IN VITRO DIRECT AND INDIRECT ORGANOGENESIS AND PLANT REGENERATION OF KENAF (Hibiscus cannabinus L) VAR. KB6

Transcription

1 IN VITRO DIRECT AND INDIRECT ORGANOGENESIS AND PLANT REGENERATION OF KENAF (Hibiscus cannabinus L) VAR. KB6 ZETI ERMIENA SURYA BT MAT HUSSIN MASTER OF SCIENCE 2016

2 In Vitro Direct And Indirect Organogenesis And Plant Regeneration Of Kenaf (Hibiscus cannabinus L) Var. KB6 by Zeti Ermiena Surya Bt Mat Hussin A thesis submitted in fulfilment of the requirements for the degree of Master of Science Faculty of Agro Based Industry UNIVERSITI MALAYSIA KELANTAN 2016

3 THESIS DECLARATION I hereby certify that the work embodied in this thesis is the result of the original research and has not been submitted for a higher degree to any other University or Institution. OPEN ACCESS I agree that my thesis is to be made immediately available as hardcopy or on-line open access (full text). EMBARGOES I agree that my thesis is to be made available as hardcopy or on-line open access (full text) for a period approved by the Post Graduate Committee. CONFIDENTIAL (Contains confidential information under the Official Secret Act 1972)* RESTRICTED (Contains restricted information as specified by the organization where research was done)* I acknowledge that Universiti Malaysia Kelantan reserves the right as follows. 1. The thesis is the property of University Malaysia Kelantan. 2. The library of Universiti Malaysia Kelantan has the right to make copies for the purpose of research only. 3. The library has the right to make copies of the thesis for academic exchange. SIGNATURE SIGNATURE OF SUPERVISOR I/C/PASSPORT NO. Date: NAME OF SUPERVISOR Date:

4 ACKNOWLEDGEMENTS In the name of Allah Taala, The Most Gracious, with God s help and His amazing grace, I m finally completed this dissertation after going through the four years of challenging and stressful period. Nevertheless, this period is so meaningful because it gives me an opportunity to step foot into the realm of true research work. I would like to take this opportunity to express my appreciation to their efforts and kindness. First and foremost, I thank my chairperson supervisory committee, Dr. Dwi Susanto for his invaluable guidance, suggestions, and help throughout the course of this study. I also wish to express my deep sense of gratitude and sincere thanks to Dr Mohammed Arifullah and Dr. Fatimah Kayat who kindly provided me with their knowledge, guidance, and advice in carrying out this study as well as completion of this thesis. They also struggled to setup new laboratory for tissue culture in Jeli Kampus to give the best tissue culture laboratory to us. I also would like to million thank to all staff members at tissue culture laboratory, especially to En. Suhaimi from Faculty of Agro Based Industry, Universiti Malaysia Kelantan, which is person I considered as a good helper and very supportive. I also want to thank my labmates, Mr. Ahmed, Husna, Ilfah Husna and Vikram, I owe a special word of thanks for their ready help and timely courtesy. Not forgetten my Jeli Penthouse group which act as a spirits around me and also their endless care, help and moral support given to me, I d really appreciate our memorable moments. Last but not least, I would like to express my deepest gratitude to my beloved family especially to my parents, my lovely husband Mohd Hamka and my dear uncle Dato Pahamin A. Rajab for their unstinting love, endless encouragement, concern, patience and sacrifices which had helped me in undertaking and completing this study. I could not have asked for better without them, My study would not have been possible. ALHAMDULILLAH! Zeti Ermiena Surya Bt Mat Hussin

5 TABLE OF CONTENTS PAGE THESIS DECLARATION ACKNOWLEDGMENT TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS ABSTRACT ABSTRAK i ii iii vi ix xii xiv xv CHAPTER 1 INTRODUCTION 1.1 Uses of kenaf (Hibiscus cannabinus L) Objectives of the study 5 CHAPTER 2 LITERATURE REVIEW 2.1 Plant tissue culture Micropropagation Kenaf Plant describtion Stem (Stalk) Leaf Inflorescence Fruit & seed Kenaf variety KB A brief review on in vitro studies of kenaf 19 CHAPTER 3 MATERIALS AND METHODS 3.1 Materials Plant collection 22

6 3.1.2 Chemical, glassware and equipment list In vitro propagation method Washing and storage of glassware Preparation & composition of nutrient media Preparing the sterile transfer hood Culture condition and subculture Experimental protocol for kenaf (Hibiscus cannabinus L) Direct shoot organogenesis Indirect shoot organogenesis Acclimitization procedure Statistical analysis 32 CHAPTER 4 RESULTS AND DISCUSSION 4.1 Shoot tip culture Direct shoot organogenesis from shoot tip explant Indirect shoot organogenesis from shoot tip explant Node culture Direct shoot organogenesis from node explant Indirect shoot organogenesis from node explant Leaf culture Direct shoot organogenesis from leaf explant Indirect shoot organogenesis from leaf explant Petiole culture Direct shoot organogenesis from petiole explant Indirect shoot organogenesis from petiole explant Acclimitization The effect of humidity 76

7 CHAPTER 5 CONCLUSION 5.1 Summary & Conclusion Recomendation for future research Schematic sketch of micropropagation protocol for kenaf var. KB6 82 REFERENCES 83 APPENDIX 92 Appendix A-Table A.1 92 Appendix B-Figure B Figure B.2 94

8 LIST OF TABLES NO. PAGE 2.1 Previous study on kenaf propagate through tissue culture 21 technique 3.1 Distribution of plant growth regulator (PGR) BAP and IAA 29 (mg/1) in MS medium for direct organogenesis shoot induction 3.2 Distribution of PGR NAA and IAA (mg/1) MS medium for root 30 induduction 3.3 Distribution of PGR 2,4-D and Kinetin (mg/l) in MS medium to induce callus in indirect organogenesis Distribution of PGR BAP and IAA (mg/l) in MS medium for shoot 31 induction from callus 4.1 The percentage multiple shoot, average number of shoot 35 induction average of shoot length and average number of leaves for shoot tip culture using MS medium with different concentration of BAP and IAA (mg/l) 4.2 Percentage and average of root induction, root length, root number 37 from regenerated shoot using NAA (mg/l) verses IAA (mg/l) 4.3 The percentage pf explants forming callus and cullusing morphology 41 from shoot tip induction of callus using MS medium different concentration of KN (mg/1) and different concentration of 2,4-D (mg/1) 4.4 Frequency of callus generate into multiple shoot, average of shoot induction per explants and average of shoot length from shoot tip callus using MS medium with different concentration of BAP (mg/l) and IAA (mg/l) Rooting induction of regenerated shoot from shoot tip callus on 43 MS medium with various concentration of NAA and IAA (mg/1)

9 4.6 The percentage of multiple shoot, average number of shoot induction 47 average of shoot length and average number of leaves for node cultureusing MS medium with different concentration of BAP (mg/l) and different concentration of IAA (mg/l) 4.7 Percentage and average of root induction, root length, root 48 number from node culture regenerated shoots using MS medium and different level of NAA (mg/l) and MS medium and different level of IAA (mg/l) 4.8 The percentage of explants forming callus and callusing morphology 51 from node induction of callus using MS medium different concentration of KN (mg/l) and different concentration of 2,4-D (mg/l) 4.9 Frequency of callus generate into multiple shoot, average of 52 shoot induction per explants and average of shoot length from node callus using MS medium with different concentration of BAP (mg/l) and different concentration of IAA (mg/l) 4.10 Rooting induction of regenarated shoot for node callus using 54 MS medium with different level of NAA (mg/l) and MS medium and different level of IAA (mg/l) 4.11 Frequency of shoot induction, percentage of root form, average 57 number of root per explants, average of root length in induction of shoot stage from leaves culture using MS medium with different concentration of BAP and IAA (mg/l) 4.12 The frequency explants forming callus and callus morphology, 62 in indirect organogenesis from leaves culture using MS medium with different concentration of KN and 2, 4-D (mg/l) 4.13 The frequency of callus generate into multiple shoot (%), average 63 of shoot induction per explants and average of shoot length from leaves culture callus using MS medium with different concentration of BAP and IAA (mg/l) 4.14 Rooting induction of regenerated shoot for leaves callus using 64 MS medium with different level of NAA (mg/l) and MS medium and different level of IAA (mg/l) 4.15 Percentage of explants forming callus and callus morphology 70

10 from petiole culture induction of callus using MS medium with different concentration of KN and 2, 4-D (mg/1) 4.16 The frequency of callus generate into multiple shoot (%) average of shoot induction per explants and average of shoot length (cm) from petiole culture callus using MS medium with different concentration of BAP and IAA (mg/l) Rooting induction of regenerated shoot for petiole callus using 71 MS medium with different level of NAA (mg/l) and MS medium and different level of IAA (mg/l) 4.18 Different high of plantlets, the percentage of explants survive and the different number of explants leaves for acclimitization process from effect of humidity 74

11 LIST OF FIGURES NO. 2.1a Leaves of kenaf was picked fresh from farm 2.1b Canabis sativa or Marijuana leaf identical to kenaf's leaf (Source from Wikipedia) PAGE a Flower of the kenaf freshly picked from farm b Center of the flower, red in colour c Pollen grain under the microscope (source from Ahmed et al, 2014) a The wedges-shaped seed of kenaf, seed sample collected from MARDI Telong 3.1 Preparation of stock solutions and volumes taken from the stocks to prepare 1 liter of MS medium. Gamborg and shyluk (1981), Gamborg (1982), 1991) and Dodds and Roberts (1982) in (Aurifullah, 2006) 4.1a Single initiation of shoot tip explants on MS medium on 1st day of inoculation, rapid observation was done on first 10 days to observe the contamination 4.1b Single shoot initiation of shoot tip explants on MS medium with 0.05 mg/l BAP and 0.01 mg/l IAA after 2 weeks 4.1c Shoot proliferation on MS medium with 0.05 mg/l BAP and 0.01 mg/l IAA after 3 week d Multiple shoot production MS medium with 0.05 mg/l BAP 38 and 0.01 mg/l IAA after 5 weeks 4.1e Induction and elongation of multiple shoot on MS medium with 0.05 mg/l BAP and 0.01 mg/l IAA after 6 weeks 4.1f Elongation of single plantlet and rooting of in vitro raised shoot on MS basal medium on 8 weeks 4.2a Shootip explants were inoculated in MS medium with KN and 2, 4-D on first day

12 4.2b Callus induction for shoootip explants in MS medium with 0.2 mg/l 2,4-D after 10 days c Shoot induction from callus in MS medium with 0.1 mg/l BAP 45 and 0.03 mg/l IAA after 20 days 4.2d Shoot elongation and multiplication on MS medium with 0.1 mg/l BAP 45 and 0.03 mg/l IAA after 5 weeks 4.2e Single shoot elongation & rooting in MS basal medium on 45 2nd week in rooting medium 4.3a Direct organogenesis of node explants in MS medium with 0.1 mg/l 49 BAP after 6 days 4.3b Shoot proliferation & petiole fall down from node explants 49 in MS medium with 0.5 mg/l BAP and 0.05 mg/l IAA after 14 days 4.3c Multiplication of shoot in MS medium with 0.5 mg/l BAP 49 and 0.05 mg/l IAA after 5 weeks 4.3d Elongation of single plantlet and rooting of in vitro raised shoot 49 on MS medium basal on 8 weeks 4.4a Indirect organogenesis of node explants in MS medium 55 with 0.2 mg/l 2, 4-D on 1st 7 days start to swollen at the bottom 4.4b Callus induction from nodal explants on MS medium 55 with 0.2 mg/l 2,4-D after 12 days 4.4c Shoot induction from pieces of callus on MS medium 55 with 0.1 mg/l BAP after 3 weeks 4.4d Shoot induction from nodal callus on MS medium 55 with 0.1 mg/l BAP after 5 weeks 4.5a Direct organogenesis of leaves on MS medium with BAP and IAA in first week b Root form from leaves direct organogenesis on MS medium 59 with 0.05 mg/l BAP and 0.01 mg/l IAA after 9 days 4.5c Abudant of root from leaves culture on MS medium 0.1 mg/l BAP 59 with 0.05 mg/l IAA after 4th weeks

13 4.6a Indirect organogenesis of leaves on MS medium with KN and 2, 4-D first week of inoculation b Callus induction of leaves indirect organogenesis on MS medium 66 with 0.2 mg/l 2, 4 D after 14 days 4.6c Callus induction of leaves indirect organogenesis on MS medium 66 with 0.2 mg/l 2, 4 D after 25 days 4.6d Shoot induction from callus in MS Medium with 0.3 mg/l BAP 66 and 0.03 mg/l IAA after 20 days 4.7a Indirect organogenesis of petiole in MS medium 75 with KN and 2, 4-D on 1st day of inoculation 4.7b Callus induction after 15 days on MS medium with 0.2 mg/l 2,4-D c Callus induction after 10 days on MS medium 1.0 mg/l KN 75 with 0.1 mg/l 2,4-D 4.7d Initiation of multiple shoot from callus after 25 days on MS medium 75 with 0.1 mg/l BAP and 0.02 mg/l IAA 4.7e Elongation and rooting of multiple shoot after 47 days on MS medium 75 with 0.1 mg/l BAP and 0.02mg/l IAA 4.8a Combination of sand, coco pit and vermiculite with 3:2:2 ratios b Plantlets were covered with the hole plastic containers on 78 1 st day of acclimatization 4.8c The plantlet survive after 9 days of acclimatization under shaded roof d Field plant of kenaf plantlet was successfully tested after 15 days in 78 shaded roof. The plantlets were 40 days

14 LIST OF ABBREVIATIONS µm Micron μg Microgram μm Micrometer µl Microlitre % Percent C Degree Celsius 2,4-D 2,4-Dichloro-phenoxy acetic acid BAP Benzyl-6-aminopurine C2H5OH Ethyl Alcohol cm Centimeter Fig. Figure g Gram h Hour HgCl2 Mercuric Chloride IAA Indole-3-acetic acid KIN Kinetin KNO3 Potassium Nitrate m Meter M Molarity min Minute ml Millilitre mg Miligram mg/l Miligram/litre mm Millimetre MS Murashige and Skoog s Medium n Sample size NAA Naphthalene-3-acetic acid PGR Plant growth regulator/s s Second Sp Species

15 Direct and indirect organogenesis and plant regeneration of an industrial plant kenaf (Hibiscus cannabinus L) var. KB6 ABSTRACT As an important fiber crop, many potential applications of kenaf are being identified and developed in 21 century, especially in developed countries such as America, Japan, and France and Malaysia as well. The present study report a protocol for the efficient in vitro propagation of kenaf (H.cannabinus L) to initiate multiple shoot from mother plant part (shoot, petiole, node and leaf) through direct and indirect organogenesis using MS medium + BAP + IAA for direct organogenesis and MS medium + KN+2,4-D for indirect organogenesis to get callus and after 8 weeks, the calli were put in the MS medium + BAP +IAA for shoot induction. The highest number of shoots produced from node explants part via direct organogenesis (16.33/explants) in MS medium mg/l BAP mg/l IAA. The highest percentage explants forming callus and callus generate into shoot also from node explants part which was induced 75% callus from explants and 73.33% of callus turn into shoot in MS medium mg/l BAP. Several subcultures were drived in order to enhance the multiplication rate. The treatments have their significant different with others. The shoots then were transferred to the root induction medium. Shoots showed the vigorous roots in the MS basal medium. The in vitro rooted plantlets were acclimatized in sand+ coco pit + vermiculite with ratio 3:2:2 and were covered with hole container for 0-15 days to test the effect of humidity to plantlets. The 9 th -15 th days explants covered with container showed 100% of survive. Survive well rooted plantlets were transferred to the field. Plants grew well into maturity without any remarkable morphological variations within the treatments.

16 Regenerasi Tumbuhan Secara Langsung & Secara Tidak Langsung Tumbuhan Industri, Kenaf (Hibiscus cannabinus L) var. KB6 ABSTRAK Sebagai salah satu tanaman yang penting, banyak aplikasi potensi kenaf sedang dikenalpasti dan dibangunkan di abad 21, terutama di negara-negara maju membangun seperti Amerika, Jepun, dan Perancis dan juga Malaysia. Laporan kajian ini adalah untuk mengkaji protokol yang terbaik dalam pembiakan in vitro kenaf (H.cannabinus L) dengan menggunakan eksplan dari pelbagai bahagian pada induk kenaf variasi KB6 (pucuk, daun, nod dan daun nod) melalui organogenesis langsung dan tidak langsung dengan menggunakan MS medium + BAP + IAA untuk organogenesis langsung dan MS sederhana + KN + 2,4-D untuk organogenesis tidak langsung untuk mendapatkan kalus dan selepas 8 minggu, kalus telah dimasukkan ke dalam medium MS + BAP + IAA untuk mendapatkan pucuk. Bilangan tertinggi pucuk dihasilkan dari nod eksplan bahagian melalui organogenesis langsung (16.33 / eksplan) dalam MS medium mg / l BAP 0,05 mg / l IAA. Peratusan tertinggi eksplan yang membentuk kalus dan kalus berpotensi mengeluarkan pucuk juga dari nod eksplan sebahagian yang disebabkan 75% kalus daripada eksplan dan 73.33% daripada kalus bertukar menjadi pucuk di MS medium mg / l BAP. Beberapa subkultur telah dilakukan bagi meningkatkan kadar pengeluaran pucuk. Setiap rawatan mempunyai perbezaan yang signifikan dengan rawatan lain. Pucuk kemudian dipindahkan ke medium induksi akar. Pucuk mengeluarkan akar yang baik dalam medium asas MS. In vitro anak pokok berakar telah dipindahkan ke medium pasir + coco pit + vermikulit dengan nisbah 3: 2: 2 dan ditutup dengan bekas berlubang untuk 0-15 hari untuk menguji kesan kelembapan ke atas anak pokok. Anak pokok yang ditutup dengan bekas selama 9-15 hari menunjukkan 100% daripada bermandiri. Anak pokok yang berakar dipindahkan ke tanah lapang di luar kawasan rumah hijau. Anak pokok tumbuh dengan baik matang tanpa apa-apa perubahan morfologi yang luar biasa dalam rawatan.

17 CHAPTER 1 INTRODUCTION 1.1 Uses of kenaf (Hibiscus cannabinus L) Kenaf (Hibiscus cannabinus L.) is widely known as an important family of Hibiscus for modern needs. The reason behind it is apparently because it produces high biomass, and also able to adapt well with local environment. Kenaf has been adopted in many countries ever since, including Malaysia. This unique plant is still new in Malaysia and has been cultivated generally in the eastern part of the country, especially Kelantan. Initially, kenaf is to replace tobacco industry since it is no longer supported by the government. Far in Africa, kenaf is used in anaemic therapy (Charles et al., 2002). This plant can rise up to cm height as young as 2-3 months. U.S. Department of Agriculture reported that kenaf can produce up to 10 tons of dry fiber per acre per year. That is 3 to 5 times greater than the production of Southern pine trees, in which it would take 7 to 40 years before it can be harvested (McLean et al., 1992). As known in the botanic field, kenaf is classified in various types in which each of them can perform at their best in certain locations, conditions and other variables. These groups of kenaf are known to produce different flowering schedule. Some may be harvested earlier than the other and some later. The good part of it is the flowering can last up to 3 to 4 weeks or more, with each individual bloom can last only for one whole day (Charles et al., 2002).

18 Kenaf s flowers would eventually bloom at the end of growing season. The process takes place when the flower drops off and leave a seed pod behind. The seeds, however, will not get matured in many parts of U.S. Although some of the plant s varieties are known to bloom early, its biomass production is still not tangible enough to provide adequate amount of fiber economically. This is because of their African origin require an additional days of frost free condition for the seeds to germinate. Because of this particular, kenaf is not suitable for massive plantation across the country like a normal weed. It also presents some interesting challenges for developers to ensure a consistent supply of seed for next year s crop (McLean et al., 1992). The stem of the plant consists of two recognizable types of fiber. The first layer of fiber is called bast. It is made up of roughly 40% of the stem s total dry weight. The refined bast fiber measures at 2.6mm and is similar to the fiber commonly used to make paper. The core is the second layer. It is made up of 60% of the stem s total dry weight. Its refined fiber measures at approximately 6mm and is comparable to hardwood tree fiber which is used in wide range of paper products (Rowell and Cook, 1998). Harvesting kenaf is not difficult as it can be processed in a common mechanical fiber separator. It is very similar to a cotton gin. While the two fibers are separated through the mechanical separator, they are allowed to be processed independently, in which it provides raw materials for a wide range of products, such as paper, particle board, bioremediation aids and animal bedding (McLean et al., 1992). According to Duke 1983, kenaf was propagated by seed, but seed availability is limited (Sullivan, 2003). Seed production strategies are affected by the cultivar, locationespecially latitude, and cultural practices. The first issue to address is the cultivar photosensitivity, whether the cultivar is an ultra-early, an early to medium, or a late

19 maturing cultivar (Webber III & Bledsoe, 2002). Researchers and seed producers have reported that the strategy for maximizing harvestable kenaf seed is very different than the production approach in maximizing kenaf fiber yields (Mullens, 1998; Webber III & Bledsoe, 2002). Kenaf seed has high oil content (Mohamed et al., 1995) and seed viability decreases over time when stored at higher relative humidity and higher temperatures (Webber III & Bledsoe, 2002). Propagation by seed is not recommended due to the heterogeneous nature of the seedlings owing to its cross pollination. Thus, any kenaf variety will decrease in purity, leading to the increment of seed yields and reduction of fiber yields (Ramesh, 2016). The application of plant tissue culture as a micropropagation technique has become an important biotechnological tool which offers additional advantages such as the rapid multiplication rate (Edwin et al, 2008). Therefore, in vitro propagation methods would be important to propagate this crop for high and stable cellulosic fiber production, as large numbers of genetically identical plants and in a relatively short time for the industrial plantation. Axillary buds are widely used for micropropagation as they have entire rudimentary vegetative shoot and can be induced to develop into plants easily which are similar to the parental type (Zapata et al., 1999; Srivatanakul et al., 2000). The development of tissue culture and regeneration techniques are the most important procedures that must be established in order to improve kenaf by genetic transformation procedure (Banks et al., 1993). There are few reports on the in vitro propagation of the Hibiscus genus, with kenaf being the most studied species in terms of organogenesis regeneration. According to McLean et al. (1992), organogenesis of kenaf via callus culture was not reproducible and resulted in very low regeneration efficiency and the induction of heritable mutation. Preliminary research on shoot regeneration of

20 kenaf callus used the shoot apex still unsuccessful though direct shoot regeneration without a callus phase has been achieved (Zapata et al., 1999; Srivatanakul et al., 2000), while other authors reported the formation of multiple shoot from young shoot and cotyledons (Khatun et al., 2003; Herath et al., 2004). A protocol for plant regeneration from callus using segments of hypocotyls and cotyledons has been established for the H. siryacus (Jenderek & Olney, 2001). The establishment methods for sterilization from mother plant also have not been proposed by any researcher due to high contamination in culture. Using seeds as a means of plant propagation is simple and can be produced by farmers, but disadvantage are the progeny may be genetically different from the parents and some plant have the dormancy period. So, in vitro propagation is the one of the alternative techniques to solve these problems and can be used for mass propagation of seedlings. That s why this experiment was proposed.

21 1.2 Objectives of the present study Therefore, the aims of this research were to: (a) To analyses the effect of different plant growth regulator on direct and indirect organogenesis of kenaf var. KB6 (b) To develop a protocol for kenaf var KB6 (Hibiscus cannabinus L) in vitro regeneration using different explants like shoot tips, nodes, leaves and petioles

22 CHAPTER 2 LITERATURE REVIEW 2.1 Plant tissue culture Plant tissue culture has gone through a long developmental history to achieve its current state. The history and milestones of tissue culture development has been elaborated in many reviews (Gamborg, 2002; Thorpe, 2007; Vasil, 2008). Plant tissue culture is an essential component of Plant Biotechnology. Plant cell and tissue culture has already contributed significantly to crop improvement and has great potential for future (Mehbooba et al., 2011). Research efforts in plant cell and tissue culture have increased dramatically worldwide in recent years including efforts in developing nations. Plant cell and tissue culture is defined as the capability to regenerate and propagate plants from single cells, tissue and organs under sterile and controlled environmental conditions (Murashige, 1974). According to George & Debergh (2009), tissue culture techniques are now being widely applied for improvement of field crop, forest, and horticulture and plantation crop for increased agricultural and forestry production. Today tissue culture technology is being exploited mainly for large scale production or micropropagation of elite planting material with desirable characteristics. This technology has now been commercialized globally and has contributed significantly towards the enhanced production of high quality planting material. Recently emphasis has been on genetic transformation, especially for (1) increased production of secondary metabolite for example, Bourgaud et

23 al., 2001, was studied about secondary metabolite from hairy roots callus and other organ culture of Plumbago indica L. The work on in vitro production of secondary metabolites also has been reviewed by (Ramachandra & Ravishankar, 2002). Many researchers have reported the production of secondary metabolites by cell cultures where the synthesis and yield of compounds approached or exceeded the levels found in natural plant resources (Eran & David, 2000; Nakashima et al., 1997), (2) production of alkaloids pharmaceutics, nematocidal compounds, and also some novel compounds not found in the whole plants, regeneration of plant resistant to herbicides, disease, and pests (Leslie & Johan, 2000) (3) scale up of cultures in bioreactors, Jacqueline & John (1999), was build a research about hairy root culture by setup the bioreactor culture, (4) plants with different morphological traits and (5) transgenic crops for production of recombinant vaccines and anti-microbial antibodies (Peter & Stoger, 2011). These developments have far-reaching implications in the improvement of medicinal plants as well (Bajaj, 1990). 2.2 Micropropagation Micropropagation is a rapid multiplication of a selected plant using in vitro culture techniques and it s developed in past 35 years (Aneesha, 2015). In vitro clonal propagation provides true to type plants of a selected genotype using in vitro culture techniques (Thorpe, 2007). Micropropagation is also used to promote germplasm storage for maintenance of disease-free stock in controlled environmental condition and in long term via cryopreservation (Nukari et al, 2009). Micropropagation also gives the rapid production of high quality, disease-free and uniform planting material (Ahloowalia et al,

24 2004). The plants can be multiplied under a controlled environment, anywhere, irrespective of the season and weather, on a year-round basis (Bourgaud et al., 2001). Production of high quality and healthy planting material of ornamentals, and forest and fruit trees, propagated from vegetative parts, has created new opportunities in global trading for producers, farmers, and nursery owners, and for rural employment (Ahloowalia et al, 2004). One of the most exciting and important aspects of in vitro cell and tissue culture is the capability to generate and propagate plants from cultured cells and tissues. The simplest type of in vitro plant propagation is the stimulation of axillary bud development (Edwin et al., 2008). This technique exploits the normal ontogenetic route for branch development by lateral (axillary) meristems. The axillary buds are treated with hormones to break dormancy and produce shoot branches (Zhu et al, 2015). The shoots are then separated and rooted to produce plants. Alternatively, the shoots are used as propagules for further propagation (Aneesha, 2015). Plant regeneration from cultured tissue can also be achieved by culturing tissue section lacking a preformed meristem (adventitious origin) or from callus and cell cultures (de novo origin) (De Filippis, 2014). Axillary buds are preformed meristems. In contrast, adventitious regeneration events occur at unusual sites of a cultured tissue such as the internode, leaf blade, cotyledon, or root elongation zone, where meristem do not naturally occur (Edwin et al, 2008). According to Nukari et al. (2009), adventitious plant regeneration often is dependent upon the presence of organized explants tissue. In comparison, de novo (literally, to arise new ) plant regeneration occurs from callus and cell cultures in the absence of organized explants tissues. Whether adventitious or de novo in origin, plant regeneration can occur by one of two processes. Organogenesis is the formation of individual organs, such as shoots or

25 roots. Somatic embryogenesis is the formation of a bipolar structure containing both shoot and root meristems, and developing in a manner similar to zygotic embryos (Sara et al, 2002). According to Edwin et al (2008), most plant species are capable of plant regeneration by either organogenesis or somatic embryogenesis, but very few species are capable of both. Some species are easy to regenerate from callus or cell cultures, while others regenerate only by an adventitious process. The choice of plant species and the goal of the research will determine the plant regeneration or propagation procedure (Vasil, 2008). When available for the same species, the different regeneration approaches may yield different propagation rates (Chu, 1992). Axillary bud proliferation and culture of individual nodes are the techniques most widely used in commercial micropropagation and which show the least variation among the propagated plants (Ngezahayo & Liu, 2014). In contrast, adventitious shoot organogenesis and regeneration of plants from callus by organogenesis or by somatic embryogenesis show the most variation, as well as higher propagation rates (Gest, 2004). Direct (adventitious) somatic embryogenesis and repetitive embryogenesis show the best balance of high propagation rates with relatively few off-types (Gamborg, 2002). There are several defined steps in a typical micropropagation system (Murashige, 1974). The first step is the initiation of a sterile culture of the explant (Stage I). The second step is the multiplication of shoots or other propagules from the explants (Stage II). Adventitious shoot proliferation is the most frequently used multiplication technique in micropropagation systems (Chu, 1992). The culture media and growth conditions used in Stage II systems are optimized for maximum rates of multiplication. The third step is the development of roots on the shoots to produce plantlets (Stage III). Specialized media