In vitro clonal propagation of Crataeva magna (Lour.) DC, a tree of medicinal importance

1. In vitro clonal propagation of Crataeva magna (Lour.) DC, a tree of medicinal importance Nishritha Bopana and Sanjay Saxena TERI University, New Delhi INDIA

2. Plant description • Family: Capparaceae • Habit : A moderate-sized deciduous tree • Habitat: Grows in semi-arid regions in India • Bark: Grey & horizontally wrinkled • Leaves: Trifoliate • Flowers: White or cream in many flowered terminal corymbs • Fruits: Multiple seeded which are embedded in a yellow-fleshy pulp • Secondary metabolite : Lupeol (isolated from the stem bark)

3. Medicinal applications • Mainly used in Ayurveda to treat a variety of urinary disorders - Known to prevent stone formation in the kidney & promote their discharge - Used in the treatment of prostrate enlargement • Anti-arthritic • Hepatoprotective • Cardio-protective

4. Current status • Shrinking of natural populations due to multiple usages & rising demand • The problem has aggravated as the active compound is primarily extracted from the bark in limited quantity • Species is now categorized as rare or vulnerable in its natural habitat

5. Problems with conventional propagation • Poor seed viability • Low germination frequency • Young floral buds develop insect galls (due to oviposition of Aschistonxy crataeva & A. baranni)and drop down prematurely hampering seed production

6. In vitro techniques for propagation can be of immense value in large-scale multiplication of the species thereby offsetting the pressure on natural populations and conserving the species

7. Micropropagation Clonal propagation under aseptic conditions is a proven means of producing disease-free superior quality planting material Applied for multiplication of : • Elites (genotypes containing higher amount of active principle) • Species which are difficult to regenerate by conventional methods • Species where conventional methods are inadequate to meet the demand of quality planting material • Rare or endangered species for their conservation

8. Three main approaches for the propagation of medicinal plants through tissue culture are: • Axillary shoot proliferation (regeneration from explants having a pre-existing meristem) • Somatic embryogenesis • Organogenesis (adventitious shoot formation) Axillary shoot proliferation is the most preferred pathway as it is least prone to genetic variations that may occur during the in vitro process

9. Major stages in micropropagation • Initiation • Shoot multiplication • Rooting • Hardening and transplantation

10. Prior art on micropropagation of C. magna Shortcomings in existing reports include: • Complex initiation procedure • Fragile shoots • Low multiplication rate • Low rooting frequency • Poor transplantation success • Lack of clonal fidelity studies

11. Initiation of aseptic cultures Single node segments from a C. magna tree Quick rinse in 70% ethanol Teepol wash (10min) Washing under running tap water for 15 min 0.1% HgCl2 for 10min 3 washings in sterile RO Culturing of explants on MS basal + 3% sucrose and 0.8% agar

12. Initiation • 98% cultures were aseptic • Bud-break was 100%

13. Shoot multiplication In vitro shoots (from 2-3wk old cultures) excised from nodal segment Cultured on MS medium + 3% sucrose + 0.2% gelrite

14. Effect of various cytokinins

15. Effect of combination of cyokinins with auxins

16. Effect of sucrose concentration

17. Effect of gelling agents

18. MS medium supplemented with BAP (2.66µM), kinetin (1.39µM), IAA (1.14µM), sucrose (3%) & gelrite (0.2%) yielded a multiplication rate of 5.5 fold on a recurrent basis after three passages

19. Effect of MS and modified MS medium on rooting

20. Effect of different auxins on rooting

21. Effect of other growth adjuvants (PG, AC)

22. Optimal rooting medium: MS 1/2 + 11.42µM IAA + 9.8µM IBA + 0.46µM kinetin + 198.25µM phloroglucinol

23. Hardening and Field transplantation Rooted plants washed free of agar & transplanted in polythene bags with soil and agropeat (1:0, 1:1, 2:1, 3:1 & 0:1) (v/v) Plants reared in greenhouse (28 ± 2°C) (RH: 80– 85%) Plants transferred to polyhouse Open nursery Field transplantation

24. 100% survival in all potting mixtures More than 500 plants have been produced till date

25. Assessing clonal fidelity of micropropagated plants • Leaves from randomly selected tissue cultured plantlets collected at (at 4th, 8th, 12th, 16th & 20th passages in vitro) & from hardened plants (at polyhouse stage & field level) • Total DNA extracted following a modified Cetyl Trimethyl Ammonium Bromide (CTAB) DNA extraction procedure • Inter Simple Sequence Repeat (ISSR) marker assay employed

26. M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 DNA amplification obtained with primer UBC 840; 1kbp DNA ladder (lane M); mother plant (lane 1); micropropagated plants at 4th (lane 2 and 3), 8th (lane 4 and 5), 12th (lane 6 and 7), 16th (lane 8 and 9), and 20th (lane 10 and 11) passages; plant at polyhouse stage (12); plant at field stage (13) and outlier (lane 14) ISSR studies have confirmed clonal uniformity of tissue cultured raised plants

27. CONSERVATION STRATEGY Natural Resource Base Chemoprofiling (HPLC, GC) Molecular characterization (AFLP, RAPD, ISSR) Elites Cell culture for production of sec. metabolites Conventional propagation Micropropagation Clonally uniform plants (RAPD, ISSR) Industrial use Commercial plantations Conservation

28. Thank you • NISHRITHA BOPANA TERI University, Darbari Seth Block, Habitat Place, Lodhi Road, New Delhi 110 003, INDIA Phone: 91-11-24682100, Fax: 91-11-24682144 E mail: nishritha@gmail.com • SANJAY SAXENA, PhD The Energy and Resources Institute (TERI) Darbari Seth Block, Habitat Place, Lodhi Road, New Delhi 110 003, INDIA Tel: 91-11-24682100, Fax: 91-11-24682144 E mail: sanjays@teri.res.in