The process employed to obtain healthy citrus plants

Step 3: Establishment of the cell culture and plant regeneration

In this phase, the explants are dissected, the

female reproductive parts are removed and placed

onto a nutrient medium which stimulates cell

division. Cell division transforms the female

reproductive parts to a clump of unorganised

cells, called a ‘plant callus’. The plant callus is then

transferred to a different nutrient medium with

different growth hormones that stimulate the

development of a plant embryo and later to a plantlet

with roots, a shoot and leaves.

Step 4: Transfer to soil and acclimatisation

Once the regenerated plantlet reaches an ideal size, it is

either planted into sterilised soil in plastic pots or mini-

grafted onto certified disease-free rootstock seedlings.

The regenerated plantlets are then allowed to

acclimatise slowly, in order to grow and adapt to

external environments.

Reference: 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.

Photography: Chris Leone Ganado

Step 2: Sterilisation of all equipment and biological material

The successful execution of sanitation and

micro-propagation techniques strictly require a clean

environment free from bacteria and fungi, since

these can contaminate the explants. The collected

explants are sterilised using alcohol, distilled water

and bleach under controlled conditions, the nutrient

medium and vessels for plant growth are sterilised

in an autoclave, while the tools are sterilised in a dry

heat steriliser.

Plant callus with developing embryo

PLANT HEALTH DIRECTORATE

The resilience of the local citrus industry is

dependent on the maintenance of a healthy

citrus tree population. This can be achieved by the

conservation and promotion of old local varieties

of citrus trees and by adopting preventive

measures to control the introduction of devastating

diseases, such as the Citrus Tristeza Virus.

The conservation of old local varieties of citrus

trees is important as these have been selected

by farmers for their desirable characteristics

that make them adaptable and resistant to the

local environment. Having been cultivated for

hundreds of years, they also form part of our

living traditional heritage and may serve as a

source of useful genetic material for the future.

Through the EAFRD Project on the Study and Sustainable

Conservation of Varieties of Local Plants, the Plant

Health Directorate has embarked upon activities to

identify local varieties of citrus trees through clonal

selection, which were then sanitised via a technique

called ‘Somatic embryogenesis’, as a means of

ensuring the continued existence of such varieties

which are at risk of extinction. This technique is ideal

because it produces disease-free plants that are

genetically identical to the parent tree.

Sanitation via somatic embryogenesis involves a number of steps:

Step 1: Selection and collection of plant material

Traditional knowledge from experts and farmers is

essential to identify local citrus varieties. During

the project, three orange and three lemon varieties,

suspected to be of local origin, were studied.

These trees were found in the old citrus groves

at San Anton Gardens and in private gardens

bearing old trees in Santa Venera and Siġġiewi.

The plant material collected for sanitation,

which are referred to as ‘explants’, were closed

flower buds.

Address:

Plant Health Directorate, Plant Biotechnology Centre,

Annibale Preca Str, Lija LJA 1915, Malta.

Website: www.planthealth.gov.mt

Telephone: 22926535

Email: plant.health@gov.mt

Regenerated plantlets during acclimatisation

Parliamentary Secretariat for agriculture,fiSherieS and animal rightS