TOPOLINS VIABLE ALTERNATIVE GROWTH REGULATORS IN MICROPROPAGATION? JOHANNES VAN STADEN, ADEYEMI O. AREMU & STEPHEN O. AMOO

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1 TOPOLINS VIABLE ALTERNATIVE GROWTH REGULATORS IN MICROPROPAGATION? JOHANNES VAN STADEN, ADEYEMI O. AREMU & STEPHEN O. AMOO Research Centre for Plant Growth and Development School of Life Sciences University of KwaZulu-Natal Pietermaritzurg Private Bag X01, Scottsville 3209, South Africa

2 World Population Growth

3 Food Security Issues

4 Climate Change Gloal warming Catastrophic effects

5 Plant Biotechnology Selective reeding Seed development and culture Plant hormone physiology Germplasm conservation Transgenic plants Genetic engineering Plant tissue culture

6 Plant Tissue Culture Benefits Rapid turn-over Clonal regenerants Secondary metaolite manipulation Exchange of clean germplasm/plant material across orders Conservation of plant species

7 Historical Perspectives Origin of plant tissue culture Schleiden and Schwann's 1838/9 The theory of totipotency 1902 Tomato root culture (White 1934) Cell differentiation and proliferation - White 1939 Gottlie Haerlandt Plant Physiologist Google images - Gautheret Noecourt 1939 Foundation for a surge in research

8 Historical Perspectives Coconut milk - Van Overeek et al. 1941, 1942 Kinetin - Hall and deropp 1955 Zeatin - Miller 1961; Letham 1963 Isoprenoid or aromatic ased on structural features H CH 2 OH HN CH 2 C C HN CH 2 1 N N 3 N 7 N 9 Zeatin CH 3 1 N N 3 N 7 N 9 Benzyladenine (BA)

9 Historical Perspectives Aromatic CKs were considered synthetic Later found to exist naturally in plants lower quantity [6-(2-hydroxyenzylamino)-9-β-D-riofuranosylpurine] otr Horgan et al. 1973, 1975 Further search for more potent aromatic CKs continued The discovery and application of topolins Strnad et al onwards

10 Some Useful References Horgan et al Tetrahedron Lett 14: Horgan et al Phytochemistry 14: Kamínek et al J Plant Growth Regul 6: Werrouck et al Physiol Plant 98: Strnad et al Phytochemistry 45:

11 Role of Cytokinins Cell division promoters Influence physiological and developmental plasticity Affect growth processes differently (Holu et al. 1998) - Isoprenoid - continuation of the cell cycle - Aromatic - developmental processes (morphogenesis) Define the pattern of growth in conjunction with auxins - Auxin-CK ratio very important Different plants respond differently to various CKs - Variation in the physiology of the plant - Variation in CK metaolism

12 Topolin & BA Structures 6-Benzyladenine (BA) meta-topolin (mt) meta-topolin rioside (mtr) meta-methoxy topolin rioside (MemTR)

13 Physiological Advantages Hydroxylation for O-glucoside formation Reversile sequestration of metaolites Protection against formation of N9-glucoside Reduction of anormalities Promotion of rooting Improved shoot proliferation

14 Topolins in Aloe polyphylla mt Zeatin BA MemT MemTR Control (no cytokinin)

15 Topolins in Aloe polyphylla Numer of shoots Control c BA a mt a MemT MemTR Zeatin Effect of cytokinin types on incidence of hyperhydricity Total numer of shoots Normal shoots Hyperhydric shoots

16 Topolins in Aloe polyphylla Total numer of shoots Numer of ig shoots

17 Topolins in Aloe ferox No. of shoots per explant Control BA mt mtr a a a c Cytokinin concentration (µm) Effect of cytokinin types on shoot multiplication

18 Topolins in Aloe ferox No. of normal shoots per explant Control BA mt mtr d c c c a a Cytokinin concentration (µm) Effect of cytokinin types on normal shoot production

19 Topolins in Aloe ferox Control BA mt mtr a a Anormality index c Cytokinin concentration (µm) Effect of cytokinin types on anormality incidence

20 Topolins in Barleria greenii Anormality index = numer of anormal shoot/total shoot produced Anormality index ( 10-1 ) Control 1 µm 3 µm 5 µm 7 µm 0 BA Kin mt mtr MemTR Cytokinin Effects of different cytokinins on shoot anormalities

21 Topolins in Williams Banana

22 Topolins in Harpagophytum procumens

23 Topolins in Harpagophytum procumens Effect of topolins on shoot-tip necrosis

24 Topolins in Huernia hystrix Mean no. of shoots produced per explant No PGR 0.0 µm NAA 2.5 µm NAA 5.0 µm NAA 10.0 µm NAA Control mt BA Cytokinin type Interaction of auxin and cytokinins on shoot production

25 Topolins in Merwilla plumea Rooting frequency (%) Control 0.25 µm 0.5 µm 1.0 µm 2.0 µm 20 0 mtr mtthp TDZ Cytokinin type and concentration Effects of different cytokinins on rooting frequency

26 Topolins in Merwilla plumea TDZ mtr mtthp Control

27 Topolins in Merwilla plumea Ferulic acid (μg/g DW) content c Control c mt a mtr MemTTHP BA c 2iP Ferulic acid

28 Topolins in Merwilla plumea c c a a c p-coumaric acid p-coumaric acid (mg/g DW) content Control mt mtr MemTTHP BA 2iP

29 Topolins in Pelargonium sidoides 20 Control 0.5 µm 1.0 µm No. of shoots/explant µm 5.0 µm 0 Control BA mt mtr memtr MemTR Kinetin Cytokinin

30 Highlights In Other Species

31 Highlights In Other Species Species Cytokinins Parameters Countries tested Hordeum vulgare BA, mt Seed germination, Turkey rooting Petunia hyrida BA, MemTR Histogenic staility Belgium Pinus sylvestris BA, mt, TDZ, Z Shoot multiplication Spain Rosa hyrida BA, mt, MemT, MemTR, FmTR Anti-senescing effect Belgium, Czech Repulic Uniola paniculata BA, mt Shoot multiplication, rooting and acclimatization USA

32 Topolins less effectiveness Species Cytokinins tested Parameters Eucomis zamesiaca BA, ip, mt, Z Bul induction Agave difformis BA, ip, KIN, mt, TDZ Shoot multiplication Dierama erectum BA, KIN, mt, Z Shoot multiplication Malus domestica BA, BAR, mtr, TDZ Rooting and acclimatization competence Pinus pinea BA, BPA, mt, TDZ Shoot multiplication Saccharum officinarum CPPU, mt, TDZ Shoot multiplication Sorus torminalis BA, mt, MemTR Shoot multiplication

33 Major highlights in micropropagation Effect of topolins on shoot multiplication and elongation Effect on rooting and ex vitro acclimatization Effect of topolins on countering in vitro anormalities Effect on hyperhydricity Effect on early senescence and shoot-tip necrosis Effect on histogenic staility

34 Concluding Remarks Choice of cytokinin is very crucial in micropropagation Topolins are gaining wider acceptance gloally Increasing role in alleviating physiological disorders Potential in up-regulating secondary metaolite content Structure (presence of -OH group) confers oserved advantages Further studies are on-going especially on their role under stress conditions

35 Acknowledgements Collaorators Funding odies