Micropropagation Suggested reading: Vasil, ch. 3, Bhojwani, ch.

. 16 Defn asexual (vegetative) propagation of plants in vitro Criteria for commercial micropropagation
rapid multiplication vs. conventional introducing new cultivars ornamental foliage plants and some woodies species that are otherwise difficult to propagate (e.g., orchids)

Micropropagation (contin) Criteria for commercial micropropagation

propagation of "pathogen-free" plants (e.g., potatoes, bananas, strawberries, raspberries, etc.) rare or hard-to-find plants (i.e., allows a lab or nursery to offer a unique specialty)

Key to commercial feasibility is balancing positives and negatives of micropropagation

Micropropagation (contin) Positives and negatives of micropropagation

positives rapid multiplication rates low space requirement negatives labor costs high overhead (equipment, facilities, supplies) loss by contamination danger of variation

Micropropagation (contin) Stages of micropropagation

stage I initiation or establishment of an aseptic culture preferred explant type: shoot tip or ax. bud medium: high salts for herbaceous spp, low salts for woodies incubation: temp 24-26 C, photoperiod for veg. growth at 40-80 mols/sec/m2 light intensity

Micropropagation (contin) Stages of micropropagation

stage I problems contamination browning of the explant dormant buds (woodies only) stage II multiplication obtaining max. no. of propagules (tissue pieces or clumps used to generate future plantlets) the goal: stimulate axillary shoot proliferation from meristems located in axils of primordial leaves

Micropropagation (contin) stage II (contin)

cytokinin:auxin ratio adjusted to stimulate shoot production (usu. high cytokinin:auxin ratio) can usu. use the same medium for stage I and II problems vitrification a glassy appearance to leaves acclimation to stage II it takes some time to get to a rapid growth stage generation of callus (potential for mutations)

Micropropagation (contin) stage III rooting (in vitro) or regeneration of roots on shoots from stage II cultures
cytokinin is usually removed; auxin may or may not be added to the medium this stage is usu. skipped with many plants for woodies, a pretreatment may be substituted

stage IV acclimatization survival and establishment after transfer to soil (ex vitro)

Micropropagation (contin) stage IV problems to overcome

infectious diseases in soil desiccation ways to overcome it high humidity chamber intermittent mist (alternating high/low humidity mist under poly

Production of pathogen-free plants Suggested reading: Bhojwani ch. 15 Background

most vegetatively propagated plants are infected with 1 or more systemic pathogens (usu. viruses) yield increases from 30 to 300% can be achieved by replacement with virus-indexed plants distribution of viruses is uneven in plant tissues before "meristem culture" was widely adopted, heat therapy was used (but some viruses aren't susc.)

Production of pathogen-free plants Basic procedure

shoot tip or meristem-tip dissected and plated on a MS-based medium shoot tip must be less than 1 mm long low conc. of auxin/cytokinins are used the goal: to get growth of a shoot free of virus particles culture 4-6 weeks or until shoot grows out root shoot and transfer to greenhouse

Production of pathogen-free plants Other conditions are sim. to micropropagation virus particles may be present in meristem tissue, but are often not present in the shoot that grows out Hypotheses to explain why meristem culture works:
viruses depend on vascular system to move efficiently higher metabolic activity in the meristem perhaps interferes with virus replication

Production of pathogen-free plants Hypotheses

"virus inactivating system" is most active in the meristem high endogenous auxin level in shoot apex may inhibit virus multiplication

Methods of detecting viruses

goal: indexing or multiplication of plants verified free of a specific virus visual virus symptoms

Production of pathogen-free plants Methods of detecting viruses

sap transmission, by using an indicator plant serological test (antigen-antibody reaction) such as ELISAs EM test visually detecting virus particles in cells

Maintenance of indexed stock

isolation from infected plants during multiplication procedure test and multiply in vitro

Production of pathogen-free plants Maintenance of indexed stock

procedure place stage IV plantlets in screened greenhouse use 1st generation TC plants as a nuclear (stock) cutting block propagules from cutting blocks are retested production block random samples tested

Production of pathogen-free plants Productivity testing

check phenotypic integrity check for abnormal growth compare yield with conventionally propagated plants vigor is usu. much better potential for virus-indexed plants to be more susc. to other diseases

Production of pathogen-free plants Other means of obtaining specific pathogen-free plants

chemicals that eliminate viruses callus culture virus-resistant transgenic plants transformation and regeneration of a coat-protein gene results in plants that resist virus infection papaya ringspot virus cp gene of ringspot virus has been transferred into papaya transgenic cultivars