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Update on the action A 1.4 “Seed conservation at low temperature (-18°C), application of cryopreservation protocols”

The LIFE4FIR project is currently working on the project action A 1.4 ‘Seed conservation at low temperature (-18 ° C), application of cryopreservation protocols’.

The activities relate the developing of techniques for the conservation at low temperatures of seeds, pollen, isolated embryos and lines of embryogenic callus of Abies nebrodensis. These activities are functional to the implementation of the sperm bank and cryobank.

 

X-ray screening of seeds.

The vain seed problem is the main obstacle in the germination of Abies nebrodiensis seeds. X-ray analysis is a technique developed by IBE-CNR for the rapid selection of seeds with embryo and empty seeds of Abies nebrodensis (Figure 1). This procedure represents a mass screening of the seeds to be used for germination tests or to be stored at -18 ° C.

Figure 1 and Figure 2. Mature seeds of Abies nebrodiensis observed under X-rays for the selection of seeds with embryo (top frames) and empty seeds (bottom frames)

Figure 3. Mature seeds of Abies nebrodiensis with embryo, seen using X-rays

Prove di germinazione e coltura

In vitro germination tests of seeds stored at -18 ° C and embryos extracted from mature seeds and kept in a refrigerator (4 ° C) were carried out at the CNR-IBE and Unipa-Saaf laboratories, placing the isolated embryos on filter paper and then on different culture substrates (Figures 4,5 and 6).

Figure 4. Isolation of mature embryos from Abies nebrodensis seeds

Figure 5. In vitro germination of an Abies nebrodensis embryo

Figure 6. Germination and in vitro development of Abies nebrodensis embryos

 

In vitro culture tests of immature embryos isolated from cones collected in July are underway at the two laboratories, in order to obtain direct or indirect embryogenesis. The embryogenic callus, obtained from the excision of the embryos extracted from the seeds of the immature cones from plants 6, 8, 10, 15, 16 and 27 shows a rather slow growth. Various culture media containing different basic salts and plant growth hormones are being tested. A better growth of calluses seems to be favored by the contribution of cytokinins.

Germination tests are also underway on Abies nebrodensis pollen samples stored at low temperatures (Fig. 7).

Figure 7: Abies nebrodensis pollen grain in the germination phase

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Remote sensing via drone and monitoring activities of the LIFE4FIR project

The remote sensing via drone of the LIFE4FIR project

At the beginning of October 2020 the survey of the Abies nebrodensis natural are was carried out using a drone equipped with multispectral cameras. Remote sensing allows the monitoring of the health of trees and habitat through the measure of the radiation reflected by the vegetation.

During the drone flight, panoramic shots of the habitat and of the individual trees of the Abies nebrodensis population were acquired.

Tree n. 8

Trees n. 10 ed 11

Tree n. 12

Trees n. 16 e 17

Tree n. 19

Phytosanitary monitoring and laboratory activities of LIFE4FIR project

Phytosanitary surveys were carried out in November 2019 and in October 2020. Trees were subjected to a careful visual examination to evaluate their vegetative condition based on observations of the crown shape and transparency, turning foliage, presence of declining, desiccated or damaged parts, occurrence of lesions.

Laboratory analysis for LIFE4FIR monitoring activities

Laboratory analysis of collected samples were conducted through: observations under stereomicroscope, isolation and culture of fungal colonies and their genetic characterization through PCR amplification and sequencing of target loci for their identification. This allowed identification of the fungal microflora associated with the observed alterations and to detect the possible presence of harmful pathogens.

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Autumn update on LIFE4FIR project activities

The activities of the LIFE4FIR project continue, despite the significant difficulties related to the current COVID-19 pandemic.

In particular, in October 2020 the beneficiaries of the project met in Sicily, in order to hold the periodic coordination meeting. In addition to the updating for the 12 months of the project, being in Sicily the beneficiaries had live visits to the sites involved in the LIFE4FIR project.

Here are some photos taken during these activities.

During the technical and financial monitoring meeting held last October 12th in the restored rooms of the ancient library at the Botanical Garden of Palermo.

 

The LIFE4FIR group during the visit to the Abies nebrodensis Museum set up inside the town hall of Polizzi Generosa. The museum will host the cryobank foreseen in the project.

 

Approaching specimen no. 12 during the monitoring of the vegetative and sanitary conditions last October.

 

Taking samples from specimen no. 12 for laboratory observations and analyses.

 

Transit on the stony ground between the Vallone Madonna degli Angeli and Monte Scalone.

 

In front of tree n. 1, the most remote of the population.

 

The fruiting that occurred in 2020 was particularly abundant. The cones collected from the individual treeswere placed in boxes at the Piano Noce nursery, awaiting extraction of seeds.

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Summer update on the activities and results achieved by LIFE4FIR

The activities for the protection of the specimens of Abies Nebrodensis and their repopulation foreseen in the LIFE4FIR project continue also in these hot summer months.

Here are some new photos taken by the beneficiaries during field trips and laboratory activities.

Adult specimen of Abies Nebrodensis with damaged top

 

Damages by wild animals on adult specimens

 

Specimen affected by defoliation

 

Specimen showing scattered desiccation

 

Specimen whose top has been removed to eliminate the almost desiccated part

 

Nursery-grown seedlings with desiccation and chlorosis

 

Branch with desiccation observed in the laboratory

 

Detail of alteration observed under the stereoscope in the laboratory

 

Collaborator at the laboratory counter engaged in the isolation of fungal colonies

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The progress of the LIFE4FIR project continues!

Despite the continuing difficulties associated with COVID-19, the activities of the LIFE4FIR project have not stopped. In the last few months, there have been both field actions (such as in situ observation of trees in their habitat and of plants in the nursery and operations during the pollination phase) and monitoring and lab analysis actions. Following there are some photos taken by the beneficiaries during these recent activities.

Damage caused by wild animals on adult specimen of Abies Nebrodensis

 

Adult specimen of Abies Nebrodensis with defoliated and damaged crown

 

Adult specimen of Abies Nebrodensis with bare foliage in its lower part due to the shadow of beech trees

 

Adult specimen of Abies Nebrodensis with scattered desiccations

 

Seedlings raised in the nursery with desiccation, defoliation and chlorosis

 

Desiccation and defoliation of plant branches in the laboratory

 

Needles and twigs with alterations, observed with stereoscope in laboratory

 

Isolation of fungal pathogens in the laboratory

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Isolation and hand pollination of strobiles of Abies nebrodensis

Despite the difficult health situation, the activities of the LIFE4FIR project continue for defending the latest specimens of Abies nebrodensis.

Given the favorable seasonal trend, the action aimed at the isolation and hand pollination of the strobiles of the firs subjected to the protection measures started: on one side, barriers have been mounted in order to avoid contact with foreign pollen, coming from other varieties of fir trees present in the nearby reforestation areas; on the other side, pollen selected from Abies nebrodensis trees is collected in order to proceed with the following targeted hand pollination of other trees of the same species.

Below is a brief interview with Peppuccio Bonomo and Gaetano La Placa, during these field operations.