| dc.description.abstract | The goal of this thesis was to enhance the effectiveness of the conservation of the potato in vitro bank, by finding more efficient ways to maintain the bank and to introduce new varieties. A different method for virus detection was examined (Recombinase Polymerase Amplification on potato skin cDNA samples), slow growth mediums were tested on different potato varieties, and meristem culturing was carried out to eradicate viruses from infected plant material.
First, an initial slow growth test experiment with ten potato varieties was executed. Apical and axillary shoot cuttings were subjected to mediums with 0 mg/L, 0,5 mg/L, 1 mg/L, 3 mg/L or 5 mg/L Abscisic acid (ABA). The apical shoot cuttings had a lower survival rate. The ABA successfully reduced growth, but the high ABA mediums (3 mg/L and 5 mg/L ABA) had a lower survival rate. After nine months on the mediums the survival rate was higher on the 3 mg/L and 5 mg/L ABA mediums.
Second, a bigger slow growth experiment was done including ten potato varieties and 12 different mediums. The mediums both varied in sucrose and ABA concentration. The experiment showed that sucrose and ABA both stunt the growth of potatoes in vitro. The medium with the best characteristics seemed to be 3% sucrose and 1 mg/L ABA, as this medium gave no abnormal growth (contrary to what is seen on the high sucrose mediums), no fall in early survival (compared to what was seen on high ABA medium), low variance in growth between varieties (compared to the other sucrose treatments) and gave considerable growth reduction. This medium would allow the varieties to be transferred to a new medium after more than four months in the potato in vitro bank, lessening the work needed to maintain the potato in vitro bank.
Four virus-infected varieties were meristem cultivated to virus free material. Only one of these, Blåpotet, was completely cleaned of viruses. The potato virus Y (PVY) (Species Potato virus Y, genus Potyvirus, Family Potyviridae) was cleaned from Kvit Rund Kvam. Åkerøy was not successfully cleaned for viruses, but some meristems survived and grew into plants. Only the shoot tip in vitro cultures of Early Rose survived.
Primers were designed to test for PVY using Recombinase Polymerase amplification (RPA). Three different primer pairs were tested; “PVY1” which used primers from two different papers (Cassedy et al., 2022; Y. Wang et al., 2020), “PVY2” from a paper by Ying Wang and Ruhao Chen (Y. Wang et al., 2020), and “PVY3”, a primer pair designed by me. The primer pair that gave the least extra bands and streaks of nucleic acid when visualized on gel was PVY1. The RNA samples extracted from Potato skins were however of too low quality to be used for regular PVY testing. Better sampling of potato skins and improvement of the RNA extraction is needed. RPA on potato skin cDNA samples cannot be reliably used for virus testing, until the protocol is changed. This could be done by using a RNA extraction kit optimized for tuber samples. | |
