Seminar
Conservation and Protection of Local
Plants
14th October, 2015
Plant Health Directorate
A Study on Citrus Somatic Embryogenesis:
A Protocol for use in Citrus Sanitation
Mr. Christopher Leone Ganado
Seeds and other Propagation Material Unit (SPMU)
Rural Development Programme for Malta
2007 – 2013
Axis 2 – Improving the Environment and the Countryside
Project Part-financed by the European Union
The European Agricultural Fund for Rural Development
Co-financing Rate: 80% European Union, 20% Government of Malta
Europe Investing in Rural Areas
MINISTRY FOR SUSTAINABLE DEVELOPMENT,
THE ENVIRONMENT AND CLIMATE CHANGE
Veterinary and Phytosanitary Regulation Department
Plant Health Directorate, Lija, MALTA
1. Overview
1. Overview
2. Introduction
3. Materials & Methods
4. Results & Conclusions
5. Limitations
6. Benefits
7. References
Rural Development Programme for Malta
2007 – 2013
Axis 2 – Improving the Environment and the Countryside
Project Part-financed by the European Union
The European Agricultural Fund for Rural Development
Co-financing Rate: 80% European Union, 20% Government of Malta
Europe Investing in Rural Areas
2. Introduction - 1
Aims & Motivation:
- Decrease imports
- decrease pest introductions
- decrease risk of CTV and other viral introductions
- Conserve local citrus germplasm
- Create stock of sanitized local varieties for propagation purposes
Research question:
- Current studies indicate that specific climatic conditions are required
- One expert in the field indicated that locally available light intensities should suffice
although these differ slightly
- Success greatly dependent on the plants genotype
- Attempts carried out to replicate results utilizing local citrus genotypes under available
conditions.
Rural Development Programme for Malta
2007 – 2013
Axis 2 – Improving the Environment and the Countryside
Project Part-financed by the European Union
The European Agricultural Fund for Rural Development
Co-financing Rate: 80% European Union, 20% Government of Malta
Europe Investing in Rural Areas
2. Introduction - 2
- Most common local citrus species reported to be:
- sweet oranges, namely Tarocco, Valencia and Washington navel
- lemons
- Citrus cultivars grown in the Maltese Islands:
- bergamotte
- fifteen lemon varieties
- four limes
- twelve common orange varieties
- thirteen different Seville orange varieties
- pomelos
- shaddock
- Local citrus genetic resources comprise a mix of modern and old citrus varieties
- Malta is unique in that it can boast having old varieties which are still productive
Rural Development Programme for Malta
2007 – 2013
Axis 2 – Improving the Environment and the Countryside
Project Part-financed by the European Union
The European Agricultural Fund for Rural Development
Co-financing Rate: 80% European Union, 20% Government of Malta
Europe Investing in Rural Areas
2. Introduction - 3
- Continued presence of old varieties is only possible due to the lack of severe citrus
diseases in Malta
- Origins of the local citrus industry estimated to date back to A.D. 870 during the period of
Arab rule
- It is reported that the Mediterranean area is of importance for citrus diversity
- Although research was carried out to identify local varieties no attempts were made for
their conservation
- Historically Maltese citrus was of utmost economic importance as citrus fruits were sought
after throughout Europe
- Annual producer price index for citrus fruits for the years 2007 through 2012 were 138.6,
110.2, 147.4, 144.9, 112.1 and 143.81 respectively
- Total citrus production for the crop year:
- 2008/2009 amounted to 1,900 tonnes + 13,900 tonnes imported
- 2009/2010 amounted to 1,900 tonnes + 8,300 tonnes imported
- 2010/2011 amounted to 2,200 tonnes + 10,400 tonnes imported
Rural Development Programme for Malta
2007 – 2013
Axis 2 – Improving the Environment and the Countryside
Project Part-financed by the European Union
The European Agricultural Fund for Rural Development
Co-financing Rate: 80% European Union, 20% Government of Malta
Europe Investing in Rural Areas
2. Introduction - 4
- Total quantity of ancient citrus trees registered in the Maltese Islands amounted to 2,738:
- 2,292 registered by sixty Maltese growers
- 398 registered by seven Gozitan growers
- 48 registered by a single farmer on Comino
- 50% of trees registered in Malta situated towards the north-west region:
- mainly Siġġiewi, Rabat, Attard, Naxxar and Balzan
- Over 60% of trees registered in Gozo located in Xagħra
- Genetic erosion of local citrus trees is a reality, mainly due to the phytosanitary risk posed
by the introduction of CTV
- through purchases/imports from abroad due to a lack of locally produced citrus trees
- Genetic characterisation and morphological studies of local citrus varieties is lacking,
therefore it is difficult to apply for protection for local varieties or to promote them within
the farming community
- CTV discovered locally in 2005 in Wardija (Malta) and in 2007 in Daħlet Qorrot (Gozo)
Rural Development Programme for Malta
2007 – 2013
Axis 2 – Improving the Environment and the Countryside
Project Part-financed by the European Union
The European Agricultural Fund for Rural Development
Co-financing Rate: 80% European Union, 20% Government of Malta
Europe Investing in Rural Areas
3. Materials & Methods - 1
Study carried out on nine varieties all of which are suspected to be of local origin. These include:
- Six lemon trees
- three in a pre-study,
- three in the main
- Three orange trees
- in the main study
Seedless local lemons
Seedless local lemon
Orange flowers
Rural Development Programme for Malta
2007 – 2013
Axis 2 – Improving the Environment and the Countryside
Project Part-financed by the European Union
The European Agricultural Fund for Rural Development
Co-financing Rate: 80% European Union, 20% Government of Malta
Europe Investing in Rural Areas
Photos by Christopher Leone Ganado
3. Materials & Methods - 2
Procedure entailed:
- Collection of closed flower buds
- Surface sterilized in 2% sodium hypochlorite or 2% household bleach
- Excision of pistils
- either left whole, split into two equal parts both with and without ovaries,
or cut into thin transverse cell layers
- Pistils cultured onto MS nutrient medium
- fortified with sucrose, malt extract and agar in the presence and/or
absence of plant growth regulators
- Cultures maintained in a climatic controlled growth room
- constant temperature of 24±1 °C
- 16-hour photoperiod
- photosynthetic photon flux of between 44 and 49 μmol m-2 s-1
Rural Development Programme for Malta
2007 – 2013
Axis 2 – Improving the Environment and the Countryside
Project Part-financed by the European Union
The European Agricultural Fund for Rural Development
Co-financing Rate: 80% European Union, 20% Government of Malta
Europe Investing in Rural Areas
3. Materials & Methods - 3
Orange flower buds
Sterilization
Flower bud dissection Pistil dissected into 2
Rural Development Programme for Malta
2007 – 2013
Axis 2 – Improving the Environment and the Countryside
Project Part-financed by the European Union
The European Agricultural Fund for Rural Development
Co-financing Rate: 80% European Union, 20% Government of Malta
Europe Investing in Rural Areas
Photos by Christopher Leone Ganado
3. Materials & Methods - 4
Photos by Christopher Leone Ganado
Culture initiation
Culture incubation
Callus
Rural Development Programme for Malta
2007 – 2013
Axis 2 – Improving the Environment and the Countryside
Project Part-financed by the European Union
The European Agricultural Fund for Rural Development
Co-financing Rate: 80% European Union, 20% Government of Malta
Europe Investing in Rural Areas
4. Results & Conclusions - 1
- Callus obtained from many explants
- lemon explants quicker to respond
- Somatic embryos obtained from four lemons + a single orange
- first lemon somatic embryos after 40 days
- first orange somatic embryo after 89 days
- Majority of somatic embryos obtained on nutrient medium lacking growth
regulators
- Somatic embryos germinated and rooted on nutrient medium lacking
growth regulators
- Acclimatized in glasshouse utilizing compost, peat and perlite (3:2:5)
- Over 2500 plantlets transferred to glasshouse for acclimatisation
Rural Development Programme for Malta
2007 – 2013
Axis 2 – Improving the Environment and the Countryside
Project Part-financed by the European Union
The European Agricultural Fund for Rural Development
Co-financing Rate: 80% European Union, 20% Government of Malta
Europe Investing in Rural Areas
4. Results & Conclusions - 2
Somatic embryo formation Somatic embryo germination
Regenerated lemon plantlets
Acclimatized lemon plantlets
Photos by Christopher Leone Ganado
Rural Development Programme for Malta
2007 – 2013
Axis 2 – Improving the Environment and the Countryside
Project Part-financed by the European Union
The European Agricultural Fund for Rural Development
Co-financing Rate: 80% European Union, 20% Government of Malta
Europe Investing in Rural Areas
4. Results & Conclusions - 3
Rural Development Programme for Malta
2007 – 2013
Axis 2 – Improving the Environment and the Countryside
Project Part-financed by the European Union
The European Agricultural Fund for Rural Development
Co-financing Rate: 80% European Union, 20% Government of Malta
Europe Investing in Rural Areas
4. Results & Conclusions - 4
Rural Development Programme for Malta
2007 – 2013
Axis 2 – Improving the Environment and the Countryside
Project Part-financed by the European Union
The European Agricultural Fund for Rural Development
Co-financing Rate: 80% European Union, 20% Government of Malta
Europe Investing in Rural Areas
4. Results & Conclusions - 5
Rural Development Programme for Malta
2007 – 2013
Axis 2 – Improving the Environment and the Countryside
Project Part-financed by the European Union
The European Agricultural Fund for Rural Development
Co-financing Rate: 80% European Union, 20% Government of Malta
Europe Investing in Rural Areas
5. Limitations
- Seasonal variations
- Sample sizes
- Specific explants required
- Incomplete flower buds
- Explant contamination
- No somatic embryogenesis
Incomplete flower
Bacteria
Mould
Photos by Christopher Leone Ganado
Rural Development Programme for Malta
2007 – 2013
Axis 2 – Improving the Environment and the Countryside
Project Part-financed by the European Union
The European Agricultural Fund for Rural Development
Co-financing Rate: 80% European Union, 20% Government of Malta
Europe Investing in Rural Areas
Rural Development Programme for Malta
2007 – 2013
Axis 2 – Improving the Environment and the Countryside
Project Part-financed by the European Union
The European Agricultural Fund for Rural Development
Co-financing Rate: 80% European Union, 20% Government of Malta
Europe Investing in Rural Areas
6. Benefits
Benefits related to trade:
- Healthy material free from the major viruses including:
- the main graft transmissible diseases
- cachexia, concave gum, cristacortis, exocortis, impietratura, infectious
variegation, psorosis and tristeza - Trees produced are pre-basic
- white label with a purple line
7. References
ATTARD, D., 2008. Maintaining Malta's Protected Zone for the Citrus Tristeza Virus and its importance for Maltese
Citriculture. Bachelor of Science (Hons) edn. Malta: Universita' degli studi di Perugia, Facolta' di agraria & University of
Malta, Institute of agriculture.
BLONDY, A., SAVOYE, L. and SAVOYE, J.N., 2003. Le commerce des oranges entre Malte et la France au XVIIIe siècle.
Editions Bouchene et Fondation de Malte.
BORG, J., 1922. Cultivation and diseases of fruit trees in the Maltese Islands. Govt. Printing Office, Malta.
GALEA, J., 2008. Local citrus genetic resources of the Maltese Islands. Bachelor of Science (Hons) edn. Malta: Universita'
degli studi di Perugia, Facolta' di agraria & University of Malta, Institute of agriculture.
GATT, M.B. and D'ONGHIA, A.M., 2002. Serological investigations on the main citrus viruses in Malta. In: Proceedings of
the Mediterranean Network on Certification of citrus; 1998 - 2001. Options Méditerranéennes, Series B Studies and
research, 43, pp. 97-99.
LEONE GANADO, C., 2014. A study on citrus somatic embryogenesis: A protocol for use in citrus sanitation. Master of
Science, Malta: University of Malta, Institute of earth systems.
NSO, 2012. Annual Publication: Agriculture and Fisheries 2011. Marsa, Malta: National Statistics Office.
NSO, 2013. News Release - Producer/Input Agricultural Price Indices: Q4/2012. 043/2013. Marsa, Malta: National Statistics
Office.
PLANT HEALTH DIRECTORATE, MINISTRY FOR SUSTAINABLE DEVELOPMENT, ENVIRONMENT AND CLIMATE
CHANGE, 2013. Ancient fruit tree register 2009 - 2013.
Rural Development Programme for Malta
2007 – 2013
Axis 2 – Improving the Environment and the Countryside
Project Part-financed by the European Union
The European Agricultural Fund for Rural Development
Co-financing Rate: 80% European Union, 20% Government of Malta
Europe Investing in Rural Areas