Assuring Reduced Variation in Commercial Crops (In vitro mass propagation experiences from Barbados and Caribbean plant tissue culture laboratories) Plants as Providers of Fine Chemicals, Conference presentation, August 2012, School of Chemistry, Bangor, Wales1 Collin Scantlebury University of the West Indies Cave Hill Campus BARBADOS

UAPS – Unit for Advanced Propagation Systems, Wye College, University of London at Wye near Ashford, Kent, UK BDD – British Development Division ODA – Overseas Development Administration Plants as Providers of Fine Chemicals, August 2012, School of Chemistry, Bangor, Wales2 “Plant tissue culture and its applications in plant biotechnology”

Applications of plant tissue culture to phytochemistry Via in vitro mass clonal propagation each single plant cell potentially can generate an entire new plant and thousands of clones Introduction of genetic/phenotypic variability in tissue cultured plants and associated different chemicals Direct gene cloning and host cell modification to increase access to target chemicals (molecular biology techniques) Concerted precautions to ensure clonal fidelity Concerted precautions to ensure clonal fidelity Plants as Providers of Fine Chemicals, August 2012, School of Chemistry, Bangor, Wales3

Caribbean Agricultural Research and Development Institute; University of the West Indies, Cave Hill Campus, St. Michael, Barbados (Diamond Valley, St. Philip, lab) Ministry of Agriculture. Graeme Hall, Christ Church, Barbados. (Home Tissue Culture Laboratory, The Home Agricultural Station, St. Philip). Plants as Providers of Fine Chemicals, August 2012, School of Chemistry, Bangor, Wales4

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6 - A single plant from a remote forest -Limited numbers in germplasm or genebank the athlete plant - Research (field and laboratory studies) driven identification of a particular superior plant species or variety (the athlete plant) - Insufficient numbers for scientific field studies with replicated plots

Plants as Providers of Fine Chemicals, August 2012, School of Chemistry, Bangor, Wales 7 - Annual threat of hurricanes Going bananas on the banana plantations - Thousands of suckers required /acre ( /hectare) - Only sword suckers for planting (uniform) - Uniformly growing and fruiting plants for better forecasting of harvesting - Not seed-propagated like corn, wheat etc. - Crop comprises a single variety not mixtures - Black Sigatoka disease limits movement of germplasm across international borders

Plants as Providers of Fine Chemicals, August 2012, School of Chemistry, Bangor, Wales8

In vitro mass propagation process Weaned and hardened vitro plants ready for field planting Multiplying banana cultures Plants as Providers of Fine Chemicals, August 2012, School of Chemistry, Bangor, Wales9

macro & micro nutrients, vitamins, amino acids, sucrose, plant hormones, phytagel/agar, deionized H 2 O, HCl, NaOH, (ethanol, NaOCl, fungicides, detergent/tween surfactant) cytokinins auxins Plants as Providers of Fine Chemicals, August 2012, School of Chemistry, Bangor, Wales10 IAA IBA NAA BAP Kinetin TDZ - thiaduzuron

Swelling and greening of meristematic buds within first week of explant initiation Single shoot formation form (meristematic) bud or eyes Single shoot ready for subculture or rooting (or splitting down the middle) Plants as Providers of Fine Chemicals, August 2012, School of Chemistry, Bangor, Wales11

Plants as Providers of Fine Chemicals, August 2012, School of Chemistry, Bangor, Wales12 Other Plant TC Applications for Phyto-chemistry Cell and callus cultures for induced variation Cell culture for genetic modification Cell culture for more ready access to target chemicals Anther culture for polyploids Somatic embryos for artificial seed

Plants as Providers of Fine Chemicals, August 2012, School of Chemistry, Bangor, Wales13 Safeguards for Reduced Variation Each explant can produce thousands of offshoots but be conservative In vitro multiplications rate may be variable so don’t stress the system Use judicious levels of plant hormone (cytokinin, auxin, gibberillin) Start clonal propagation process with proven mother plant (athlete plants) Require field studies to ensure true- to- type-ness and not epigenetic/ephemeral phenotypic characteristic or environmental effects being observed Field surveys of vitroplants are required to verify frequency of off-types/sports Use preformed meristems (nodal and shoot tip explants) rather than leaf, stems, anthers etc. which involve callus stages Selection of weaned and hardened plants (size, production dates, iso-lining)