in both explants (Table-3). Increasing the concentration of L-glutamine increased the culture response and highest number of shoots (53.4) and culture response were noted at 137µM concentration. The cotyledon explants responded most favorably than the nodal explants. Hence, a concentration of 137 µm was used to enhance multiple shoot induction during shoot induction and subculture experiments. The enhancement of growth rate and shoot induction by L-glutamine could be explained on the basis that L-glutamine provides a readily available source of nitrogen, the implication being that the formation of necessary carbon skeleton or the reduction of nitrate to ammonia is a limiting factor in the cells ( Gamborg, 1970). Locy and Wehner (1982) who 49

4 reported that asparagines and L-glutamine stimulated cucumber shoot tip growth in culture better than the MS nitrogen sources (Ammonium and nitrate). Selvaraj (2002) reported that adding L-glutamine (137 µm) improved the shoot production in the shoot tip culture of Cucumis sativus L. Vasudevan et al. (2004) reported in cucumber that supply of L-glutamine produced highest number of shoots. The present study correlated with earlier statements in cucumber Effect of subculture Subcultures of shoots were carried out in MS medium containing BAP (4.44 µm), NAA (1.07 µm) and L-glutamine (137 µm) at 25 days intervals for five times. Slight increase in the number of shoots per explant was noticed in the first two subcultures from both explant sources. After two subcultures there was no further improvement in the number of shoots. After two subcultures the maximum number of shoots recorded from cotyledon explant was 38.4 (Table-1) and 33.8 from nodal explants respectively (Table-2). The effect of subculture in producing multiple shoots was advocated for many cucurbit species; Cade et al. (1987) for cucumber; Barnes (1979) for watermelon; Singh et al. (1996) for melon cultivars. Repeated transfer of explants in media containing BA resulted in rejuvenation of explant tissues (Shekhawat et al., 1993). This promoted activation and conditioning of meristems (Boulay, 1985). In present investigation, first two subcultures of shoots were found fruitful and further subcultures had not increased number of shoots. Similar observation was recorded in Cucumis sativus L. (Selvaraj, 2002). 50

5 4.2. IIRECT ORGANOGENESIS In vitro plant regeneration of cucumber species is possible using different culture techniques (Malepszy, 1988). In cucumber, organogenesis and somatic embryogenesis is achieved by using different explants; cotyledons (Jia et al., 1986; Trulson and Shahin, 1986), leaves (Oryzek and Malepszy, 1985; Seo et al., 2000, Punja et al., 1990) and hypocotyls (Nishibayashi et al., 1996). Till date no report is available in the organogenesis of West Indian Gherkin. Three different explants, cotyledon (2 day old), hypocotyl (15 day old) and leaf (20-day old) from in vitro seedlings of West Indian Gherkin have been used to regenerate shoots via indirect organogenesis i.e., regeneration through callus Callus induction from cotyledon explants Callusing ability of cotyledon explants derived from 2days old in vitro seedlings was evaluated on MS medium supplemented with individual treatments of auxins (2,4-D and NAA) or their combination with BAP. Callusing occurred at the cut end of the proximal half of the explant after 7 days of culture initiation. The orientation of proximal half of the cotyledon played an important role in callus induction. The cotyledon explant placed with their adaxial surface touching the medium produced callus all over the cut ends. However the cotyledon explants placed vertically produced lower amount of callus. The distal half of the cotyledon did not respond to callusing in any growth regulator treatment (data not shown). In individual auxin treatment, 2, 4-D at 2.26 M evoked maximum callus induction response (86.2%) and produced yellowish green compact callus (Table-4). NAA at 2.67 M also responded in callus induction (80.4 %) but the callus was yellowish brown compact and non-organogenic. Organogenic, green compact callus was, produced (88.2%) from cotyledon explants when cultured in NAA (2.67 M) and BAP (4.44 M) combination. Further two 51

6 subsequent subcultures in the same medium changed them in to green nodular calli. The green nodular calli have the regeneration potentiality and was selected, sub-cultured for shoot regeneration. Although the combinations of 2, 4-D/BAP responded to callus induction, the calli produced in these treatments were yellow friable, brownish green friable or brownish green compact and non-organogenic. The other auxin like IAA and IBA did not respond to the production and multiplication of callus culture. In earlier studies it is observed that the cotyledonary explant was the most suitable explant for callus production (Ziv, 1992). The experiments with Cucumis anguria L. also support this statement and cotyledon is considered as one of the best explant materials for callus induction. Our observations revealed that callus induction from the cut ends of proximal half of the explants was achieved in NAA /BAP combination. Selvaraj et al. (2007) also reported that the NAA/BAP combination was useful to produce green nodular calli from the proximal end of cucumber cotyledon. Earlier observations from the leaf and petiole explants from cucumber proved that NAA/BAP combination was fruitful for callus induction. Our experiments showed that that the callus induction was occurred from the proximal end and not from the distal end. Gambley and Dodd (1990) suggested that cytokinin induced activation of totipotent cells were present only in the proximal half of the cotyledons of cucumber Callus induction from Leaf explant The leaf bits were cut into pieces and inoculated in MS medium either with auxin (NAA /2, 4-D) alone or in combination with BAP. After two weeks, the leaf explants bulged and develop callusing from cut end and around the wounds of the explant. Brown or yellow compact calli were formed from the explants cultured in presence of any one of the auxins. Maximum 52

7 callusing frequency (92.6%) was observed from the experiments with NAA (2.67 M) and BAP (4.44 M) combination (Table-4). In this combination the callus was green and compact in the initial stage and turned in to nodular after one or two subcultures. However the 2, 4-D and BAP combination produced brown or yellow compact callus. Seo et al. (2000) reported that NAA and BAP combinations produced the callus from the leaf explants of cucumber. Selvaraj et al. (2002) also reported that callus formation was successful in the same combination in cucumber. In accordance with these BAP and NAA combination favored callus induction and proliferation. In the present study, in contrary to this Fadia and Mehta (1976), Coutts and Wood (1975, 1977) reported callus induction from leaf explants of cucumber when the medium was amended with coconut milk, 2, 4-D and kinetin or BAP Callus induction from hypocotyl explants Callusing efficiency of hypocotyl explants was tested in MS medium containing auxin alone and in combination with BAP. After two weeks of culture, callus was initiated from cut ends of the hypocotyl in MS medium containing either auxin alone or in combination with cytokinin. The callus induction frequency was low in both 2, 4-D or NAA. The combination of 2, 4-D (1.13 M) with BAP (4.44 M) produced nodular green compact calli with callusing response of 86.2%. NAA and BAP combination produced yellowish compact callus with 60 % response (Table-4). Ziv and Gadasi (1986) also obtained friable and compact nodular callus from hypocotyl explants of cucumber in the presence of 2, 4-D and BAP. Selvaraj et al. (2006a) also achieved callus induction in 2, 4-D and BAP medium from the hypocotyl explants of Cucumis sataivus L. In the present investigation, organogenic callus was induced by same growth regulator 53

8 combination. In contrast to this Rajasekaran et al. (1983) induced green compact callus from hypocotyl explants in MS medium containing NAA and BA combinations Regeneration of shoots from callus cultures The nodular green compact calli derived from hypocotyl (2, 4-D at 1.13 M with BAP 4.44 M); cotyledon and leaf explants (NAA at 2.67 M with BAP 4.44 M) twice subcultured in the same callus induction medium with 20 days of intervals then transferred to the shoot regeneration medium. After two subcultures in the same regeneration medium with two weeks interval, the green compact nodular callus produced small shoot buds which later differentiated into shoots. The other types of calli (friable, yellow or brown) failed to regenerate shoots irrespective of the medium used. The calluses cultured in dark did not respond to shoot regeneration attempts. The shoot regeneration medium (MS medium + Zeatin (0.912 M)) either with NAA (1.34 M) and BAP ( M) combination or with 2, 4-D (1.13 M) and BAP ( M) were tested for shoot regeneration from cotyledon, hypocotyl and leaf derived calluses. The other concentrations of auxins and cytokinins either alone or in combinations were failed to regenerate shoots. The Zeatin (0.912 M) and L-glutamine (137 M) were added to all the regenerations medium because in the absence of Zeatin, shoots were not regenerated and supplement of L- glutamine improved the shoot regeneration from the callus cultures. However increasing the Zeatin concentration beyond (0.912 M) reduced the soot formation. Hence the Zeatin (0.912 M), NAA (1.34 M) and 2, 4- D (1.13 M) concentrations were kept constant and varying concentrations of BAP ( M) were tested. 54

9 Among the combinations tried, the NAA (1.34 M) and BAP (8.88 M) combination were found better for leaf and cotyledon derived callus and 2, 4-D (1.13 M) with BAP (8.88 M) combination for hypocotyl derived callus. Maximum culture response was (76.8%) found in hypocotyl derived callus followed by the cotyledon derived callus (75.6%) and leaf explant callus (72.4%) (Table-5). And also the maximum number of shoots (38.6) was regenerated from hypocotyl derived callus followed by the cotyledon derived callus (36.2) and leaf derived callus (32.6) (Table-5). The morphological nature of shoots derived from all the above said combinations were alike and no differences were observed. Zeatin a potential cytokinin has been used in tissue culture for the induction of shoots from various plant species. Punja et al. (1990) and Selvaraj et al. (2006a) used Zeatin to induce adventitious shoot regeneration from cucumber explants. Zeatin is very effective in inducing cucumber shoot regeneration only at a very low concentration. At a higher concentration shoot induction frequency was reduced (Selvaraj et al., 2007). Similar kinds of observations was recorded in our experiments. Wehner and Locy (1981) observed low yield of shoots from the cotyledon explants in BAP and NAA combination. Punja et al. (1990) recorded 38% regeneration frequency in cotyledon and highest frequency (75%) in leaf explant. But our experiments revealed that the hypocotyl explants recorded the highest number of shoots followed with cotyledon and leaf. Similar observations were reported in cucumber (Selvaraj, 2002). Nishibayashi et al. (1996) and Raharjo et al. (1996) achieved shoot formation in NAA/BA and 2, 4-D/BA combinations. In agreement with these reports the present investigation also showed the shoot regeneration in the same combinations. Our study also reported that low auxin and high concentration of cytokinin is required for the shoot regeneration from callus. 55

10 The differential response of different genotypes and explants to varied auxins and cytokinins indicate a great deal of variations in the percentage of shoots produced. This phenomenon was already reported by Wehner and Locy (1981) and Kim et al. (1998) for cucumber. Addition of L-glutamine to the regeneration medium improved the shoot regeneration efficiency. Similar observations were reported in the shoot regeneration in cucumber (Vasudevan et al., 2004; Selvaraj et al., 2007). Raharjo and Punja (1992), Chee (1990a) and Punja et al. (1990) reported that the pre-culture of callus for 2-3 weeks in dark is required for shoot regeneration. However our study indicates that the exposure to light did not affect the regeneration efficiency. Bergervoet et al. (1989) and Trulson and Shahin (1986) recommended repeated subcultures for higher shoot regeneration. But our study indicates that two subcultures of callus in shoot regeneration medium were sufficient for shoot regeneration. The present study proved that the Cucumis anguria L. can be regenerated via indirect regeneration and hypocotyl and cotyledons are being the most favourable explant SHOOT ELONGATION The shoots (1.0 cm length) regenerated via direct and indirect organogenesis from different explants like cotyledon, nodal, hypocotyl and leaf explants when sub-cultured continuously its shoot proliferation medium failed to elongate. Hence, they were transferred to shoot elongation medium (SEM) containing either GA 3 alone or in combination with BAP (4.44 M) (Table-6). The shoots grown in the medium with GA 3 alone were shown to be slender, weak and with narrow leaves, (data not shown). On the other hand, MS medium with BAP and GA 3 combination produced healthy and normal shoots. Hence in the present study different concentrations of GA 3 56

11 ( M) were tested against 4.44 M BAP. Maximum shoot elongation and percentage of response were recorded in GA 3 (1.44 M) and BAP (4.44 M) concentration from all explant derived shoots. The maximum shoot length 6.6 to 8.2cm was achieved in this concentration (Table-6). At higher GA 3 concentrations (>1.44 M), basal callusing occurred alone with inhibition of shoot elongation. Fortunato and Mancini (1985) achieved shoot elongation and rooting in cucumber by transferring the shoots to a medium with IAA (1.0 ppm), 2-ip (1.0 ppm) and GA3 (0.025 ppm). Whereas, Barnes (1979) induced shoot elongation and rooting in medium containing IAA in watermelon. However the present study we have used different media combinations for shoot elongation and rooting. Compton et al. (2001) reported inhibition of shoot elongation in Cucumis hystrix at higher NAA (> 0.5 mg/l) concentration. Stipp et al. (2001) induced shoot elongation in medium containing only BA (0.2 mg/l). Tabei et al. (1991) observed the influence of auxin type in the shoot elongation of melon. In their study, NAA did not promote shoot elongation while 2,4-D promoted shoot elongation in fewer shoots. But in the present study maximum shoot elongation was achieved in MS medium fortified with BAP and GA 3 combination ROOTING A ACCLIMATIZATION Shoots regenerated via callus from cotyledon, hypocotyl and leaf explants as well as shoots regenerated from cotyledon explants by direct organogenesis failed to root when transferred to MS medium lacking growth regulators. Addition of IBA was found useful in inducing root development from elongated shoots. Though other auxins like NAA and IAA also favoured rooting, the efficiency of IBA was superior (data not shown). Hence different concentrations of IBA (0.98 to 3.94 M) were employed to induce roots. Both the percentage of response and maximum 57

12 number of roots (25.2 roots/shoot) were observed with 2.46 M IBA concentration (Table-7). In general, the response of rooting of shoots derived from callus culture was similar to that of rooting of shoots derived from direct organogenesis. Although the promotive effective of auxins in eliciting rooting response has been well established (D Silva and D Souza, 1992), their type and level in the nutrient medium were found to vary from tissue to tissue and species to species (Rao and Padmaja, 1996). NAA and IBA have been commonly used for in vitro rooting of cucumber plants. Barnes (1979) achieved rooting in watermelon shoots by IAA amended MS medium. IBA was used to induce rooting of shoots in cucumber (Misra and Bhatnagar, 1995), melon (Singh et al., 1996) and Momordica dioica (Shiragave and Chavan, 2001). Addition of NAA to rooting medium improved rooting of Cucumis hystrix micro shoots (Compton et al., 2001). The present study also confirmed the role of auxin in inducing rooting of regenerated shoots. Well rooted plants at various stages of height obtained from rooting medium were transferred to small pots filled with sterilized garden soil, sand and vermiculite (1:1:1 v/v/v) for hardening (Fig-2and 3). These plants were maintained under 80% relative humidity in the growth chamber at 25 ± 2 o C. The individual pots with single plant are partially covered with the polythene bag to maintain high humidity. When the plants had shown signs of new leaf growth, the polythene covers were removed. 45% of plants survived during the hardening process (data no shown) and these plants were established successfully in the field after four weeks of acclimatization. Our results showed that the efficiency of hardening and acclimatization was about 45%. Similar kinds of results were obtained in acclimatization by Compton et al. (1993) for watermelon and Compton et al. (2001) for C. hystri. 58

13 The present investigation clearly reveals that Cucumis anguria L. can be regenerated through direct and indirect organogenesis. The results obtained in our experiments are comparable to other species of cucumber. Among the different explants tested, cotyledon is the best explant source for regeneration of plants and can be utilized for genetic transformations studies because of their easy handling and culture practice. 59

14 Table-1 Effect of BAP, Kn and NAA on adventitious shoot production from two days old cotyledon derived from mature seeds of Cucumis anguria L. on MS medium. Growth regulator (µm) Kn Kn + NAA BAP BAP + NAA BAP + Kn BAP+Kn+NAA Percentage of explants responded 48.2 u 54.6 sa 60.4 n 68.0 j 71.0 ga 61.6 m 73.8 e 79.2 d 52.0 sc 53.2 sb 57.4 r 64.8 l 71.0 g 80.6 c 70.2 i 61.0 ma 86.2 b 91.2 a 70.8 h 72.4 f 67.6 k 60.2 na 51.4 sd 48.2 qa 40.0 w 55.8 s 64.0 nb 51.0 t No. of shoots after 15 days of initial culture (per explant) 4.4 o 6.2 na 9.0 l 10.8 k 13.6 i 10.2 kb 14.0 h 19.8 d 10.0 kc 6.4 n 8.2 m 12.4 ja 16.0 g 22.8 c 19.0 db 11.2 k 23.6 b 29.4 a 18.0 ca 19.4 da 17.2 f 14.0 ha 12.6 j 10.2 ka 9.0 la jc e 12.4 jb No. of shoots after first sub culture (per explant) 5.2 p 7.8 o 11.2 ma 13.4 f 18.0 h 13.2 fe 17.2 i 21.4 e 12.2 l 7.6 oa 9.4 na 14.8 k 20.2 ga 26.0 ba 23.0 c 14.2 kb 26.2 b 34.2 a 20.0 gb 21.2 ea 20.4 g 16.2 j 14.0 ka 11.6 m 9.8 n 14.8 k 22.0 d 13.2 fa No. of shoots after second sub culture (per explant) 6.0 r 9.2 pa 13.4 m 15.6 k 21.2 f 14.2 lc 18.2 ha 23.6 da 14.4 lb 8.6 q 10.8 p 17.4 i 24.8 d 30.2 b 27.2 c 17.0 j 29.0 ba 38.4 a 22.2 e 23.4 da 21.8 g 18.4 h 16.4 ja 12.0 n 11.2 o 17.2 ia 26.2 ca 14.8 la Each value represents the treatment means of 3 replicates with 50 explants Values with the same letter within columns are not significantly different according to Duncan s Multiple Range Test (DMRT) at 5 % level

15 Table-2 Effect of BAP, Kn and NAA on adventitious shoot production from nodal explants derived from 15 day old in vitro seedlings of Cucumis anguria L. on MS Medium. Growth regulator (µm) Kn Kn + NAA BAP BAP + NAA BAP + Kn BAP+Kn+NAA Percentage of explants responded 47.2 r 53.0 o 57.6 m 67.4 i 70.8 f 60.4 l 73.2 e 77.6 d 50.0 pc 46.8 s 52.4 p 62.4 ja 69.0 ga 80.2 c 70.4 fb 60.0 la 85.0 b 90.6 a 69.6 g 70.6 fa 68.2 h 61.4 k 50.6 pb 49.4 q 41.2 t 56.0 n 63.2 j 50.8 pa No. of shoots after 3 weeks of initial culture (per explant) 4.0 o 5.4 na 7.4 l 7.2 la 11.4 ib 9.8 j 11.6 ia 12.0 h 8.2 ka 5.6 n 6.8 m 9.4 jb 15.2 fa 21.0 b 18.6 d 9.8 ja 20.2 c 24.8 a 15.4 f 15.2 fb 16.4 e 11.2 ic 8.4 k 6.8 ma 6.2 mb 9.2 jc 13.8 g 11.6 i No. of shoots after first sub culture (per explant) 5.0 q 6.8 pa 9.6 na 10.6 m 14.2 ka 10.0 ma 14.8 k 16.2 i 11.6 la 4.6 r 6.8 p 11.2 ld 17.8 h 23.2 d 21.4 e 11.6 lc 24.0 c 30.2 a 17.4 ha 19.0 f 18.2 g 15.0 j 11.8 l 9.6 n 7.4 o 11.6 lb 27.2 b 11.0 le No. of shoots after second sub culture (per explant) 5.8 r 8.4 qa 10.8 o 13.2 ka 18.4 g 11.8 n 16.8 i 19.0 fb 12.8 m 5.8 ra 8.4 q 14.2 k 19.6 fa 26.0 ba 23.4 c 15.2 j 26.2 b 33.8 a 19.6 f 20.2 e 17.6 h 14.2 ja 13.8 l 9.6 p 9.0 pa 14.0 jb 21.6 d 11.0 na Each value represents the treatment means of 3 replicates with 50 explants.

16 Values with the same letter within columns are not significantly different according to Duncan s Multiple Range Test (DMRT) at 5 % level. Table-3 Effect of L- glutamine in MS medium supplemented with BAP (4.44µM) and NAA (1.07 µm) on shoot multiplication from cotyledon, Nodal explants of Cucumis anguria L. on MS medium. L- glutamine (µm) Explant responded % No. of shoots/explant Cotyledon* Nodal* Cotyledon* Nodal* f 58.4 e 70.4 d 88.2 b 96.8 a 85.4 c 54.2 f 60.4 e 68.2 d 82.4 b 92.6 a 80.2 c 38.0 f 39.4 e 42.8 d 47.0 b 53.4 a 46.6 c 33.0 e 34.6 d 36.4 c 39.0 b 44.0 a 36.2 ca *All the datas collected after two subcultures Each value represents the treatment means of five replicates Values with the same letter within columns are not significantly different according to Duncan s Multiple Range Test (DMRT) at 5 % level

17 Table-4 Comparative effect of various concentrations of NAA and 2, 4-D individually and in combinations with BAP on callus induction from(2 day old) Cotyledon, Leaf (20 day old) and Hypocotyl explant of 15 day old in vitro seedlings of Cucumis anguria L. PGR ( M) Callus induction frequency (%) Cotyledon Nature of callus Leaf Nature of callus Hypocotyl Nature of callus NAA , 4-D NAA+BAP j 80.4 b 76.6 da 68.2 ga 70.6 f 76.2 d 74.4 e 66.2 h 76.6 d 88.2 a 78.2 c 64.2 i YBC YBC YGC GF BGC YBC YGC GF YBC GC GF BGC 56.4 k 78.0 c 72.4 da 66.2 h 64.4 ia 70.8 e 56.2 ka 48.4 l 86.2 ba 92.6 a 86.4 b 70.0 e YBC BGC BC BGF BGC YBC YGC YGF YBC GC BGC YGC 52.4 k 70.2 da 66.4 f 50.2 l 62.4 g 68.2 e 60.6 h 54.2 ia 54.8 i 60.0 j 52.2 ka 46.4 m YF WC GBC BGF BC YGC BGC GF YF YC BGC YGC 2,4-D+BAP g 78.2 ba 66.2 ha 58.6 k BGC BGC BGF YBF 66.8 g 72.6 f 64.4 i 58.2 j YBC YGF BGC BGC 80.4 b 86.2 a 76.0 c 70.2 d YBC GC BGC BC B-Brown, Com-Compact, G-Green, Y-Yellow Each value represents the treatment means of five replicates Values with the same letter within columns are not significantly different according to Duncan s Multiple Range Test (DMRT) at 5 % level

18 Table-5 Effect of different concentrations of BAP with NAA (1.34 M) and 2, 4-D (1.13 M), Zeatin (0.912 M), TDZ (0.908 M) on shoot regeneration from cotyledon, hypocotyl and leaf derived calluses of Cucumis anguria L. on MS medium fortified with L- glutamine (137.00). PGR ( M) Culture showing response (%) Number of Shoots / Callus explant NAA BAP Cotyledon Hypocotyl Leaf Cotyledon Hypocotyl Leaf , 4-D BAP e 68.2 ba 75.6 a 64.4 c 54.2 ga 56.2 f 64.2 ca 68.4 b 60.6 d 54.4 g 60.4 f 68.4 ca 70.6 b 62.2 e 58.4 ga 58.6 g 66.4 d 76.8 a 68.6 c 56.4 h 54.2 g 70.4 b 72.4 a 68.2 c 52.0 ha 56.4 f 62.2 d 70.2 ba 60.2 e 52.4 h 20.2 g 28.4 c 36.2 a 26.6 d 18.2 h 16.2 j 24.2 e 30.6 b 22.4 f 18.0 i 16.2 i 22.4 e 30.2 b 20.6 f 19.4 g 18.6 h 26.2 c 38.6 a 25.4 d 20.2 fa 18.2 g 26.4 d 32.6 a 22.2 e 20.6 f 20.2 fa 28.6 c 30.4 b 26.2 da 16.4 h Each value represents the treatment means of five replicates Values with the same letter within columns are not significantly different according to Duncan s Multiple Range Test (DMRT) at 5 % level

19 Table-6 Effect of GA 3 along with BAP (4.44 M) on elongation of shoots regenerated from 2 day old cotyledon and nodal, hypocotyl 15 day old and 20 day old leaf explants of in vitro seedlings of Cucumis anguria L. on MS Medium. PGR ( M/l) Percentage of response Mean shoot length (cm) GA 3 SRC SRN C L H SRC SRN C L H h 78.6 f 87.2 e 95.4 b 97.2 a 92.6 c 88.4 d 74.2 g 70.2 h 76.8 f 88.2 d 94.6 b 95.4 a 93.2 c 80.8 e 72.2 g 80.4 f 84.2 e 88.8 c 96.4 b 98.2 a 86.4 d 78.2 g 66.6 h 82.2 f 86.6 d 90.4 c 94.8 b 97.6 a 84.8 e 68.2 g 56.4 h 74.2 f 80.6 e 86.4 d 94.2 b 95.4 a 88.6 c 66.4 g 58.8 h 2.8 g 3.2 f 4.8 d 5.6 ca 7.2 a 6.8 b 5.6 c 4.2 e 2.8 h 3.2 g 4.8 d 6.2 b 6.6 a 6.0 ba 5.2 c 4.4 f 3.6 h 4.8 g 5.4 f 6.0 c 8.2 a 7.2 b 6.4 d 5.8 e 2.6 g 3.6 f 4.6e 5.8 c 7.0 a 6.6 b 5.2 da 5.4 d 4.2 db 5.8 c 4.6 da 5.4 cb 6.8 a 6.2 b 5.6 ca 4.8 d SRC- Shoots directly regenerated from cotyledon explants SRN- Shoots directly regenerated from nodal explants Cot Cotyledon, L Leaf, H - Hypocotyl Each value represents the treatment means of five replicates Values with the same letter within columns are not significantly different according to Duncan s Multiple Range Test (DMRT) at 5 % level

20 Table - 7 Effect of IBA on induction of roots from the regenerated shoots cultured on MS medium containing BAP (4.44 M). Plant growth regulator s (µm) Percentage of response Number of roots/shoot Root length (cm) IBA SRC SRN C L H SRC SRN C L H SRC SRN C L H g 44.2 g 48.2 f 44.2 g 40.2 g 10.2 h 8.4 ea 12.6 f 12.2 f 8.4 da 10.2 f 7.2da 8.2 f 8.4 d 7.2 e f 86.6 b 90.2 a 84.4 c 80.2 d 74.4 e 64.4 e 78.2 c 86.6 a 82.2 b 68.0 d 46.4 f 60.8 e 76.4 c 88.6 a 82.0 b 76.2ca 68.4 d 58.4 e 76.0 c 84.2 a 78.6 b 66.4 d 50.2 f 52.8 e 74.6 c 80.2 a 76.4 b 58.0 d 42.2 f 14.6 e 18.4 c 25.2 a 20.4 b 16.8 d 12.2 f 10.2 da 14.6 b 18.4 a 12.2 c 10.4 d 8.6 e 16.8 d 18.2 b 20.6 a 17.4 c 14.6 e 10.2 g 14.6 d 16.4 b 18.2 a 15.4 c 13.0 e 11.2 g 10.2 e 13.8 b 15.8 a 13.2ba 10.0ca 8.4d 12.4 e 14.2 c 17.4 a 14.6 b 12.8 d 10.2 f 10.4ca 12.8 b 14.2 a 12.2 ba 10.8 c 7.6 d 12.4 d 16.2 b 16.6 a 16.0ba 13.4 c 10.2 e 10.6 c 12.8 b 14.4 a 12.2ba 10.2ca 8.2da 8.4 d 10.0ba 12.2 a 10.6 b 9.4 c 8.0 da SRC- Shoots directly regenerated from cotyledon explants SRN- Shoots directly regenerated from nodal explants C Cotyledon, L Leaf, H - Hypocotyl Each value represents the treatment means of five replicates Values with the same letter within columns are not significantly different according to Duncan s Multiple Range Test (DMRT) at 5 % level

21 Fig-2 Direct regeneration of shoots from 2 day old cotyledon explants derived from mature seeds of Cucumis anguria L. a. De-coated seeds of Cucumis anguria L. b. Cotyledon Explants c. Shoot induction in MS+BAP (4.44µM) +NAA (1.07 µm) +L Glutamine (137 µm) d, e & f. Shoot proliferation in MS + BAP (4.44 µm) + L-glutamine (137 µm) g. Shoot elongation in MS + BAP (4.44 µm) + GA 3 (1.44 µm) h. Rooting in MS + BAP (4.44 µm) + IBA (2.46 µm) i. Acclimatized plant 57

22 58

23 Fig-3 Direct regeneration of shoots from Nodal explants derived from 15 day old in vitro seedlings of Cucumis anguria L. a. Nodal explant of Cucumis anguria L. b & c. Shoot induction in MS medium with NAA (1.07 µm) + BAP (4.44 µm) + L-glutamine (137 µm) d, e & f. Shoot proliferation in MS medium with BAP (4.44 µm) + L- glutamine (137 µm) g. Shoot elongation in MS medium with BAP (4.44 µm) + GA 3 (1.44 µm) h. Rooting in MS medium with BAP (4.44 µm) + IBA (2.46 µm) i. Acclimatized plant 59

24 60

25 Fig-4 Different types of calluses obtained from cotyledon (a1-d1), hypocotyl (a2-d2) and leaf (a3-d3) explants derived from in vitro seedlings of cucumis anguria L. on MS medium containing different concentrations and combinations of auxins and cytokinins. a1, a2 and a3 - Yellow friable callus b1 - Yellowish green friable callus c1 and c2 - Brownish green friable callus d1, d2 and d3- Green compact callus b2 - White friable callus b3 - Brownish green compact callus c3 - Yellowish brown friable callus 61

26 62

27 Fig-5 Indirect regeneration of shoots from cotyledon explants of Cucumis anguria L. a. Cotyledon explant cultured on MS medium supplemented with NAA (2.67 µm) +BAP (4.44 µm) b. Greenish compact nodular callus developed from cotyledon cultured on MS medium supplemented with NAA (2.67 µm) +BAP (4.44 µm) c. Differentiation of calli into shoot bud d. Adventitious shoot development from cotyledon derived callus cultured on MS medium supplemented with NAA (1.34 µm) + BAP (8.88 µm) + Zeatin (0.912 M) e, f & g. Multiple shoot Proliferation in MS + BAP (4.44 µm) + L-Glutamine (137 µm) h. Elongation of shoots in MS + BAP (4.44 µm) + GA 3 (1.44 µm) i. Rooting of elongated shoot in MS + BAP (4.44 µm) + IBA (2.46 µm) j. Acclimatized plant 63

28 64

29 Fig-6 Regeneration of shoots from Hypocotyl derived callus of Cucumis anguria L. a. Hypocotyl explant cultured on MS + 2, 4-D (2.26 µm) +BAP (4.44 µm) b. Greenish compact nodular callus in MS + 2, 4-D (2.26 µm) +BAP (4.44 µm) c. Initiation of shoot bud d. Initiation of adventitious shoots from Hypocotyl derived callus MS + 2, 4-D (1.13 M) +BAP (4.44 µm) + TDZ (0.908 M) e. Multiple shoot Proliferation in MS + BAP (4.44 µm) + L-Glutamine (137 µm) f. Elongation of shoots in MS + BAP (4.44 µm) + GA 3 (1.44 µm) g. Rooting of shoots in MS + BAP (4.44 µm) + IBA (2.46 µm) h. Acclimatized plant 65

30 66

31 Fig-7 Regeneration of shoots from Leaf derived callus of Cucumis anguria L. a. Leaf explant b. Green compact nodular callus cultured on MS + NAA (2.67 µm) +BAP (4.44 µm) c. Initiation of shoot bud d. Initiation of adventitious shoots from leaf derived callus in MS + NAA (1.34 µm) +BAP (4.44 µm) + Zeatin (0.912 µm) e, f & g. Multiple shoot Proliferation in MS + BAP (4.44 µm) + L-Glutamine (137 µm) h. Elongation of shoots in MS + BAP (4.44 µm) + GA 3 (1.44 µm) i. Rooting of elongated shoot in MS + BAP (4.44 µm) + IBA (2.46 µm) J. Acclimatized plant 67

32 68

33 4.5. HISTOLOGICAL STUDIES The calli derived from cotyledon, hypocotyl and leaf explants were sectioned, treated with three histochemical stains and examined under microscope to analyze their anatomical changes during the course of cytodifferentiation into shoots. Indeed these parts of study on histochemical comparison provide a detailed understanding on the differentiation of callus and shoot bud initiation. Three stains were employed i.e., Toluene blue (TBO), Ruthenium Red (RR) and Coomassie Brilliant Blue (CBB) in order to find out the variation in physical and chemical nature of callus derived cells from the three different sources. The TBO was used to distinguish the cause structure of callus chemically by three colorations (Carboxylated polysaccharide-pink; Lignin-blue and Phenolic compound bluish green). The RR was applied to find out the growth stage of cells by red colored pectinized cell walls as the pectinization depend upon the differentiation of particular cells within the callus mass. The CBB was used to find out the total proteins which were stored and accumulated in the callus cells. The histochemical studies on callus differentiation and shoot initiation showed clear differentiation in all the three explants. In the leaf explant the callus initiation originated from surface of epidermis, most often inter-cellular spaces where the stomatal complexes were present. The anatomy of direct shoot regeneration in Cucurbitaceae has not been studied previously (Gaba et al., 1999). The cross sections of proximal region of cotyledon explants revealed that initiation and development of several meristematic regions (Fig-8). Gaba et al. (1999) has described the presence of meristematic protuberances which do not develop into shoot buds, instead forming leaf primordial into many larger leaves covering the proximal end of melon (cv. Galia) cotyledon explants. Similar observations of aberrant organogenic pathway has been reported by Stipp et al. (2001) in melon var. indorus. However, in the present investigation, adventitious shoot buds with leaf primordia were observed on cotyledon explants which subsequently developed 69

34 into normal shoots (Fig-5d). Many such buds developed on the epidermis which later development in to shoots. The hypocotyl and cotyledon explants produced the callus and then the callus cells aggregated and united to produce the shoot buds. It was observed that both the explants were not initially produced any bud like structure as observed in the leaf explant. Uniformally, the callus initiation was noticed from all directions of the explant but the size of the cells of the callus varied in hypocotyl and cotyledon derived calluses. The hypocotyl derived callus contained more number of long sized cells when compared to cotyledon explants (Fig-9). However, the compactness of callus shown to the prominent in cotyledon derived callus with higher number of shoot buds as compared to hypocotyl derived callus. Another interesting observation noted in cotyledon derived callus was that the nodular structures appear before the onset of shoot initiation in many regions of the callus. Whereas the shoot buds initiation of hypocotyl was observed only in marginal cells of the callus (Fig-9). The overall structure and development of shoot bud after the initiation not showed much variation in all these explants (Fig-6). The initiation of the regeneration process in melon is similar in vitro to that of many other species. Mendoza et al. (1993) and Wright et al. (1986) have reported that adventitious shoot meristems formed directly from explants in vitro is often initiated by cell divisions beginning in the epidermal or sub-epidermal layers. In our observation, a higher population of callus found to be meristematic and adventitious shoot meristems were developed predominantly from epidermal region (Fig-9 e). Histological examinations of organogenic callus derived from leaf explants revealed that initiation of meristemoids from callus started after four weeks of subculture. In the initial stage of shoot differentiation the callus cells showed a higher accumulation of storage foods like starch. This deposition was exhibited clearly as the cortex region of the cross sections deeply stained with TBO (Fig-10 c, d and e). 70

35 Furthermore, staining with Rithudium Red authenticated the development of thick cell wall structures with higher pectinization (Fig-10 f and g). The observations of our present study are correlative with the earlier reports (Torrey et al., 1971; Khatoon et al., 2006). As a result of morphogenetic changes, young adventitious shoot buds surrounded by leaf primordial might have originated from these meristemoids (Fig-7c). 71

36 Fig-8 Histological analysis on cytodifferentiation of cotyledon derived calli into shoots. a. Cross section of undifferentiated, friable callus, without much stored foods and cell wall thickening stained with TBO. 20X b,c&d. Cross section of differentiating procambial calli with gradual pectination in cell walls stained with Ruthidium Red. 20X e &f. Cross section of differentiating callus tissue with well developed vascular elements and polysaccharides stained with TBO. 20X g. Cross section of fully differentiated callus tissue showing the shoot apex (dome) with thick cell wall, stained with TBO. 20X

37

38 Fig-9 Histological analysis of shoot induction from hypocotyl derived callus (cross section of callus tissue). a. Primary callus with undifferentiated cells. 20X b & c. Differentiating callus tissue stained with Ruthidium Red. 20X d. Shoot primordia from peripheral layer. 20X e. Dome formation (Shoot bud initiation). 20X

39

40 Fig-10 Histological analysis of shoot induction from leaf derived callus (cross section of callus tissue). a, b. Friable callus with undifferentiated cells. 20X c, d & e. Callus undergoes cytodifferentiation moderately stained with TBO due to the onset of cell wall thickening and storage of complex nutrients. 20X f, g & h. Development of epidermis from the differentiated cells. 20X. Close up view of epidermis (g) i. Origin of shoot primordia from the peripheral layer. 20X j. Dome formation (Shoot bud initiation). 20X

41

42 4.6. PRELIMINARY AGROBACTERIUM MEDIATED TRANSFORMATION Cucumber transformation is more than a decade old. The transformation of cucumber was first achieved by Trulson et al. (1986) and then the transgenic plants were regenerated from different explants like cotyledons and hypocotyls. At present different marker, reporters and various types of transgenes were introduced in to the Cucumis sativus L. Direct regeneration using proximal half of cotyledon explants has already been employed successfully to recover phenotypically normal transgenic cucumbers (Tabei et al., 1998; Vengadesan et al., 2005; Selvaraj et al., 2010). However there were no reports on transformation of West Indian Gherkin (Cucumis anguria L.). Among the available genetic transformation systems, Agrobacterium - mediated gene transfer is considered as more efficient for the stable integration of genes into plant genome. Cotyledon has been selected as regeneration system for transgenic recovery as it avoids interviewing callus during adventitious bud development. Based on the available reports in other species of cucumber, the present investigation focused to standardize the cotyledon based genetic transformation in West Indian Gherkin Plant material Selection of proper explants is one of the most important factors in genetic transformation. Explant with high regeneration potential is a desired character for transformation experiments. Different kinds of explants were used in cucumber transformation experiments. Schulze et al. (1995) used leaf derived embryogenic suspension culture for genetic transformation in Cucumis sativus L. Hypocotyl explants has been used in Agrobacterium - mediated transformation of Cucumis sativus L. (Nishibayashi et al., 1996). Most of the transformation studies in cucumber species used cotyledon as explant source (Tabei et al., 1991; Mohiuddin et al., 1997; Dabauza et al., 60

43 1997; Gambley and Dodd, 1990). Direct regeneration using proximal half of cotyledon explants has already been employed successfully to recover phenotypically normal transgenic cucumber (Tabei et al., 1998; Ganapathi and Perl-Treves, 2000; Selvaraj, 2002; Soniya and Das, 2002; Prem Anand and Perl Treves, 2005; Vengadesan et al., 2005; Vasudevan et al., 2007a). The current investigation used cotyledon explants from mature seeds. The regeneration efficiency and transformation rate was found higher than our earlier experiments in Cucumis sativus L. (Vengadesan et al., 2005; Vasudevan et al., 2007a; Selvaraj et al., 2010). The reason for high regeneration rate is the cotyledon explants have many small potential areas which response showed better in organogenesis (Han et al., 2005). Gambley and Dodd (1990) noted that the use of this system which produces multiple shoots from numerous adventitious buds with minimal peripheral callus production so that the transformation of many of these individual buds is possible Pre-culture of explants The cotyledon explants were pre-cultured in regeneration medium prior to infection and co-cultivation. Different pre-culture timings from 1-5 days has been studied. The control experiments without pre-culture of cotyledon explant observed 1.0 GUS positive shoots. Slight increase in the number of GUS positive shoots were noted in 1 day and 2 days preculturing of explants (Fig-12). Higher numbers of GUS positive shoots were noted in 3 days pre-cultured explants. However pre-culture of cotyledon explants for 4 days and 5 days showed reduction in recovery of GUS positive shoots. In contrary to this Selvaraj et al. (2010) reported that 5 days pre-culture for Cucumis sativus L. transformation. The reason for this decrease is pre-emergence of shoot primodia from the explants before infection. Cucumis sativus L. required a minimum of 5 days or more preculture before infection (Vengadesan et al., 2005; Vasudevan et al., 2007a). 61

44 Most of the cucumber transformations by earlier workers have not studied the effect of pre-culture of explants but they used three to seven day old seedlings as the donors for cotyledon explants (Chee, 1990a; Chee and Slightom, 1991; Soniya and Das, 2002; Sapountzakis and Tsaftaris, 1996). On the other hand, Tabei et al. (1998) used 1 day old cotyledon explants for transformation experiments. Pre-culture of explants is a critical factor to achieve high frequency of transformation. It makes the tissues competent enough to withstand the bacterial infection and increased the production of GUS positive shoots. Pre-culturing of explants prior to infection with Agrobacterium enhanced the transformation frequency in woody fruit species such as plum (Mante et al., 1991) and apricot (Laimer et al., 1992). Sangwan et al. (1992) have also reported that in Arabidopsis thaliana explants, the number of putatively competent cells for transformation was greatly increased by a pre-culture treatment on a medium rich in auxins. In contrary to this, Cervera et al. (1998) reported that the pre-culture of explants drastically reduced the regeneration of GUS positive shoots Sensitivity of cotyledon explants to PPT Sensitivity of cotyledon explants were analyzed in order to find out the appropriate concentration of selection agent and to control the development of non transgenic shoots or escapes. Different concentrations of phosphinothricin (0.5 to 2.5 mg/l) have been employed during shoot bud regeneration. Control experiments without PPT produced the shoots normally. The number of shoots produced in 0.5 mg/l PPT concentration has been reduced and further reduction has been noted in 1 to 2.0 mg/l PPT concentrations (Fig-13). At 2.5 mg/l concentration the shoot formation has been completely arrested. This concentration was used for the selection of transformed shoots with minimal escapes. The same concentration has stopped the root formation from the shoots. Our earlier observations in 62

45 Cucumis sativus L. proved that PPT was a good selection agent (Vengadesan et al., 2005; Vasudevan et al., 2007a). Recently Selvaraj et al. (2010) reported that 55 to 59% of escapes and 33 to 37% chimeras were found when kanamycin was used for selection of cucumber transformation. Kanamycin selection of transformed shoots in cucumber has been adopted (Chee, 1990b; Raharjo et al., 1996; Tabei et al., 1998; Prem Anand and Perl-Treves, 2005). In muskmelon 30% (Fang and Grumet, 1990) and 75-90% (Dong et al., 1991) of escapes has been noticed after selection on mg/l kanamycin. A strong selection agent is important for the transformation of cucurbits, especially one that regenerates via direct organogenesis from cotyledon explants (Gaba et al., 1999). Our results in Cucumis anguria L. supported the use of PPT as a selection agent Agrobacterium infection and co-cultivation Co-cultivation period and sonication of explants greatly improved the transformation efficiency. One day co-cultivation with or without sonication have not produced any GUS positive shoots. Increase in co-cultivation period and sonication time has increased the transformation efficiency. The three day co-cultivation period with 60 seconds sonication have produced significantly higher number of GUS positive shoots (Table-8). At 3 days co-cultivation with 60 seconds sonication period produced many fold increase in the number of GUS positive shoots as compared experiments done without sonication. However increasing the co-cultivation period beyond 4 days and sonication period 90 seconds have shown decrease in transformation efficiency due to over growth and leaching of Agrobacterium. Bacterial-induced stress is seems to affect/inhibit regeneration. A decrease in transformation rate was accountable due to high bacterial and longer than optimal co-cultivation period (Fillatti et al., 1987). However, Cucumis sativus L. required less time (2 days) of co-cultivation (Selvaraj et al., 2010). Earlier reports in Cucumis 63

46 sativus L. supported two to three days of co-cultivation period (Vengadesan et al., 2005; Vasudevan et al., 2007). Few reports in Cucumis sativus L. revealed that long time co cultivation period (2-5 days) was found effective for infection and transgenic plant production (Chee and Slightom, 1991; Prem Anand and Perl-Treves, 2005). Using SAAT with immature cotyledons of soybean, Trick and Finer (1997) also observed an increase in transient GUS expression with longer duration of sonication. The efficiency of SAAT may be even further increased by vacuum infiltration (Bechtold et al., 1993). Although explants responded to a wide range of SAAT durations, was often damaged by the longer SAAT treatments and the tissue culture response was reduced. This result was in agreement with the earlier reports of soybean transformation by Finner et al. (1991). Acetosyringone is a phenolic compound produced during wounding of plant cells, and in previous studies has been shown to induce transcription of the virulence genes of Agrobacterium tumefaciens (James et al., 1993). The beneficial role of acetosyringone has been demonstrated in the genetic transformation of monocot and dicot plants. Table-9 shows that addition of 100μM acetosyringone to Agrobacterium culture had significant improvement in number of GUS positive plants over the Agrobacterium culture without acetosyringone. Although addition of acetosyringone increases the transformation efficiency, the concentrations beyond 100μM have reduced the transformation efficiency because of the overgrowth and leaching of Agrobacterium. Several fold increase in transformation efficiency has been noted when 50 μm acetosyringone is used in cucumber transformation (Selvaraj et al., 2010). 3 day co-cultivation, 60 seconds SAAT and 100µM acetosyringone showed positive cumulative effects on Cucumis anguria L. transformation. 64

47 Shoot regeneration, elongation, rooting and hardening The shoots regenerated after co-cultivation and washing, when placed in shoot regeneration medium contained BAP (4.44 µm), NAA (1.07 µm) and L-glutamine (137 µm) along with Cefotaxime (300 mg/l) and PPT (2.5 mg/l) resulted in the proliferation of new shoots that survived in the selection pressure. The shoot regeneration was observed within 3 weeks from the day of inoculation in regeneration medium. Presence of L - Glutamine in the regeneration medium improved shoot regeneration efficiency. Similar results were noted in earlier transformation experiments with cucumber transformation (Selvaraj et al., 2010). After differentiation of shoot buds in medium containing PPT, explants were transferred to GA 3 containing medium for elongation. After two weeks the shoots were elongated up to 5cm in length and subjected to root induction in half strength MS medium containing IBA and PPT 2.5mg/l. The shoots were transferred to rooting medium with same selection pressure to (2.5 mg/l PPT) restrict the growth of escapes. The shoots survived in this medium started rooting within a week time and completed the rooting within 4 weeks. All the fully grown plants were acclimatized in growth chamber. A total of 94 transgenic plants were regenerated and 90 plants survived in hardening process from the total of 300 explants used for transformation experiments (Table-9). These plants were healthy and no morphological differences were observed from control seed raised plants. In cucumber species kanamycin and phosphinothricin has been used as a selection agents. Earlier reports in cucumber and muskmelon revealed that use of kanamycin produces more escapes (Trulson et al., 1986; Tabei et al., 1994; Valles and Lasa, 1994). Our earlier experiments also revealed that Kanamycin selection produced escapes and chimeras, further selection in PPT reduced the same (Vengadesan et al., 2005; Selvaraj et al., 2010). However, Chee (1990a) and Sarmento et al. (1992) used kanamycin as the 65

48 sole selection agent to regenerate transformants in cucumber. The present study showed very less number of escapes and proved that the PPT is the best selection agent in Cucumis anguria L. The plants produced in this study were morphologically normal and healthy Assay for β-glucuronidase activity Histochemical staining was used to study the factors affecting the transformation experiment. Histochemical GUS activity showed blue colour staining in shoots. The non transformed control shoots did not show blue colour staining. GUS histochemical assay allows a very rapid and sensitive screening of the transformed cells and tissues by visual observation. While evaluating various parameters during standardization of the transformation protocols, the choice of histochemical marker was realized since the effort of the different treatments could be assessed in the target tissues even before the plantlets grow and mature (Hinchee et al., 1988). In the present study, high level of GUS activity was observed due to the expression of GUS gene in dividing cells. This property is regulated by the promoter that drives the expression of the GUS gene. Nehra et al. (1990) reported a similar intense GUS activity under the CaMV35S promoter in meristematic region of strawberry. CaMV35S promoter expresses in all plant parts, its expression being reported to be organ specific as it was more pronounced in immature leaves than mature leaves (Jefferson et al., 1987). Further its expression is dependent upon the phase of the cell cycle and cell types (Nagata et al., 1987). In our recent study in cucumber gfp driven by CaMV35S expressed in leaf, stem and root (Selvaraj et al., 2010) promo gene has been expressed. In the present study, cotyledon explants, leaves and shoots exhibited stronger GUS expression. Our observation suggests that the cut surface of the cotyledon explants contain potential meristematic tissues that are susceptible to Agrobacterium, whereas cotyledon explants with proximal end showed lesser GUS activity as their region contained less 66

49 transformation competent cells. No endogenous GUS expression was detected in the shoots of non transformed cotyledon explants Molecular conformation of transformants PCR analysis The putative transformed shoots were checked for the presence of transgene in genomic DNA. The DNA isolated from transformed plant (T 0 ) and non transformed control plants were used as template DNA for PCR amplification of npt II gene. The size of the amplified band corresponded to the expected size of 690 bp and indicated the integration of npt II fragments in the transformants (Fig-16; lanes 3-6). No amplification was observed in non transformed control plants (Fig-16; lane-1 and 7) Southern hybridization The stable transformation of T 0 transgenic Cucumis anguria L. plant was further confirmed by southern blot. Southern blot analysis was carried out on total genomic DNA isolated from the leaves of kanamycin resistant shoots (T 0 ) and non transformed control plants. PCR amplified npt II fragment probe (Fig-16; lane-l) was hybridized to putative transformed plant DNA samples digested with Hind III enzyme and non-transformed control plants. All the transgenic shoots showed integration of npt II gene. Signals detected from the blot hybridized with the single digestion with Hind III enzyme confirmed that the presence of two copies in one putative transformants and other three putative transformants with single copy number indicates the random insertion of transgene in the genome (Fig-16; lanes 3-9). The absence of the same signal in lanes loaded with samples from non transformed control plants (Fig-16; lane-1 and 2) proved that the same transgenes are not available in the genome of Cucumis anguria L. 67

50 The plants regenerated from the transformation experiments proved the expression of bar gene by the survival of shoots in PPT selection pressure, expression of GUS gene by showing blue colour in GUS histochemical staining, integration of npt II genes by showing the amplication of npt II fragments and by producing signals from the southern blot probed with npt II gene. The present study proved that Cucumis anguria L. is a suitable plant for Agrobacterium - mediated transformation and the genes transferred to this plant express clearly. Hence this protocol will be suitable to transfer candidate genes to improve this crop species. 68

51 Table 8 Effect of Co-cultivation period and sonication timing on Transformation efficiency of Cucumis anguria L. using cotyledon explants. Co-cultivation period (days) Sonication time (seconds) Number of GUS positive shoots 0.8 j 1.8 g 2.8 e 2.0 f 1.0 i 3.8 d 7.4 a 4.0 c 1.2 h 2.6 eb 5.6 b 2.8 ea Transformation efficiency (%) 1.6 k 3.6 h 5.6 e 4.0 g 2.0 j 7.6 d 14.8 a 8.0 c 2.4 i 5.2 f 11.2 b 5.6 ea Each value represents the treatment means of five replicates with 50 explants per experiments. Number of GUS positive shoots Transformation efficiency = x 100 Total number of explants co-cultivated Values with same letter within columns are not significantly different according to Duncan s Multiple Range Test (DMRT) at 5% level Not determined due to overgrowth of Agrobacterium.

52 Table 9 Effect of Acetosyringone in Cucumis anguria L. transformation in 3 day co- cultivation period with 60 seconds of sonication. Acetosyringone concentration µm 0 (control) Number of GUS Positive shoots 4.4 d 6.2 b 9.8 a 5.4 c Transformation efficiency (%) 14.6 d 20.6 b 32.6 a 18.0 c For each co-cultivation period 30 explants were used The total number of GUS positive shoots obtained after 20 days of co-cultivation Values with same letter within columns are not significantly different according to Duncan s Multiple Range Test (DMRT) at 5% level

53 Fig-11 Linear map of plasmid vector pme 524 present in the Agrobacterium tumefactions strain EHA105 used for the transformation experiment.

54 Fig-12 Effect of pre-culture period on the protection of GUS positive shoots after co-cultivation of cotyledon explants of Cucumis anguria L. for five days with Agrobacterium strain EHA105 in MS medium. For each pre-culture period 100 explants were used and co-cultivated for 3 days. The experiments were repeated for five times and the numbers of GUS positive shoots are the average of three experiments.

55 Fig-13 Sensitivity of cotyledon explants of Cucumis anguria L. to various concentrations of PPT in MS medium containing 4.44 M BAP and M TDZ. For each concentration 100 explants were used.

56 Fig-14 GUS expression patterns from Sonication method. a. Control shoots b. 30; c. 40; d. 50; e. 60 seconds sonication treated shoots showing GUS expression.

57 Fig-14

58 Fig-15 Agrobacterium-mediated transformation of 2 day old cotyledon Explants. a, b, c. Infected and co-cultivated cotyledon explants on MS basal medium d. Multiple shoot formation in MS + BAP (4.44 µm) + NAA (1.07 µm) + L- Glutamine (137 µm) + Cefotaxime (300 mg/l) e. Selection of regenerated shoots in MS medium containing BAP (4.44 µm) +L- Glutamine (137 µm) + PPT (2.5 mg/l) + Cefotaxime (300 mg/l) f & g.gus positive shoots h. Elongation of selected shoot in MS + BAP (4.44 µm) + GA 3 (1.44 µm) + PPT (2.5 mg/l) + Cefotaxime (300 mg/l) i. Rooting of elongated shoot in MS + BAP (4.44 µm) + IBA (2.46 µm) + PPT (2.5 mg/l) + Cefotaxime (300 mg/l) j &k. Rooted plantlets maintained in growth chamber for acclimatization l. Acclimatized plantlets transferred into field condition

59 Fig-15

60 Fig-16 PCR and Southern confirmation of transformed shoots (EHA105). PCR confirmation of putatively transformed plants to detect the presence of npt II coding region Lane L Lane 1 & 7 Lane 2-6 Marker (100bp DNA ladder) DNA samples from non-transformed shoots (control) -DNA samples from putative transformants (Arrow indicates the amplification of npt II gene at 690 bp) Southern blotting Southern hybridization of genomic DNA isolated from the leaves of hardened putatively transformed plants Lane L-Labelled probe DNA (npt II fragment digested with PstI restriction enzyme) Lane 1-2- Control plant (DNA isolated from non- transformed plant) Lane 3-9-DNA samples from transformed shoots

61 Fig bp

62 4.7. CHITINASE TRANSFORMATION Cucumis anguria L. is not having resistance against the leaf spot disease. It is difficult to find the resistance source for breeding. In this case a transformation technique seems to be a useful method to produce novel cultivars with disease resistance. It is known that plants have defense systems which involve pathogenesis-related proteins, e.g; chitinase (Legrand et al., 1987; Nishizawa and Hibi, 1999) and β-1, 3 glucanase (Kombrink et al., 1988). The introduction of rice Chitinase (RCC2) has been found successful in Cucumis sativus L. (Tabei et al., 1998; Kishimoto et al., 2003). The current research study is aimed to produce Cucumis anguria L. with chitinase gene (Chi-42) resistant to leaf spot disease Agrobacterium strain EHA 105 harboured the plasmid with Chi-42 gene driven by CaMV 35S promoter was used for infection (Fig-17). A total of 250 Cotyledon explants were infected in five different experiments. After the selection with 2.5mg/L PPT, a total of 103 plants were survived in elongation and rooting process (Table-10). All the survived plants were subjected to PCR with chitinase primer. Among these 75 plants showed PCR positive to the chitinase primer (Fig- 19). The average transformation efficiency was 30% in chitinase transformation experiments. Five plants selected from five different experiments (T 0 ) were subjected to southern blot with chitinase probe digested by Hind III enzyme. Signal from the southern blots revealed that 3 plants (Fig- 19; lanes 3-5) are having single copies and 2 plants (lanes 6-7) having 2 copies of chitinase gene. The control plant without Agrobacterium infection did not show any signal. Hence, it is confirmed that the chitinase gene (Chi-42) was integrated in to genome of Cucumis anguria L. 69

63 The further experiments like screening the plants with pathogen challenge and progeny analysis (Segregation of transgene pattern in T 1 generation) are in progress. 70

64 Table - 10 Transformation efficiency of cotyledon explants of Cucumis anguria L. using Chi-42 in the presence of 100 µm Acetosyringone with 3days co-cultivation. Experiment No. Number of plants regenerated and survived Number of PCR + ve shoots Transformation efficiency (%) e 16 b 15 c 14 d 17 a 26 e 32 b 30 c 28 d 34 a Total For each experiment 50 explants were used. Number of PCR positive shoots Percentage of transformation efficiency = x 100 Total number of explants infected

65 Fig-17 Fungal Chitinase (Chi-42-4C)

66 Fig-18 Agrobacterium - mediated Chitinase gene (Chi-42) transformation of 2 day old cotyledon explant. a, b. Infected and co-cultivated cotyledon explants cultured on MS basal medium c, d, e and f. Selection of regenerated shoots in MS medium containing BAP (4.44 µm) + L-Glutamine (137 µm) + PPT (2.5 mg/l) + Cefotaxime (300 mg/l) g. Elongation of selected shoots on MS medium containing BAP (4.44 µm) + GA 3 (1.44 µm) + PPT (2.5 mg/l) + Cefotaxime (300 mg/l) h. Rooted plantlet on MS medium with BAP (4.44 µm) + IBA (2.46 µm) + PPT (2.5 mg/l) + Cefotaxime (300 mg/l) i & j. Rooted plantlets transferred to growth chamber k. BASTA spray on control plantlet l. Acclimatized plantlets in field condition

67 Fig-18

68 Fig-19 PCR and Southern confirmation of transformed shoots (Chi-42). PCR analysis of genomic DNA to detect the presence of the Chi-42-4C coding region Lane 1 100bp DNA ladder Lane 2 DNA sample from control plant Lane 3 7 DNA from putative transformants showing amplification of the predicted 840 bp Chi-42 specific sequence Southern blotting Lane 1 - Linearized nopaline based binary vector used for transformation Lane 2 - Control plant (DNA isolated from non transformed plant) Lane3-7 - DNA samples from Transformed shoots

69 Fig-19

70 4.8. ADVENTITIOUS ROOT CULTURE The adventitious root cultures were tried in MS medium either with IBA either alone or in combination with IAA or NAA. Other auxins applied individually or in combination did not show positive response. Four types of explants i, e., cotyledon, hypocotyl, leaf and shoot nodes were tried. Among these only cotyledon and leaf explants responded positively. Hence, these two explants were used for root culture experiments. After 20 days of culture rooting was achieved in MS medium with IBA alone, or IBA/IAA or IBA/NAA combinations. The cotyledon explant when cultured in IBA/NAA combination recorded the highest number of roots (9.2) and percentage of response (96.7%) (Table-11). The leaf explant recorded the highest number of roots (7.8) and percentage of response (88.9%) in the same combinations. Although IAA and IBA combinations also showed rooting but it is lower than the individual IBA treatment. As best results was observed at 2.46 μm IBA, this concentration was kept constant for combination treatments of IAA (1.14 μm) and NAA (1.07 μm). IBA and NAA ( μm) combination produced maximum number of roots (113.42) with cm root length was observed from the cotyledon explant derived cultures. Similarly the highest numbers of roots (106.32) with cm shoot length were observed from the leaf explants derived cultures in the same concentrations (Table-12). Although the IBA (2.46 μm) and IAA (1.14 μm) combination produced roots in the subculture, the number of roots were lower than the medium containing IBA alone. Similar trend in biomass and growth index experiments were observed. The maximum biomass (fresh weight-6.52g; dry weight-0.68g) and growth index 1.88 were recorded in cotyledon explants derived cultures in IBA and NAA combination (Table-13). The highest biomass (fresh weight-7.62g; dry weight 0.74g) with 2.05 growth index was noted in leaf derived roots cultures at IBA with NAA combination. Hence the IBA (2.46 μm) and NAA (1.07 μm) combination may be considered as best hormonal combination and 71

71 cotyledons are the most suitable explant for root culture studies of Cucumis anguria L. IBA and IAA promoted more root elongation compared with NAA in Hyoscyamus maticus root cultures (Biondi et al., 1997). Similarly IBA was more effective for lateral root induction in adventitious root culture of Panax ginseng when compared with NAA (Kim et al., 2003). On the other hand Taylor and Van Staden (1998) have ascertained that NAA was the most effective auxin for tomato lateral root elongation. In our observations the combination of IBA and NAA yielded best results and addition of IAA reduced the root production. Various growth regulators influence in different way in root induction, elongation and biomass production (Gyulai et al., 1993; Biondi et al., 1997; Vanhala et al., 1998; Balestri and Bertini, 2003). The addition of auxins into the culture media induced an increase of fresh mass in Cucumis anguria L. Kim et al. (2003) ascertained that addition of IBA in the medium was more effective for root biomass production compared with NAA. A decrease of root biomass in lobelia culture at higher concentrations of IAA and NAA has been determined by Balvanyos et al. (2001). They have also ascertained that the most effective concentration for biomass production was 10 μm NAA and 25 μm IAA. However, Vanhala et al. (1998) has observed that IAA and NAA did not significantly influence the biomass production in Hyoscyamus cultures. But our experiments revealed that IBA (2.46 μm and NAA 1.07 μm) combinations were suitable for biomass production HAIRY ROOT CULTURE BY AGROBACTERIUM RHIZOGENES Optimization of Agrobacterium infectivity When strain R1000 was used, the explant and wounded site influenced root induction (Fig-21). Roots appeared within 20 days in excised cotyledon explants of Cucumis anguria L. About 96% of transformation efficiency was observed in 72

72 cotyledon explants with 19.8 number of roots and 13 cm root length (Table-14). Roots never appeared on the control cotyledon explants. The inductions of transformed roots were significantly correlated with bacterial strain and explants. These results demonstrated that Cucumis anguria L. is susceptible to strain R It has been reported that the virulence of Agrobacterium strains varies among plant species (Hobbs et al., 1989; Bush et al., 1991) and that the transformation efficiency of plant species can vary between bacterial strains (Godvin et al., 1992; Hu and Alfermann, 1993) (Table-14; Fig-21). After 3 days of co-cultivation, the explants were washed with cefotaxime and inoculated in MS hormone free medium. During the first week of culture the transformed cotyledon explants produced roots and the non transformed explants have not produced any roots. Putative transformed hairy roots were induced at a high rate at the proximal end of the cotyledon. Within twenty days all the transformed cotyledons produced hairy roots. Transformation rates were assessed from the explants after 20 days of culture on MS medium containing 300 mg/l Cefotaxime. The number of hairy roots per cotyledon explant vary from 17.5 to 19.8 with root length of 10 to 13cm. Hairy root cultures were established from primary roots (0.2g) formed at each wound sites in MS liquid medium lacking growth regulator. The roots grow rapidly and developed lateral roots within 4 weeks in MS liquid medium supplemented with 200 mg/l cefotaxime. Within 45 days, 99 % of cultures showed maximum number of hairy roots (132) with 18 cm length from cotyledon derived hairy roots (Table-15), (Fig-21). In general, all of the transformed roots exhibited a loose and dispersed morphology with long primary roots showing lateral branching and the absence of extensive root hairs (Fig-21). Within 45 days, the roots gradually changed from white to yellowish white. Non transformed roots under similar conditions showed neurosis and poor growth associated with the absence of lateral branching. When transformed roots were transferred to MS basal liquid 73

73 medium without antibiotics, they showed vigorous growth. Similarly root clones showed a higher degree of branching and rapid plagiotropic growth (Tepfer and Tempe, 1981). The first successful cucumber transformation was reported by Trluson et al. (1986) using hypocotyl explants with A. rhizogenes containing nos-npt II gene. They have regenerated transgenic cucumber plants from roots of hypocotyl explants infected with A. rhizogenes. Ping et al. (1998) obtained hairy roots after 10 days of inoculation of Cucumis sativus L. cotyledon explants with the strain of A.rhizogenes R1000 and R1601. But our result was contrary to Ping et al. (1998). We have obtained hairy roots from cotyledon explants of Cucumis anguria L. after 20 days of inoculation with the strain A. rhizogenes R1000. So the R1000 strain was found more suitable to Cucumis anguria L. than Cucumis sativus L. Matsuda et al. (2000) reported that 10 to 14 hairy roots were formed from single cotyledon explants of Cucumis melo L. Our results are comparable and we found cotyledon of this plant species to be the best explant for hairy root production. Since cell division in the host target tissue is required for successful Agrobacterium transformation, it is therefore not surprising that wound sites associated with actively dividing cells showed higher transformation rates (Binns and Thomashow, 1988). The transformation efficiency varies in Cucumis species. About 87.5% transformation efficiency were recorded in Cucumis sativus L. (Ping et al., 1998). About 95.3% transformation efficiency were reported in Cucumis melo L. (Matsuda et al., 2000). But we have obtained higher transformation efficiency (98.4%) from cotyledon explants of Cucumis anguria L. transferred by R1000 strain. Our results revealed that the Cucumis anguria L. is more susceptible to rhizogenes strain than Cucumis sativus L. Ping et al. (1998) reported that the hairy 74

74 roots induced by R1000 divide into three phenotype. However we did not observe any phenotypic differences in Cucumis anguria L Growth studies At the end of the culture period, transformed roots varied with respect to FW, DW and GI, and they produced at least 2 fold more biomass than normal root (Table-13). This was reflected in differences in root tip elongation and lateral density. Cotyledon derived hairy roots showed the best FW biomass accumulation, 9.78 g (GI of 2.83), which was 2 fold that of the non transformed roots (Table-15). Variations in biomass accumulation between different explants derived hairy roots have been reported earlier in Duboisia leichhardtii (Mano et al., 1986), Atropa belladonna (Aoki et al., 1997) and Catharanthus reseus (Batra et al., 2004). The precise mechanisms involved in lateral roots formation are still not clearly understood, but roots transformation by A.rhizogenes are characterized by the spontaneous formation of numerous laterals (Tepfer and Tempe, 1981), an important factor contributing to their high biomass productivity. Rapid elongation is a characteristic phenotypic feature of transformed roots, and this rapid elongation in growth and lateral branching are responsible for the enhanced biomass accumulation of transformed roots. The physiological basis of these changes is still not well understood, but a similar increase in growth can be obtained by inhibiting polyamine accumulation (Ben-Hayyim et al., 1996) Confirmation of transformation using PCR All putative transformed root samples were positive for the rol A and rol B genes. The PCR products were the expected size (About 490 bp and 383 bp respectively) and identical with those of the positive control. The NT roots (Negative control) were negative for the rol A and rol B genes (Fig-22). To our knowledge this is the first report on genetic transformation in Cucumis anguria L. The transformed roots showed considerable variations in 75

75 growth and biomass accumulation. In liquid media, root cultures exhibited vigorous growth and biomass accumulation than control. The successful transformation of Cucumis anguria L. by Agrobacterium rhizogenes strainr 1000 provide ample opportunity for further investigation on the transformed root cultures for the production of Cucurbitacin and other related compound. 76

76 Table 11 Effect of different concentration and combination of auxins (IBA, IAA and NAA) on adventitious root induction from different explants (cotyledon and leaf) of Cucumis anguria L. PGR concentration and combination (μm) IBA IBA + IAA % of response Cotyledon explants Total No. of roots a 3.4± ± ± ± ± ± ± ± ± ± ± ± ± ±0.12 Root length (cm) a 5.0± ± ± ± ± ± ± ± ± ± ± ± ± ±0.37 % of response leaf explants No. of roots a 3.2± ± ± ± ± ± ± ± ± ± ± ± ± ±0.22 Root length (cm) a 3.4± ± ± ± ± ± ± ± ± ± ± ± ± ±0.25 IBA+NAA ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.75 a each value represents mean ± S.E of three replicates recorded after 21 days of culture. Each mean represented five replications with 10 explants

77 Table - 12 Proliferation of adventitious root culture from different explants of Cucumis anguria L. on MS medium fortified with IBA (2.46 μm), IBA +IAA (2.46 Μm μm) and IBA +NAA (2.46 μm μm). PGR concentration and combination (μm) Control IBA (2.46) Cotyledon derived roots % of No. of Root length response roots a (cm) a ± ±0.23 Leaf derived roots % of No. of Root response roots a length (cm) a ± ±0.32 IBA + IAA ± ± ± ±0.28 ( ) IBA+NAA ± ± ± ±0.38 ( ) a each value represents mean ± S.E of three replicates recorded after 45 days of culture. Each mean represented three replications with 50 explants

78 Table 13 Fresh weight, Dry weight and growth index of Cucumis anguria L. root cultures after 45 days of culture in the dark on media supplemented with different concentrations and combinations of IBA, IBA+IAA, IBA + NAA. PGR (μm) Cotyledon derived roots Leaf derived roots FW (g) a DW (g) a GI FW (g) a DW (g) a GI IBA ± ± ± ± IBA + IAA ± ± ± ± IBA+NAA ± ± ± ± a each value represents mean ± S.E of three replicates with recoded after 45 days of culture. Each mean represented three replications with 50 explants

79 Table - 14 Effect of co-cultivation on hairy root induction from cotyledon explants of Cucumis anguria L. using A.rhizogenes strain R1000 on MS basal medium under total darkness. Experiment no. No. of transformed explants Transformation efficiency (%) Number of roots per explant a Root length (cm) ± ± ± ± ± ± ± ± ± ±0.5 Each experiment was conducted with 50 explants Transformation efficiency (%) = No. of explants infected / No. transformed explants X 100

80 Table - 15 Proliferation of hairy root cultures and biomass production from cotyledon explants of Cucumis anguria L. on MS liquid medium with all plant growth regulators under total darkness after 45 days of culture. Experiment no. Percentage of response No. of hairy roots per explant Length of the hairy root Biomass Production FW a DW a GI ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Values are the means of 50 explants, followed by S.E

81 Fig- 20 Adventitious root culture using various explants of Cucumis anguria L. a, c. Initiation of adventitious roots from cotyledon and leaf explants cultured on MS half strength solid medium supplemented with IBA (2.46 µm) and NAA (1.07µM) b, d. Proliferation of adventitious roots from cotyledon and leaf explants cultured on MS half strength liquid medium supplemented with IBA (2.46 µm) and NAA (1.07µM)

82

83 Fig-21 Agrobacterium rhizogenes-mediated transformation of cotyledon explants. A & b. Induction of hairy roots on MS solid medium c, d & e. Multiplication of hairy roots f. Proliferation of hairy roots on MS liquid medium

84

85 Fig-22.a PCR confirmation of rol A gene in putatively transformed hairy roots. Lane L - 100bp DNA ladder Lane 1 - Plasmid DNA Lane 2 - Non-transformed roots (Control) Lane 3, 6 - Putatively transformed roots Fig-22.b PCR confirmation of rol B gene. Lane L - 100bp DNA ladder Lane 1 - Plasmid DNA Lane 4 - Non-transformed roots (Control) Lane 2, 3 & 5, 6 - Putatively transformed roots

86 490bp 383bp