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

medicinal importance Nishritha Bopana and Sanjay Saxena TERI University, New Delhi INDIA

Plant description

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Family Habit : Capparaceae : A moderate-sized deciduous tree Habitat : Grows in semi-arid regions in India

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Bark : Grey & horizontally wrinkled Leaves : Trifoliate Flowers : White or cream in many flowered terminal corymbs

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Fruits : Multiple seeded which are embedded in a yellow-fleshy pulp

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Secondary metabolite from the stem bark) : Lupeol (isolated

Medicinal applications

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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

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Anti-arthritic

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Hepatoprotective

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Cardio-protective

Current status

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Shrinking of natural populations due to multiple usages & rising demand

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The problem has aggravated as the active compound is primarily extracted from the bark limited quantity in

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Species is now categorized as rare or vulnerable in its natural habitat

Problems with conventional propagation

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Poor seed viability

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Low germination frequency

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Young floral buds develop insect galls (due to oviposition of Aschistonxy crataeva & A. baranni) and drop down prematurely hampering seed production

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

Micropropagation Clonal propagation under aseptic conditions is a proven means of producing disease-free superior quality planting material Applied for multiplication of :

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Species which are difficult to regenerate by conventional methods

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Elites (genotypes containing higher amount of active principle) Species where conventional methods are inadequate to meet the demand of quality planting material

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Rare or endangered species for their conservation

Three main approaches for the propagation of medicinal plants through tissue culture are:

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Axillary shoot proliferation (regeneration from explants having a pre-existing meristem)

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Somatic embryogenesis

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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

Major stages in micropropagation

 Initiation  Shoot multiplication  Rooting  Hardening and transplantation

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

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% HgCl 2 for 10min 3 washings in sterile RO Culturing of explants on MS basal + 3% sucrose and 0.8% agar

Initiation

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98% cultures were aseptic

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Bud-break was 100%

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

4.5

4 3.5

3 2.5

2 1.5

1 0.5

0 Effect of various cytokinins MEDIUM (µM) Multiplication fold Shoot length (cm)

5 1 0 4 3 2 Effect of combination of cyokinins with auxins MEDIUM Multiplication fold Shoot length (cm)

1 0 3 2 6 5 4 Effect of sucrose concentration Optimal proliferation medium + varied sucrose concentrations Multiplication fold Shoot length (cm)

Effect of gelling agents 6 5 4 1 0 3 2 Agar 0.8% Agar gel 0.4% GELLING AGENTS Multiplication fold Gelrite 0.2% Shoot length (cm)

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

7 6 5 1 0 4 3 2 Effect of MS and modified MS medium on rooting MS full MS 1/2 Root length (cm) MS 1/4 MEDIUM Root number MS1/6 MS1/8 Rooting frequency (%) 50 45 40 35 30 25 20 15 10 5 0

4 1 0 3 2 7 6 5 Effect of different auxins on rooting 40 30 20 10 0 90 80 70 60 50 Root length (cm) MEDIUM (µM) Number of roots Rooting percentage

6 5 4 3 2 1 0 Effect of other growth adjuvants (PG, AC) 120 100 80 60 40 20 0 Root length (cm) MEDIUM Root number Rooting frequency (%)

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

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

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

Assessing clonal fidelity of micropropagated plants

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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)

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Total DNA extracted following a modified Cetyl Trimethyl Ammonium Bromide (CTAB) DNA extraction procedure

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Inter Simple Sequence Repeat (ISSR) marker assay employed

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

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

Thank you

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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: [email protected]

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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: [email protected]