Micropropagation, diversity study and detection of antioxidants in some medicinal Zingibers Thesis submitted to the University of North Bengal For the Award of Doctor of Philosophy In Botany By Malay Bhattacharya Supervisor Dr. Arnab Sen Department of Botany University of North Bengal RajaRammohunpur, Siliguri January, 2014 This work is dedicated to my Mother DECLARATION I declare that the thesis entitled “Micropropagation, diversity study and detection of antioxidants in some medicinal Zingibers” has been prepared by me under the supervision of Dr. Arnab Sen, Associate Professor of Department of Botany, University of North Bengal. No part of this thesis has formed the basis for the award of any degree or fellowship previously. [Malay Bhattacharya] Department of Botany, University of North Bengal Raja Rammohunpur, Siliguri-734013 Date: i ABSTRACT ABSTRACT Zingiberaceae is a moderately sized the rituals, ceremonies and cuisine. family of relatively advanced Due to the strong antiseptic properties, monocotyledons. It is the largest family turmeric has been used as a remedy for of Zingiberales and is one of the ten all kinds of poisonous affections, ulcers largest monocotyledonous families in and wounds. K. galanga rhizome India. The members of this family extract are used as expectorant, occur chiefly in the tropics with the stimulant, diuretic, carminative and greatest concentration in the Indo- stomachic. It is used for the treatment Malayan region of Asia. India is of leprosy, skin diseases, rheumatism, represented by around 22 genera and asthma, cough, bronchitis, ulcers, 178 species. fever, malarial fever, nasal obstruction The study area includes the foothills of and for various other ailments. Darjeeling and the adjoining plains. In the present study of some medicinal The floral elements of the region Zingibers, three broad objectives (in include various members of the family vitro regeneration, study of molecular Zingiberaceae, of which Zingiber diversity and detection of antioxidant officinale, Curcuma longa and potential) were considered. Studies in Kaempferia galanga are used in these fields were severely lacking in alternative systems of medicine. The the perspective of local cultivars of the whole parts of these plants are genera. aromatic, but it is their underground Selected places of Jalpaiguri and rhizome, fresh or preserved, that are Darjeeling district of West Bengal valuable. Z. officinale is used were visited for collection of worldwide as a cooking spice, germplasm. The collected samples condiment and herbal remedy. The were planted in separate pots placed at dried rhizomes constitute the spice and experimental garden of Department of are esteemed for its flavour, pungency Botany for future use. As, the members and aroma. C. longa occupies an of Zingiberaceae are propagated by important position in the life of Indian rhizomes; various pathogens affecting people as it forms an integral part of the crop are transmitted through this ii ABSTRACT process. Moreover, conventional BAP (4.33), kinetin (3.44) and zeatin breeding techniques to improve the (3.58). In Z. officinale the highest crops are restricted due to extreme low average shoot height with BAP (6.90 seed set. In vitro culture offers an cm), kinetin (6.06 cm) and zeatin (4.67 alternative method for producing cm) were observed. In C. longa the variations and disease free planting highest average shoot height with BAP materials. Unfortunately, this field of (6.47 cm), kinetin (5.81 cm) and zeatin research has not been explored for the (4.43 cm) were observed, while in K. local cultivars of Zingiberaceous galanga it was BAP (4.31 cm), kinetin plants. (2.82 cm) and zeatin (2.48 cm). Among In vitro regeneration experiment using the cytokinins tried, BAP gave better rhizome sprouts revealed that, results compared to kinetin and zeatin Murashige and Skoog basal media in regeneration. supplemented with vitamins, sucrose, The regeneration of plantlets also agar and plant growth regulators was varied considerably with different superior to Gamborg B5 media. The combinations of BAP and kinetin. In Z. ideal percentage of sucrose required for officinale culture, maximum number of growth of the sprouts was 30 g/l, when plantlets per explant were obtained in the media was supplemented with 3 or the medium supplemented with 4 mg/l 4 mg/l BAP. BAP + 3 mg/l kinetin (9.60), while the Efficacy of three important cytokinins maximum plantlet height was obtained like benzyl amino purine, kinetin and in the medium supplemented with 4 zeatin were tested in different mg/l BAP + 3 mg/l kinetin (8.59 cm). concentrations and combinations. It has In C. longa culture the maximum been observed that the growth number of plantlets were obtained in regulators trigger variable responses. In the medium supplemented with 3 mg/l Z. officinale the highest number of BAP + 4 mg/l kinetin (9.20), while the shoots per explant with BAP (8.33), maximum plantlet height was obtained kinetin (5.40) and zeatin (7.20) were in the medium supplemented with 4 observed. In C. longa the highest mg/l BAP + 4 mg/l kinetin (8.36 cm). number of shoots per explant with BAP In K. galanga culture the maximum (9.08), kinetin (6.85) and zeatin (6.22) number of plantlets were obtained in were noted, while in K. galanga it was the medium supplemented with 3 mg/l iii ABSTRACT BAP + 4 mg/l kinetin (6.52), while the cultures showed maximum maximum plantlet height was obtained regeneration in the 2nd subculture. In in the medium supplemented with 2 case of K. galanga regeneration, the mg/l BAP + 4 mg/l kinetin (8.23 cm). least number of plantlets were In general plantlet regeneration was produced in the primary culture and it relatively less in the medium declined after the 1st or 2nd subcultures. supplemented with low BAP and Healthy, in vitro grown plantlets with kinetin combination. Good results were good number of roots were selected for obtained when the combined hardening. The regenerated plants Z. concentrations of BAP and kinetin officinale, C. longa and K. galanga were more than 5 mg/l. The plant showed a survival percentage of 94%, growth regulators worked better in 91% and 94% respectively. combinations than that when used Diagnostic tests were performed to alone. The plantlets rooted in the same detect the presence or absence of the medium and they were moderate to pathogen in the plants. In vitro-derived profuse in most of the combinations. plants raised under field conditions did The rate of rooting was found to be not show any disease symptoms until proportional to the number of plantlets. maturity. The rhizomes obtained from Subcultures to obtain maximum field planted tissue culture derived number of healthy shoots were plantlets on storage on sand for 6 experimented by inoculating sets of months did not show any visible root- explants in media having the same rot or shoot-rot. composition. The effects of plant Benzyl Amino Purine showed variable growth regulators on regeneration were effects on the shooting of twelve not the same in primary and secondary different C. longa cultivars. The mean cultures. Comparatively less variation number of shoots formed per explant were observed in number of shoots per were 3.075 (BAP 1 mg/l), 4.983 (BAP explant in the primary and secondary 2 mg/l), 6.891 (BAP 3 mg/l), 6.358 cultures of C. longa than Z. officinale (BAP 4 mg/l) and 5.816 (BAP 5 mg/l). and K. galanga. In all the cases of Z. Maximum regeneration potential was officinale and C. longa regeneration the observed in the cultivar CLS-2A (8.7 least number of plantlets were shoots per explant), while the lowest produced in the primary culture and the regeneration potential was observed in iv ABSTRACT the cultivar Allepy (1.8 shoots per polymorphic. The frequency of explant). polymorphism was found to be 92%. The band size ranged between 274bp to RAPD and ISSR analysis were 1758bp. Similarity coefficient among performed to analyze the genetic the 12 cultivars ranged from 0.523 to relationship among the C. longa 0.904. The lowest similarity was cultivars. In RAPD analysis, the observed between C. longa L. cv Local amplification profiles of the total -Dhupguri and C. longa L. cv Allepy, genomic DNA from the 12 cultivars of while the highest value was recorded C. longa using 14 primers resulted in between C. longa L. cv Suguna and C. production of 170 bands ranging in longa L. cv TC Assam. The between 151 and 1767 bp of which dendrogram constructed on the basis of only 10 were monomorphic. The the data obtained from ISSR analysis percentage of polymorphism was found showed three major groups viz. group I to be 94.11%. The similarity matrix (C. longa cv Local-Lataguri and Local- obtained using the Dice coefficient of Dhupguri); group II (C. longa cv similarity among the 12 cultivars (Prova, Sudarshana and PTC 13) and ranged from 0.560 to 0.857. The lowest group III (C. longa cv (Suguna, TC similarity was observed between C. Assam, Allepy, Kasturi, CLS 2A, longa cv local-Dhupguri and Roma and Suvarna and Roma) were noted. Of C. longa cv local-Dhupguri and these the first group with two local Sudarshana while the highest value cultivars of C. longa formed a distinct was recorded between C. longa cv clade from rest of the cultivars. Suguna and C. longa cv TC Assam. Three distinct groups viz. group I (C. Combined RAPD and ISSR based longa cv Local-Lataguri, Local- analysis of C. longa showed the Dhupguri, Prova, Suvarna, Suguna, highest similarity between the cultivars Kasturi and CLS 2A); group II (C. C. longa L. cv Suguna and C. longa L. longa cv Allepy, PTC13 and Roma) cv TC Assam while the lowest was and group III (C.