In Vitro and In Situ Biomass and Artemisinin Production in Worm Wood (Artemisia Annua l. Var. Chiyong)

 – In Vitro and In Situ Biomass and Artemisinin Production in Worm Wood (Artemisia Annua l. Var. Chiyong) –

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ABSTRACT

Artemisia annuahas been identified for its anti- malarial properties and millions of Nigerians are at high risk of being infected by the disease. Its propagation is hindered by shortage of seeds, germination problem and effective protocol forin vitro and in situ propagation.

The effect of some plant growth regulators (PGRs) on the in vitro regeneration of Artemisia annua using seed, leaf primordial, node explants and callus initiation were investigated in the Biotechnology Laboratory of the Department of Plant Science Ahmadu Bello University, Zaria.

Explants were surface sterilized and inoculated on Murashige&Skoog basal media supplemented with varying concentrations of plant growth regulators. Results of Analysis of Variance for the in vitro experiment indicated significant difference among the treatments (P<0.05).

In situ experiment was conducted on the farm of the Institute for Agricultural Research (IAR) ABU, Zaria. during the 2012 rainy season and 2013 dry season to determine the effect of some physical, chemical and hormone treatments as well as levels of organic and inorganic fertilizers on the growth, yield and artemisinin content in A. annua.

Results of Analysis of Variance (ANOVA) for the in situ experiment indicated no significant difference between the seasons with respect to germination, shoot and root lengths (P≥0.05).

Experiment with GA₃ and BAP, concentrations 1.5 mg/l of GA₃ and 0.5 mg/l BAP recorded the shortest days to in vitro germination. Highest regeneration percentage was observed at 2.0 mg/l of GA₃ and 0.5 mg/l of BAP. A concentration of mg/l of GA₃ and 0.5 mg/lof BAP had the best vigor.

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TABLE OF CONTENT 

LIST OF TABLES     xi

LIST OF FIGURES……………. xiii

LIST OF PLATES      xiv

LIST OF APPENDICES………. xv

CHAPTER ONE        1

• INTRODUCTION 1
  • Soil and Climate…………. 2
  • Economic Importance……………. 2
  • Propagation constraints of Artemisia annua…………… 3
  • Justification……. 4

CHAPTER TWO  

• LITERATURE REVIEW 7
  • Artemisiaannua………… 7
    • The Morphology of annua….. 7
  • Botany of Artemisia annua…………… 10
  • The significance of Artemisia annua……………… 11
    • The use of Artemisia annua in the treatment of malaria fever……… 11
    • The use of Artemisia annua in livestockindustry…………. 12
    • The use of Artemisia annua in the treatment of Cancer…… 12
  • In vitro propagation of Artemisia annua…….. 14
  • Plant Growth Promotors………. 19
  • Gibberellins…….. 22
    • Biological Activity……… 23
    • Growth Promotion…….. 24
  • Cytokinins……………. 25
    • Biological Response…………. 26
    • Organ Development……………. 26
  • In situ Propagation of Artemisia annua…………………. 27
    • Organic and inorganic Manure and the Propagation of annua…. 28
  • Artemisinin………………………… 33
  • Artemisinin Production………………. 36
    • Site of Artemisinin Synthesis…….. 43
  • Artemisinin Extraction…….. 46
• MATERIALS AND METHODS 49
  • Study Area………………………… 49
  • Experimental Materials………………. 49
    • Protocol for in vitro regeneration………….. 52
    • Surface sterilization………… 52
    • Media preparation………………. 52
    • Experimental Design and Data Analysis…. 59
  • Effective treatment for in situ germination…. 59
    • Soil Analysis………… 60
    • Nursery.. 60
  • Sowing…………………… 61
    • Watering……………. 61
    • Effect of organic and inorganic fertilizer on biomass production in situ… 62
    • Ridge Preparation………….. 62
  • Effective protocol and effect of organic and inorganic fertilizer on in vitro and in situ artmisinin production….. 63

• Sample preparation……….. 63
• Determination of Artemisinin………….. 64
• Statistical Analysis…….. 66

CHAPTER FOUR

• RESULTS 67
  • Effective protocol forinvitro regeneration……….. 67
    • Effects of GA₃ and BAP………… 67
    • Effects of BAP and NAA……….. 68
    • Effects of GA₃ and NAA…….. 68
    • Callus induction:………… 79
  • Effective treatment forin situ germination…. 85
  • Effect of Organic and Inorganic Fertilizers on biomass production in situ……. 90
  • Protocol for Artemisinn content in vitro…. 96
    • Effect of organic and inorganic fertilizer on artemisinin production in situ….. 98
    • Relative effectiveness of in vitro and in situ artemisinin …………. 100

DISCUSSION 103

• • Protocol for in vitro regeneration……. 103
  • Effective treatment for in situ germination…. 109
  • Summary…………. 120
  • Conclusion……….. 122
  • Recommendations……….. 123

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INTRODUCTION

Artemisia annua L (Wormwood) belongs to the family Asteraceae (Ferreira, and Janick, 2002), whichconsist of about 400 species (Bailey and Bailey, 1976; Bennett et al., 1982; McVaugh, 1984 El- haq et al., 1991; Klayman, 1993; Jaime and Da silver, 2003). The family is characterized by extreme bitterness of all parts of the plant (Tripathi et al, 2000; Tripathi et al., 2001; Ferreira and Janick, 2009).

Artemisia annua is a new crop in Nigeria. Its propagation  started  in  2003  while  artemisinin  extraction commenced in 2005. As  of  August  2007,  over  1,500  ha  were  prepared  for  planting  with seeds from Brazil, China and local breeds (Anonymous, 2007; National Academics, 2007).

Cultivation of A. annua spread from China to Africa, mainly Kenya,  Tanzania  and  Nigeria  in  response to the call by the World Health Organization for the use of Artemisinin-Combination  Therapies (ACT). The  most  concentrated  areas  of  Artemisia  production  are  Asia,  Europe,  and  USA and  recently  East  Africa  (Klayman,  1993).

However,  global  estimates  for  A.  annua  revealed a gradual increase in production area of 2,000 ha in 2003, 3000 ha in 2004, 9,500 ha in 2005, 26.000 ha in 2006, 14,500 ha in 2007, 4,500-5,000 ha in 2008, 6,000 ha in 2009, 15,000 ha in 2010, 17,500 ha in 2011 and 16,500 ha in 2012 (Artepal,  2008;  Hiey,  2010;  Ut,  2011).

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REFERENCES

Abdin, M.Z., Israr, M., Rehman, R.U., and Jain, S.K. (2003). Artemisinin, a novel anti-malarial drug: Biochemical and molecular approach for enhanced production. Planta Medica.69 (4):289–299.
Abobkar I. M. Saad and Ahmed M. Elshahed (2012).Plant Tissue Culture Media: In Recent Advances in Plant in vitro Culture. http://dx.doi.org/10.5772/50569. Pp 12.
Advanced  Bio-Extracts  (ABE)  (2006).―The  Accelerated  Production  of  Artemisinin  in  East  Africa, Project Summary,‖Advanced Bio-Extracts Ltd.‖ Nairobi, Kenya.
Advanced   Bio-Extracts   (ABE)   (2007).   ―Project   Status   Report,‖  Advanced   Bio-Extracts   Limited, Nairobi, Kenya.
Acton, N., and Klayman, D.L.(1985) Artemisinin, a new sesquiterpene lactone endoperoxide from
Artemisia annua.Planta Medica 47: 442-45.
Agboola, A.A. (1986).Solving the problem of soil fertility in the African semi-arid zone.Proceedings of an International Drought Symposium, Nairobi, Kenya.

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