Integrating A Tissue Culture Laboratory into a Nursery

Ian Gordon
School of Agronomy and Horticulture University of Queensland Gatton Campus

Australian Native Plants in Culture In Australian research institutions, published research on the initiation of native plants into tissue culture has not always translated into the availability of commercial volumes of plants in the nursery and flower trades.

Australian Native Plants in Culture

Lack of commercial success: Ptilotis Swainsona

Blandfordia
Platycerium

Australian Native Plants in Culture

Some success stories:

Anigozanthus
Asplenium

Nephrolepis Kimberley Queen

Australian Native Plants in Culture

There is clearly a major gulf between the small scale success in a research project needed for publication and the development of reliable systems of commercial production in plant tissue culture:

Australian Native Plants in Culture This paper aims to identify the operational issues in a plant tissue culture laboratory and how these can be integrated with commercial nursery production practices to ensure the successful commercial introduction of plants into the trade:

The Plant Tissue Culture Laboratory

Factors of importance

Microbial contamination The management of microbial contamination is the single most important factor in producing plants in the laboratory:

A hygiene program must be implemented in all sections of the laboratory facilities:

Microbial contamination Despite the title of the paper, I do not support the physical integration of the lab into the nursery: The risk of contamination increases dramatically if the lab is situated within the nursery:

Microbial contamination In the UG Gatton campus the Plant Nursery Unit and the Tissue Culture Laboratory are well separated: I see integration as the practices which are carried out in the lab are integrated with the practices used when the plants arrive in the nursery:

The Incubation Environment

A high light intensity and an appropriate light quality are needed to promote successful growth in the incubation laboratory:

Many plants require a light intensity of 5,000 lux to promote satisfactory growth:

The Incubation Environment In the laboratory at Gatton the light intensity is in the range of 7,000 to 10,000 lux: The light intensity (mid May) in the fog propagation house is in the range 15,000 to 20,000 lux:

Plantlets can acclimatise easily:

Laboratory Lighting Design Many labs use multi-tiered racks with fluorescent lights mounted on the underside of each tier of the rack: This means we are trying to direct the light through opaque plastic lids: Obviously not much light gets to plants:

Laboratory Day length & Temperature

As with most labs, we operate with a standard day length of 16 hours and 8 hour dark regime: Temperature is maintained at a standard 25oC:

A Standard Laboratory Environment This uniform lab environment should mean that cultures will grow consistently at all seasons of the year: However, some plants show altered physiological status at some times of year; Some become almost dormant during winter:

Sterile, Filtered Air A tissue culture lab will also benefit from the installation of a HEPA filtered air supply which will provide and maintain a sterile filtered air supply through the facility:

Hormonal Actions & Interactions During multiplication phases, cytokinins are used to achieve multi-branched plants: Benzyl amino purine (BAP) is most widely used: Cytokinins have an anti apical dominance effect:

Hormonal Actions & Interactions Auxins such as IBA are used to induce root formation on culture shoots:

These are the same products that the propagator uses to induce roots on stem cuttings:

Hormonal Actions & Interactions Using the correct compounds is important for success:

Using the compounds at an appropriate concentration is equally important: There are instances where too high a concentration of cytokinin causes shoot vitrification: e.g. Bananas:

Technical Expertise in Tissue Culture Members of IPPS look upon the propagator as a technically skilled professional who also has a degree of flair for plant propagation:

The lab manager should have a strong science background but also need this flairfor manipulating plants:

Technical Expertise in Tissue Culture This includes decisions on what material to use for initiation media combinations required

the hormonal balance needed

easy methods of sub-culturing

Root Formation in-vitro It is my personal preference to receive in-vitro rooted plants from the lab for deflasking in the nursery: Most soft-tissued plants will give 100% rooting: Some woody plants are less easy:

Root Formation in-vitro

Some lab managers use activated charcoal in the in-vitro rooting media: This reduces the light intensity around the root zone and thus increases the rooting percentage:

WHAT DOES THE NURSERY MANAGER WANT FROM THE LABORATORY

What does the nursery want from the lab The nursery success rate in deflasking rooted plantlets is strongly influenced by the quality of the plantlets delivered: Actively growing plantlets with some autotrophic growth will deflask more successfully than weak and spindly plantlets:

What does the nursery want from the lab Obviously there are many issues of nursery design and nursery technique which influence success: However, even the best nursery cannot achieve success with poor quality plants from the laboratory:

What does the nursery want from the lab

There is considerable evidence from the UQ Gatton nursery that deflasking success rates in winter are often better: Plantlet growth after deflasking is faster than in summer:

THE DEFLASKING PROGRAM IN THE NURSERY

The Nursery Transfer Room An enclosed transfer room with a high standard of hygiene and good temperature control is essential: It should be kept in a clean, hygienic condition: Good design will make it easier to manage hygiene:

The container to deflask into Often determined by the customer: Many of our customers want plants in 50mm round tubes: This is not necessarily the best option: Multi-celled trays are also used:

Deflasking Media A high quality, pathogen free medium is essential: It must be reproducible from batch to batch:

We use equal parts of sphagnum peat, perlite and vermiculite:

The Deflasking Operation

Deflasking is relatively simple but it must be done quickly to avoid any drying or desiccation of plantlets: As soon as a tray is completed it should be moved into a high humidity propagation environment:

The Propagation Environment

Low light intensity is essential for newly deflasked plants: The UQ Gatton fog propagation house has a light transmission in the range of 10,000 to 20,000 lux: This is 10-20% of ambient light:

The Propagation Environment A warm temperature must be maintained:

We use a bench installed warm water heating system set to maintain 25oC: A high pressure fogging system keeps humidity in the range of 80-90%:

The Propagation Environment The propagation greenhouse must have a high standard of hygiene: Regular monitoring is important to ensure that problems are detected at an early stage: A routine fungicide program is applied weekly:

Residual Hormonal Effects

A residual cytokinin effect is evident on many tissue culture produced plants: Plants such as Dieffenbachia and Dianella show a dense multi-branched growth habit as a result of the residual cytokinin effect:

Residual Hormonal Effects Many other plants show a residual auxin effect:

This manifests itself as an apically dominant single stemmed plant with no lateral branching:
The growing points of these plants must be removed to induce branching:

Ultra-Soft Tip Cuttings These ultra-soft shoot tips are loaded with auxin and with a well controlled propagation facility it is often possible to use these as soft tip cuttings to produce another batch of plants: I get good success with Actinotus and Metrosideros from soft tips:

Supplementary Liquid Feeding Two weeks after deflasking plants, we commence liquid feeding on a regular weekly cycle: A proprietary brand of liquid fertiliser with a complete range of major nutrients and trace elements is used:

Weaning off the fog Most TC produced plants are weaned off the fog environment after 2-3 weeks in the fog house: Once it is clear that root development is continuing, the plants are moved to an adjacent greenhouse with a higher light intensity and a lower humidity:

Weaning off the fog Poorly controlled weaning is where large scale losses can occur:

I have seen many examples of serious losses in nurseries where weaning is poorly controlled:
Our losses are rarely more than 10%:

Conclusion The high success rates we achieve in deflasking indicate that we have successfully integrated the practices in the laboratory with the practices in the nursery unit: Thank you for you attention: