Micropropagation of ximenia americana l from shoot tip explants

ADDIS ABABA UNIVERSITY SCHOOL OF GRADUATE STUDIES INSTITUTE OF BIOTECHNOLOGY Micropropagation of Ximenia americana L from shoot tip explants A Thesis Submitted to the School of Graduate Studies, Addis Ababa University, in Partial Fulfilment of the Requirements for the Degree of Master of Science in Biotechnology By Keredin Mohamed Addis Ababa, Ethiopia June, 2017 i ADDIS ABABA UNIVERSITY SCHOOL OF GRADUATE STUDIES INSTITUTE OF BIOTECHNOLOGY Micropropagation of Ximenia americana L from shoot tip explants A Thesis Submitted to the School of Graduate Studies, Addis Ababa University, in Partial Fulfilment of the Requirements for the Degree of Master of Science in Biotechnology By Keredin Mohamed Keredinmohamed@gmail.com Addis Ababa, Ethiopia June, 2017 ii Micropropagation of Ximenia americana L from shoot tip explants Keredin Mohamed Institute of Biotechnology, Addis Ababa University, PO Box 1176, Addis Ababa, Ethiopia Email: keredinmohamed@gmail.com ABSTRACT: Ximenia americana is a critically threatened multipurpose plant It is among the most important medicinal plants, valued primarily for its fruit, roots, leaves and bark parts This study aimed to develop micropropagation protocol for Ximenia americana using shoot tip explants The study started with seed sterilization test using different clorox concentration and exposure time Shoot tips from in vitro germinated seedlings were cultured on shoot initiation MS medium supplemented with 0.0, 0.5, 1.0, 1.5 and 2.0 mg/l BAP alone or KN alone Explants were cultured on shoot proliferation media fortified with kinetin or BAP, each at 0.0, 0.25, 0.5, 0.75, 1.25, 1.75 and 2.0 mg/l in combination with 0.0, 0.1, 0.20 and 0.40 mg/l NAA on full salt strength MS medium For rooting, half strength MS media supplemented with IBA and NAA alone each at 0.0, 0.1, 0.25, 0.5, 1.0, and mg/l were used Growth regulator free MS medium was used as control Study results showed that 73.32% germination was recorded on 35% clorox concentration within 20 minutes Highest mean shoot number per explants on initiation medium (2.06±0.15) and multiplication medium (4.16±0.17) were recorded on MS medium supplemented with 0.5mg/l BAP Best rooting was obtained on half strength MS media supplemented with 0.5mg/l IBA with 3.36±0.69 roots per shoot and 2.21±0.40 cm root length on 2.0 mg/l IBA and established in greenhouse with 100% survival The results show that, the study is important for mass propagation, rehabilitation in their natural habitat and conservation of this threatened multipurpose plant Key words: Micropropagation, Plant Growth Regulator, shoot tip, Ximenia americana iii ACKNOWLEDGMENTS Firstly I would like to thank Allah Almighty for His immense blessings that gave me the strength and patience to accomplish my work successfully I would like to express my sincere gratitude and esteem to Dr Tileye Feyissa for his helpful advices, affectionate guidance and constant support throughout this research study I am glad and extremely grateful to my mother and wife for their immense love, continuous support and encouragement in every stage of my life I am grateful to acknowledge Addis Ababa University in general and Institute of Biotechnology Program in particular for providing me all academic supports during the study period iv Table of Contents Page List of Tables vii List of Figures viii List of Abbreviations ix List of Appendix…………………………………………………………………….…………….x 1.INTRODUCTION 1.1 Background of the study LITERATURE REVIEW 2.1 Description, ecology, taxonomy and distribution of Ximenia americana 2.2 Significance and uses of Ximenia americana 2.3 Ximenia americana propagation and its challenges 2.4 Plant tissue culture and its application .7 2.5 Advantages of Micropropagation 2.6 Source of Explants and Aseptic Techniques 10 2.7 Composition of Culture Medium requirements for optimal growth of a tissue 11 2.8 Plant Growth Regulators 13 OBJECTIVE 15 3.1 General objective 15 3.2 Specific objectives 15 MATERIALS AND METHODS 16 4.1 Plant material 16 4.2 Experimental Procedures 16 4.2.1 Preparation of stock solutions 16 4.2.2 Plant growth regulators stock solution preparation 16 4.2.3 Culture media preparation 17 v 4.2.4 Explants surface sterilization and in vitro seed germination 17 4.2.5 Shoot initiation 18 4.2.6 Shoot multiplication 18 4.2.7 Rooting 19 4.2.8 Acclimatization 19 4.3 Experimental design 19 RESULTS 20 5.1 Effect of different concentration of sterilants and exposure time on X americana seed explants and in vitro germination 20 5.2 Shoot initiation 20 5.3 Shoot multiplication 23 5.4 Rooting and acclimatization 27 DISCUSSION 29 6.1 Sterilization and in vitro seed germination of X americana 29 6.2 Shoot Induction 30 6.3 Shoot multiplication 31 6.4 Rooting 34 6.5 Acclimatization 35 7.CONCLUSIONS 36 RECOMMENDATIONS 37 REFERENCES 38 10.APPENDIX 46 vi List of Tables Tables Page Table 1: Effect of different concentration of clorox and exposure time on seed explants of X americana …………………………………………….21 Table 2: Effect of different concentrations of KN and BAP on shoot induction from shoot tip of X americana……………………………………………………………………… 21 Table 3: Effect of different concentrations of KN and BAP alone on shoot multiplication of shoot tip of X americana…………………………………… .24 Table 4: Effect of different concentrations and combinations of KN and NAA on shoot multiplication of X americana………………………………………….………………………24 Table 5: Effect of different concentrations and combinations of BAP with NAA on shoot multiplication of X americana… ………………………………………………………………………25 Table 6: Percentage of rooting, mean number of roots and mean root length obtained on half MS rooting medium containing different concentrations and combinations of IBA and NAA 27 vii List of Figures Figure Page Figure 1: Effect of different concentrations of KN and BAP on shoot induction of shoot tip explants of X americana………………………………………………………………… 22 Figure 2: Shoot multiplication from shoot tip explants of X americana on MS medium with different hormone concentrations and combinations ……………………………….……26 Figure 3: In vitro rooting of X americana shoots on 1/2MS medium and acclimatization ….28 viii List of Abbreviations BAP 6-Benzyl Amino Purine IAA Idol acetic acid GA3 Gibberrellic Acid IBA Indol-3-Butyric Acid KIN Kinetin MS Murashige and Skoog basal media NAA Naphthalene acetic acid PGR Plant Growth Regulator ix List of appendix APPENDIX page Appendix 1: Full MS basal medium stock solution composition……………….………………46 x that the high proliferation rate in MS medium containing 1.0 mg/l BAP compared to the low proliferation rate in MS medium containing 1.25 mg/l BAP caused a reduction in the height of shoots length Higher concentrations of BAP had an inhibitory effect on proliferation The current results are in agreement with this author Borthakur et al (2011) investigated different concentration of BAP alone or in combination with NAA on Albizia odoratissima (Bansa) and found that the MS medium supplemented with low concentration of 0.74 mg/l BAP resulted in highest numbers of shoots per explants from apical buds of day old in vitro seedlings In both studies best result was obtained relatively at low concentration of BAP On the other hand highest shoots per explants (2.83±0 36) was recorded on MS media supplemented with 0.5 mg/l KIN But, further increase in the concentration of KIN from 0.5 to 2.0 mg/l significantly decreases from 2.83±0 36 to 1.80± 0.10 mean shoot number per explants This also shows the same trends with BAP in inhibiting shoot proliferation as concentration increases Shoot explants cultured on MS medium supplemented with 0.75 mg/l BAP in combination with 0.1 mg/l NAA exhibited both the highest mean number of shoot (3.23±09) and mean length of shoots (3.98±0 28) per explants The result from combined effect of NAA and BAP was found to vary with the concentration of NAA and BAP in two parameters of shoot growth (mean number of shoot and shoot length) When the concentration of BAP was greater than that of NAA, relatively better result was recorded This is due to the effect of cytokinines as it promotes the axillary branching or axillary bud proliferation (Vieitez and Vieitez, 1980) Explants responded positively with multiple shoots initiated when BAP levels were higher than NAA, beyond that the multiplication rate declined Propagation of Azadirachta indica via axillary buds and nodal segments was investigated by Rodriguez and Ortiz (2001) and found the best medium for shoot regeneration on 3.0 mg/l BAP The finding of these authors is also contrasting to the 32 present one Combining of BAP with NAA to MS medium was comparatively more effective than KIN with NAA for efficient shoot proliferation in shoot tip explants of Ximenia americana These results contrast with the report of (Hung and Stephen, 2011) The positive effects of combined BAP and NAA treatments in Khaya senegalensis But, (Husain and Anis, 2009) agreed with the greater effectiveness of BAP compared with kinetin than kinetin with NAA for shoot proliferation of Melia azedarach Among explants cultured on MS medium supplemented with different concentration of KIN along with NAA show significant differences Highest shoots per explants (2.64±0.05) recorded on 0.75 mg/l KIN combining with 0.1 mg/l NAA This also conform the effect of cytokinines as it promotes the axillary branching (Vieitez and Vieitez, 1980) Further, increase in the concentration of NAA from 0.1 to 0.4mg/l showed a reduction from (2.64±0.05 to 1.31±0.00) in the mean shoot number per explants Generally, the present study were showed that the explants responded positively with multiple shoots initiated when BAP alone or KIN alone were used and combining of these growth regulators with lower concentrations of NAA Using BAP in combination with NAA was comparatively more effective than using KIN in combination with NAA for efficient shoot multiplication 33 6.4 Rooting Auxins are mainly used in root induction and their effect varies with type and concentrations used in different plant species (Swamy and Singh, 2002) In the present study, the analysis of variance revealed that root number and root length varied significantly with half strength MS medium supplemented with NAA and IBA Applications of IBA alone exhibited the highest mean root number per shoot as compared to NAA and roots produced were thin The highest number of mean root per shoot (3.36±0 69) was recorded on 0.5 mg/l IBA and mean root length (2.21±0.40) was recorded on 2.0 mg/l IBA This was similar to the results of Dahab et al (2010) and Aloufa et al (2003) The number of roots produced per shoot increased when concentrations of IBA increased from 0.1 mg/l to 0.50 mg/l was applied However, further increase in the concentration of IBA to 2.0 mg/l showed a reduction in the mean root number per shoot in agreement with Dahab et al (2010) and Tileye Feyissa et al (2005) As the present data shows that using lower levels of auxin (IBA) is significantly better than higher levels for root induction and elongation Weiler (1984) also reported the inhibition of root elongation by higher concentration of growth regulators and stated ethylene deposition as the reason Auxins of all types stimulate plant cell to produce ethylene, especially when high amount of synthetic auxins are used Ethylene retard root elongation According to this author, the other reason for reduced response of root number and root length at higher concentration of auxin may be poor vascular connection of the root with the stem because of the interventions of callus Moreover, the optimum concentration is may be between 0.1 and 0.5 mg/l as the present result indicated In the present study comparison was made between the rooting hormones and IBA was found to be more effective in increasing root number and length than NAA 34 6.5 Acclimatization Rooted plantlets were transferred to polyethylene bags containing autoclaved garden soil and placed inside the growth chamber for two weeks After weeks, the covers were removed Then, they were taken into the greenhouse where 100% plants survived and established as healthy This result was much better than previous report of Aloufa et al (2003) where only 80% of the plants taken from regulator supplemented media were acclimated versus 15% of those taken from auxins free media 35 CONCLUSIONS Ximenia americana propagates by seed which is incompetent due to seed viability loss after short period, and time consuming for large scale production and conservation of this species Thus, this protocol can be suitably exploited for the large scale production of cloned plants for their rehabilitation in natural habitat, sustainable utilization of this valuable medicinal plant, propagation of genetically uniform plant for commercial purpose and conservation Thirty five percent concentration of commercial bleach clorox and 20 minutes exposure time was the best sterilization protocol for seed explants of Ximenia americana as this treatment resulted in highest percentage of survived clean explants (72.33%) BAP is the most important cytokinins for initiation and multiplication of Ximenia americana The highest mean number of shoots per explants was (4.16±0 17) on full strength MS medium supplemented with 0.5 mg/l BAP alone for shoot multiplication and half MS strength supplemented with 0.5 mg/l IBA for root induction 36 RECOMMENDATIONS Future perspectives, based on the present study, should focus on the following area: A The effect of different nutrient and physiological dormancy of the seed on in vitro germination of Ximenia americana seed with respect to low germination rate and longevity B Different rooting hormone and rooting response of multiplied shoots at each subculturing stage should be investigated C The effect of different concentrations of auxins and cytokinins on callus induction and regeneration of shoots and roots should be further studied D The effect of 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