ministry for sustainable development, the · pdf fileproject part-financed by the european...

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


2007 – 2013






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.


2007 – 2013






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


2007 – 2013






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




2007 – 2013






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)


2007 – 2013






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


2007 – 2013






Photos by Christopher Leone Ganado


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


2007 – 2013







Orange flower buds

Sterilization

Flower bud dissection Pistil dissected into 2


2007 – 2013









Culture initiation

Culture incubation

Callus


2007 – 2013






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


2007 – 2013







Somatic embryo formation Somatic embryo germination

Regenerated lemon plantlets

Acclimatized lemon plantlets



2007 – 2013








2007 – 2013






5. Limitations

- Seasonal variations

- Sample sizes

- Specific explants required

- Incomplete flower buds

- Explant contamination

- No somatic embryogenesis

Incomplete flower

Bacteria

Mould



2007 – 2013







2007 – 2013






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.


2007 – 2013






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