MICROPROPAGATION AND PIGMENT EXTRACTION OF Echinocereus cinerascens HASHIMAH ELIAS FACULTY OF SCIENCE UNIVERSITY OF MALAYA KUALA LUMPUR 2017 MICROPROPAGATION AND PIGMENT EXTRACTION OF Echinocereus cinerascens HASHIMAH ELIAS THESIS SUBMITTED IN FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSTITUTE OF BIOLOGICAL SCIENCES FACULTY OF SCIENCE UNIVERSITY OF MALAYA KUALA LUMPUR 2017 UNIVERSITY OF MALAYA ORIGINAL LITERARY WORK DECLARATION Name of Candidate: Hashimah Binti Elias Registration/Matric No: SHC110033 Name of Degree: Doctor of Philosophy (Ph.D.) Title of Project Paper/Research Report/Dissertation/Thesis (“this Work”): Micropropagation and Pigment Extraction of Echinocereus cinerascens Field of Study: (Science) Plant Biotechnology I do solemnly and sincerely declare that: (1) I am the sole author/writer of this Work; (2) This Work is original; (3) Any use of any work in which copyright exists was done by way of fair dealing and for permitted purposes and any excerpt or extract from, or reference to or reproduction of any copyright work has been disclosed expressly and sufficiently and the title of the Work and its authorship have been acknowledged in this Work; (4) I do not have any actual knowledge nor do I ought reasonably to know that the making of this work constitutes an infringement of any copyright work; (5) I hereby assign all and every rights in the copyright to this Work to the University of Malaya (“UM”), who henceforth shall be owner of the copyright in this Work and that any reproduction or use in any form or by any means whatsoever is prohibited without the written consent of UM having been first had and obtained; (6) I am fully aware that if in the course of making this Work I have infringed any copyright whether intentionally or otherwise, I may be subject to legal action or any other action as may be determined by UM. Candidate’s Signature: Date: Subscribed and solemnly declared before, Witness’s Signature: Date: Name: Designation: ii ABSTRACT Protocols were successfully established for in vitro regeneration and coloured callus production of Echinocereus cinerascens. Investigation mainly focused on the effects of plant growth regulators in rapid production of this endangered species and the optimum production of coloured callus. Additional assessments studied concerning the production of synthetic seeds, extraction of natural pigments and detection of somaclonal variation of the regenerants. Rapid production through direct in vitro regeneration gave the highest mean number of shoots, 4.37 ± 0.27, observed in MS medium supplemented with 2.0 mg/l Kinetin + 1.0 mg/l IBA which promoted the highest production of shoots after 4 months, 131 shoots in total. Nevertheless, through indirect in vitro regeneration, somatic embryos of Echinocereus cinerascens were successfully developed in two treatments of liquid medium including MS medium supplemented with 0.5 mg/l 2,4-D + 0.1 mg/l BAP + 0.5 mg/l thiamine HCl and MS medium supplemented with 0.5 mg/l 2,4-D + 0.5 mg/l BAP + 0.5 mg/l thiamine HCl, as both media promoted 100% total mean production of somatic embryos (globular, heart, torpedo-shaped and cotyledonary stage) after 4 months. Practically, the production of ideal synthetic seeds was successfully established whereby, micro shoots as the most responsive propagule were encapsulated in 3% of sodium alginate hardened in 100 mM of calcium chloride dehydrate solution for 30 minutes gave 100% of germination rate after 4 months. Complete plantlets were successfully acclimatized with the highest survival rate of 90% observed in sand, the most suitable planting substrate which possessed 3 major elements such as SiO2, Al2O3 and CaO that play important roles to support the growth of Echinocereus cinerascens. Basically, the present study indicated that 100% production of green, yellow and pink callus was obtained after 2 months in several treatments of MS medium supplemented with 2,4-D + BAP + thiamine HCl applied in combination. Interestingly, the occurrence of dramatic changes in the iii production of coloured callus was clearly observed in a conversion of green to pink callus within 2 months. Moreover, pigment extraction analysis through UV-VIS spectroscopy discovered that both regenerants and callus possessed chlorophyll a and b as the major pigment while carotenoids as the minor pigment. Meanwhile, HPLC analysis revealed individual carotenoids present in in vitro plantlets namely, neoxanthin, β-carotene, lutein and violaxanthin, whereas in callus were β-carotene and lutein only. Analysis of cytological studies clarified that there were no significant differences in cell organization and behaviour of in vitro plantlets and ex vitro plants. The evidence verified that Echinocereus cinerascens regenerated normally in vitro and grown vigorously after being transferred to the natural environment. iv ABSTRAK Protokol telah berjaya ditubuhkan untuk regenerasi in vitro dan penghasilan kalus berwarna Echinocereus cinerascens. Penyelidikan terutamanya difokuskan pada kesan penggalak pertumbuhan dalam penghasilan pesat species terancam ini dan penghasilan optimum kalus berwarna. Penilaian sampingan yang dikaji adalah berkenaan penghasilan biji benih sintetik, pengekstrakan pigmen semula jadi dan pengesanan variasi somaclonal regenerants. Penghasilan pesat melalui regenerasi in vitro secara langsung telah memberikan jumlah tertinggi purata bilangan pucuk, 4.37 ± 0.27, diperhatikan dalam media MS ditambah 2.0 mg/l Kinetin + 1.0 mg/l IBA yang menunjukkan penghasilan tertinggi pucuk selepas 4 bulan, 131 jumlah pucuk keseluruhannya. Walau bagaimanapun, melalui regenerasi in vitro secara tidak langsung, embrio somatic Echinocereus cinerascens telah berjaya dihasilkan dalam dua rawatan media cecair termasuklah media MS ditambah 0.5 mg/l 2,4-D + 0.1 mg/l BAP + 0.5 mg/l thiamine HCl dan media MS ditambah 0.5 mg/l 2,4-D + 0.5 mg/l BAP + 0.5 mg/l thiamine HCl, kerana kedua-duanya menunjukkan 100% jumlah purata penghasilan embrio somatik (peringkat berbentuk globular, hati, torpedo dan kotiledon) selepas 4 bulan. Secara praktikal, penghasilan biji benih sintetik yang ideal telah berjaya ditubuhkan di mana, pucuk mikro sebagai propagul yang paling responsif dikapsulkan dalam 3% sodium alginat dan dipejalkan dalam 100 mM larutan kalsium klorida dihidrat selama 30 minit, memberikan 100% kadar percambahan selepas 4 bulan. Anak pokok lengkap telah berjaya diaklimatasi dengan kadar kelangsungan hidup tertinggi 90% diperhatikan dalam pasir, substrat penanaman paling sesuai yang mempunyai 3 elemen utama seperti SiO2, Al2O3 dan CaO yang memainkan peranan penting untuk menyokong pertumbuhan Echinocereus cinerascens. Pada asasnya, kajian ini menunjukkan 100% penghasilan kalus hijau, kuning dan merah jambu diperolehi selepas 2 bulan dalam beberapa rawatan media MS ditambah 2,4-D + BAP + thiamine v HCl dalam kombinasi. Menariknya, berlaku perubahan dramatik dalam penghasilan kalus berwarna yang jelas diperhatikan dalam penukaran kalus hijau kepada merah jambu dalam masa 2 bulan. Selain itu, analisis pengekstrakan pigmen melalui UV VIS spektroskopi mendapati bahawa kedua-dua regenerants dan kalus memiliki klorofil a dan b sebagai pigmen utama manakala karotenoid sebagai pigmen minor. Sementara itu, analisis HPLC mendedahkan individu karotenoid yang hadir dalam anak pokok in vitro iaitu neoxanthin, β-karotena, lutein dan violaxanthin, manakala pada kalus adalah β- karotena dan lutein sahaja. Analisis kajian sitologi menjelaskan bahawa tiada perbezaan yang signifikan pada organisasi dan tingkah laku sel dalam anak pokok in vitro dan tumbuhan ex vitro. Bukti-bukti ini mengesahkan bahawa Echinocereus cinerascens telah diregenerasi secara normal in vitro dan tumbuh subur selepas dipindahkan ke persekitaran semula jadi. vi ACKNOWLEDGEMENTS IN THE NAME OF ALLAH, THE BENEFICENT AND THE MERCIFUL Alhamdulillah… all praises to Allah. This research is finally succeeded with Allah’s blessings. I would like to express my greatest appreciation to my respectable supervisor, Professor Dr. Rosna Mat Taha, for giving me full supervision with extensive knowledge, meaningful guidance and advice throughout this few years during my candidature session. Besides, I would like to thank the University of Malaya for the financial support, Postgraduate Research Fund (PPP) and all the facilities provided. To my entire family, my mother, my late father, my brothers and sister, thank you for everything. Thank you for always giving me precious advice, motivation and endless love. I love all of you so much. To my mother, Fatimah Salleh, I’m very thankful to have a mom like you. Your special understanding, steadfast support, sacrifice and prayers for me will always keep you deep in my heart. Special thanks to all seniors that are very helpful and knowledgeable, Dr. Nor Azlina Hasbullah, Dr. Asmah Awal, Kak Azani, Kak Ain, Kak Noraini, Kak Ina and Kak Fatimah. Thank you for sharing great moments, valuable experiences and being the best seniors in Lab B 2.5. To my dearest friends, Ima, Diha, Kinah, Shamrul, Anis, Shikin, Aziemah, Ummi, Ema, Mira, Diyana, Ecah and Lydia, I truly appreciate and grateful to have all of you as my friends. Your generous assistance and moral support inspired me to face every challenging moment while your priceless joy and laughter had turned up my life during sadness. Thank you so much, my friends. Last but not least, I wish to thank everyone who has helped me directly or indirectly, willing to share their expertise in order to accomplish