The invention relates to a process for the production of micro-propagated seedlings treated with micro-organisms capable of exerting phyto-stimulant and bio-control activities

1. METHOD FOR BIOFERTILIZATION
AND BIOPROTECTION OF
VEGETAL MATERIAL AND
BACTERIAL FORMULATION FOR
CARRYING OUT SAID METHOD
INVENTORS:
Annita Toffanin
Lorenzo Vettori,
Anna Russo
Cristiana Felici
Stefano Morini
PATENT STATUS: GRANTED
PRIORITY NUMBER: PI20070067
PRIORITY DATE: 01/06/2007

2. Invention
The invention relates to a process for the production of micro-propagated seedlings treated with
micro-organisms capable of exerting phyto-stimulant and bio-control activities, so as to favour a
better condition of the plants propagated in vitro and subsequently their better adaptation to the
cultivation conditions in vivo.
Numerous microorganisms beneficial to plants (Agrobacterium, Azotobacter, Bacillus, Erwinia,
Pseudomonas, Rhizobium, Bradyrhizobium, Streptomyces, Actinomyces, and Azospìrìllum) are
known to be growth promoters and/or biocontrol agents. Their use in the production of
micropropagated plants is innovative, considering that in vitro propagation requires aseptic
conditions.
The invention relates to a bacterial formulation, in particular based on Azospirillum brasilense for
such applications. These are gram-negative bacteria that live in the rhizosphere of numerous plants,
positively influencing their growth, and especially the root system, through the production of
substances similar to plant hormones (cytokinins, auxins and gibberellins) and processes such as the
fixation of atmospheric nitrogen, thus making it absorbable by the plant. The experimental tests
carried out concern:
• fungal pathogen inhibition tests (in vitro and in vivo);
• biodiversity studies on the inoculated rhizospheric soil compared to the control soil using DGGE
(Denaturating Gradient Gel Electrophoresis);
• application of the bacterial formulation during the radicalization and adaptation of clonal
rootstocks of currently marketed fruit tree species.

3. In vivo inhibition test of Rhizoctonia Spp. in the presence of
the bacterial formulation. Death of untreated control
seedlings.
Bacterial formulation
Control
Drawings
& pictures
In vitro inhibition test of Rhizoctonia Spp. in the presence of the
bacterial formulation. Comparison of the mycelial diameter of
the fungus in the presence of the formulation compared to the
control plate.
Bacterial
formulation
Control
Effect of the bacterial formulation on root
development of Mr.S 2/5 seedlings during the
acclimatisation phase.
Bacterial formulation
Control
Bacterial formulation
Control
High rooting percentage of Mr.S 2/5 explants treated
with bacterial formulation compared to control
explants. Detection at the end of the rooting phase (22
days).

4. Industrial applications
The present invention relates to a process for biofertilisation and bioprotection of plant material, in particular for propagation
applications:
- in vitro including the subsequent acclimatisation phase (post vitro);
- in seedbeds;
- in protected crops;
- in hydroponics;
- with nursery techniques in general.
The technology is suitable for in vitro propagation laboratories, in agricultural marketing and product development companies and in
nurseries in order to:
• reduce the use of generally used chemicals/pesticides;
• increase production as the technique allows the vegetative apex to overcome the dormancy state, enabling the propagated
material to quickly reach commercial size;
• increase the production efficiency of the in vitro propagation technique;
• possibly extend this method to protected crops.

5. Possible
developments
The results obtained demonstrated that the application of microorganisms in a controlled
manner and with the proposed methodology can usefully counteract the problems of
micropropagation, assuring a) more vigorous plants and better protection from pathogens in
the subsequent growth phases, b) significant increases in biomass and a reduction in
traditional fertilisers.
The proposed method allows to multiply healthy and genetically uniform plants in massive
quantities and is very useful in cloning genetically improved species that can be used to
increase agricultural and forestry productivity.
The team is continually looking for agricultural crops to test and is also open to considering
licensing out the patented technology for its exploitation by attracted companies.

6. Tech Transfer Office of University of Pisa
For more information:
Ufficio Regionale di Trasferimento Tecnologico
Headquarters: Via Luigi Carlo Farini, 8 50121 Firenze (FI)
E-mail: urtt@regione.toscana.it
For more information:
Headquarters: Lungarno Pacinotti 43/44, Pisa (PI) 56126
Web site: www.unipi.it/index.php/trasferimento
E-mail: valorizzazionericerca@unipi.it