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THESIS WEE SIN LING.Pdf THE EFFECTS OF ELICITORS AND PRECURSOR ON IN VITRO CULTURES OF SAUROPUS ANDROGYNUS FOR SUSTAINABLE METABOLITE PRODUCTION AND ANTIOXIDANT CAPACITY IMPROVEMENT WEE SIN LING, PhD THESIS SUBMITTED TO THE UNIVERSITY OF NOTTINGHAM FOR THE DEGREE OF DOCTOR OF PHILOSOPHY APRIL 2015 ABSTRACT Sauropus androgynus, commonly known as ‘sweet shoot’ is an underutilized crop known for its high nutritive values and medicinal properties. To date, scientific studies assessing the potentially important benefits of sweet shoot for use as medicinal plants are still limited, with only six studies reporting on in vitro propagation and nine articles describing the production of secondary metabolites. The present study revealed that shadehouse-grown plants contained a low yield of bioactive phytochemicals, such as phenolic (61.20 µg/10g FW), flavonoid (193.62 µg/10g FW), naringenin (128.01 µg/10g FW), quercetin (1.56 µg/10g FW) and kaempferol (274.85 µg/10g FW). Moreover, it also had very low antioxidant activity in DPPH (54.03%) and FRAP (397.56 µg/10g FW) assay. The elicitation of cultured tissues is therefore necessary to improve the production of phytochemical compounds and to increase the antioxidant capacity in sweet shoot. In this study, four different types of cultured tissues (in vitro shoot cultures, light-induced callus, dark-induced callus and somatic embryos) were selected to achieve this goal, followed by the extraction of phytochemicals from these cultured tissues treated with elicitors and precursor for better production of phytochemicals. For in vitro shoot induction, nodal explants cultured on semi-solid MS medium supplemented with 2.0 mg/l 6-benzylaminopurine (BAP) and 0.5 mg/l indole-3-acetic acid (IAA) produced the highest number of shoots (7 shoots per explant) with longer shoot length (5.74 cm). For light-induced callus induction, leaf explants grown in illuminated conditions with semi-solid MS medium enriched with 2.0 mg/l α-naphthalene acetic acid (NAA) and 1.0 mg/l kinetin showed good proliferation from the leaf explants (71.67%) with the highest callus fresh weight (4.53 g) and highest callus expansion rate (18.50 cm2). Likewise, leaf explants induced in dark condition on semi-solid MS medium containing similar hormone composition, also displayed the highest callus fresh weight of 4.54 g and callus expansion rate of 13.85 cm2. Three-month-old dark-induced callus were transferred onto liquid MS medium fortified with a different concentration of NAA and kinetin to further ii induce somatic embryos. After three weeks of callus inoculation, as high as 83.33% of embryogenic cell cultures achieved its maximum density of 5.2 ml in liquid MS medium supplemented with 2.0 mg/l NAA and 1.0 mg/l kinetin. In histodifferentiation medium (liquid MS medium containing 1.0 mg/l NAA and 0.5 mg/l kinetin), a mean number of 15.60, 14.80 and 13.20 embryos per g callus of globular, heart-shaped and torpedo-shaped embryos developed respectively after 9 weeks of embryo induction. The torpedo-shaped embryos were then inoculated into hormone-free MS medium and 90% of these embryos successfully differentiated into cotyledonary embryos after 3 weeks of maturation. These results showed a complete ontogeny of sweet shoot somatic embryo from the globular stage to heart-shaped, torpedo-shaped and cotyledonary stage. After two months of shoot initiation on MS medium supplemented with 2.0 mg/l BAP and 0.5 mg/l IAA, a relatively high percentage (>75%) of shoot proliferation occurred from nodal derived shoot (6.74 shoots per explant), light-induced callus (6.23 shoots per callus) and somatic embryos (6.45 shoots per embryo) of sweet shoot. These well proliferated shoots were then subjected to root initiation in half strength MS medium containing 1.0 mg/l IAA and high percentage of root formation was successfully achieved in 90% of the plantlets after 10 days of culture. For hardening off, the rooted plantlets were transferred to culture jars containing purified water and maintained at ambient conditions for one month. High survival rate (>76.67%) was achieved in perlite:compost mixture (1:1) after one month of acclimatization in shadehouse. To enhance the production of phytochemicals and antioxidant capacity in tissue cultures of sweet shoot, elicitor and precursor treatments served as alternative methods in influencing the biosynthetic pathway for the accumulation of phytochemicals. In this study, shadehouse-grown plants and cultured tissues of sweet shoot were treated individually with different concentrations of methyl jasmonate (MJ), salicylic acid (SA) and phenylalanine (Phe) for a treatment period of 3 weeks. Light-induced callus culture produced the highest amount of phenolic and flavonoid compounds amongst the tested plant samples with those treated with Phe producing the highest antioxidants, followed by MJ and SA. After 3 weeks of Phe treatment at 20 mg/l, the highest levels of total phenolic (246.62 µg/10g FW), total iii flavonoid (636.26 µg/10g FW), naringenin (12081.05 µg/10g FW), quercetin (134.36 µg/10g FW), kaempferol (11325.13 µg/10g FW) and antioxidant activities (97.35% for DPPH and 5941.66 µg/10g FW for FRAP assay) were detected in light-induced callus cultures of sweet shoot. Since phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) were the key enzymes for the biosynthesis of phenolics and flavonoids, both enzymatic activities were also measured in light-induced callus cultures treated with Phe. The highest PAL (101.18 mmol CA/g FW) and CHS (14.49 nkat/mg protein) enzymatic activities were also attained in light-induced callus cultures fed with 20 mg/l of Phe at week 3. Light-induced callus cultures treated with Phe produced the highest amounts of phytochemicals, antioxidant capacity and enzymatic activities, and these results were chosen to undergo Pearson’s correlation coefficient analysis, which verified the positive co-relationship seen between all of the above-mentioned parameters. These findings showed that the addition of Phe enhanced the enzymatic activities in the phenylpropanoid pathway and increased the concentrations of phenolic and flavonoid compounds (naringenin, quercetin, kaempferol) which in turn contributed to the increase in antioxidant activities in light-induced callus cultures of sweet shoot. Data from this study showed that sweet shoot has the potential to be developed as a plant-based antioxidant for the pharmaceutical industry. Additionally, this study is the first to report on the complete ontogeny of sweet shoot and the positive effects of elicitation in tissue cultures of sweet shoot. iv ACKNOWLEDGEMENTS First and foremost, I would like to express my deepest appreciation and sincerest gratitude to those who tirelessly supported me throughout my PhD studies. I would foremost like to thank my mentor, friend and teacher, Assoc. Prof Dr. Winnie Yap Soo Ping, for her faith and trust in me while giving me her unerring advice throughout my research project. I truly treasure the experience that I had under her and she has etched in me invaluable guidance, constructive suggestions, constant encouragement and eminent advice throughout my PhD years. I would also love to extend my many thanks to my co-supervisors, Dr. Khoo Teng Jin and Dr. Peter G. Alderson, who have always been very supportive in guiding me and also solving the issues with regards to my research project at any point of time. Their kind support, motivation, enthusiasm and immense knowledge have been of great value in this study. I would like to also say thanks to Dr. Chin Chiew Foan, Dr. Festo Massawe and Dr. Asgar Ali for their invaluable advice and pleasant help with my PhD. This research was also made possible with the kind help and cooperation from Dr. Mahmud bin Tengku Muda Mohamed and Dr. Yahya bin Awang, Faculty of Agriculture, University Putra Malaysia, Serdang, Selangor, Malaysia. Their valuable support in providing and propagating the plant materials were highly appreciated. Additionally, I would like to acknowledge the laboratory assistants in BRC and pharmacy lab, especially Ms. Norasyikin Azlan Hadi Tan, Mr. Foong Hoe Yinn, Ms. Siti Norazlin Muhamad Nor, Ms. Shankari Shyamala and Ms. Nurul Hasila Mohd Ithnin for their excellent assistance during my laboratory experiments. I am also indebted and grateful to Dr. Tan Boon Chin, Ms. Sarah Jane Chiu, Dr. Janet Wong Pik Ching and Dr. Wardah Mustafa, who were willing to share their knowledge and experience with me. They have been my pillars during my difficult times, juggling between my research experiment and thesis writing. I truly appreciate their efforts and cooperation to shape the complete structure of this research project. v Last but not least, I would also like to thank my beloved family, friends and Dr. Cham Weng Tarng for the constant support and enduring together the many sleepless nights. Without their constant words of encouragement and their constant push for me to excel, this thesis would not have succeeded on time. My family has been a great role model for me and has given me the strength and confidence to pull through and finish my research project. vi TABLE OF CONTENTS Content Page ABSTRACT ii ACKNOWLEDGEMENTS v TABLE OF CONTENTS vii LIST OF FIGURES xii LIST OF TABLES xvi LIST OF ABBREVIATIONS xx CHAPTER 1 INTRODUCTION 1.1 Natural products in drug discovery 1 1.2 Neglected and underutilized crop species 3 1.3 Sauropus androgynus (sweet shoot) 3 1.4 Research development on medicinal plants and 5 sweet shoot 1.5 Aims and objectives 6 2 LITERATURE REVIEW 2.1 Origin and
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