Studio della risposta a Pseudomonas syringae pv. Actinidiae (PSA) di selezioni di Actinidia deliciosa e di Actinidia chinensis ottenute da miglioramento genetico non convenzionale
Author(s)
Bevilacqua, Daniele
Date Issued
June 9, 2017
Type
Doctoral Thesis
Abstract
For current knowledge, cultivars of kiwifruit (Actinidia chinensis and Actinidia deliciosa) cultivated in the world do not exhibit resistance to the bacterial pathogen Pseudomonas syringae pv. Actinidiae (PSA). Some are more tolerant (A. deliciosa, cv Hayward) while others are much less (A. chinensis, cv. Zespry Gold). This situation, coupled with the high aggressiveness of the bacterium, has immediately caused severe production losses with a high impact on producers' crop and income.
Recently, major worldwide efforts in genetic improvement are producing some varieties with higher pathogen tolerance (A. chinensis, cv G3, Soreli, Dorì), especially in the "yellow flesh" varieties that have since been more susceptible. This work, in vivo and in vitro, has the underlying unconventional genetic approach to obtain selections that were subsequently evaluated for susceptibility to the pathogen through various artificial inoculum techniques. For plant breeding, it was decided to apply Ethyl-Methane-Sulfonate (EMS), a strong chemical mutagen capable of reacting with nucleic acids causing point mutations on DNA for nucleotide alkylation or guanine deletion. For the inoculum with the bacterium, the CREA-FRU 8.43 strain (at a minimum concentration of 106 ufc / ml) was used to evaluate the response of new individuals.
In vivo work was preceded by the genetic exploration of available material, both commercial varieties and selections present in experimental fields of CREA-FRU. The 14 selected commercial varieties were grafted onto PSA-infected rootstocks, and none of the individuals survived. Other genetic material was collected in highly cultivated and highly infected PSA fields. In particular, two Tomuri male clones were collected and multiplied, which showed strong tolerance over the other plants of the specimens. Finally, we monitored the selections present at the CREA-FRU survived as a result of the strong attacks of the 2011/2012 pathogen by identifying 19 of the approximately 1750 originally present. The two Tomuri clones, part of the 19 selections of CREA-FRU, Hayward and 1 selection made available by the University of Udine were used in subsequent breeding programs.
In the first experiment EMS was applied for 18 hours and at 3 different concentrations (0.2, 0.3, 0.4%) to about 11,000 seeds obtained by free pollination of different Actinidia species: 3600 Hayward , 3600 of RII28 and 3600 of C8 (2 selections of A. chinensis of CREA-FRU and of Udine University respectively). More than 90% of the seeds germinated and about 120 days the seedlings were infected with the bacterium by direct inoculation into the leaf peduncle. After another 120 days, the surviving plants were again inoculated by infiltration (by pressure) into the leaf mesophilic of a PSA suspension in water.
In the second experiment EMS (1%) was applied (1 or 2 h) directly on the anthers of some selections of CRA-FRU (RII33; RI8; RI14) and Tomuri. The germination and vitality of pollen extracted from the anthers was evaluated and considered suitable for the execution of controlled crossings from which about 12,000 seeds were obtained. The germination rate observed was about 10%. The seedlings obtained were inoculated as before.
At the end of this in vivo selection activity, some promising seedlings were obtained and, afterwards, were cloned in vitro, transferred to greenhouse and subjected to further evaluation. The in vitro selection work was preceded by the optimization of the adventitious regeneration protocols for Hayward and A. chinensis Soreli cultivars. The effect on the regenerative leaf capacity of the duration of the dark phase (7, 14, 21 days) and application of different cytokines (Benzyladenine, Zeatina and Tidiazuron ) were evaluated. For defining the best protocol for the 2 cultivars, the mutagenic agent was applied to the calluses obtained from leaf
fragments. The EMS (1%) was applied for 4 different times (30, 60, 120 and 240 min). The time of application of mutagenesis has affected the incidence of callus necrosis, their regenerative capacity and, above all, the formation times and the number of adventitious shoots produced. Explants collected by the regenerating calluses following mutagenesis were multiplied on culture medium and then inoculated with PSA. Inoculations were performed using a PSA suspension in water, which in the first method was deposited on the leaf wound while in the second method it was used to immerse the basal part of the sprout. To date some clones that have shown, after the first in vitro screening phase, some bacterial tolerance are being multiplied in order to complete the characterization of pathogen response both in vitro and in vivo.
Additional information
Dottorato di ricerca in Biotecnologie vegetali
Subjects
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