Mouna Al-Sabbagh
General Commission for Scientific Agricultural Research/Biotechnology Department
Aleppo University, Faculty of Agriculture
2008

abstract

A successful and detailed in vitro propagation system for rapid micropropagation of three cherry rootstocks, i.e. the wild cherry rootstock, GF.8-1 and Japanese peach rootstock, as well as P. serrulata cherry cultivar by meristem culture and axillary buds without callus phase has been developed. This work describes the successful in vitro propagation, rooting and acclimatization of the studied cherry rootstocks and cultivar. Factors affecting establishment, multiplication, in vitro rhizogenesis and acclimatization were also investigated.
The ability to recover growth of GF-8-1 meristems post cryopreservation in liquid nitrogen for two years at -196 c° could be achieved. Acclimatized plants were healthy, vigorously growing and free of any abnormalities or morphological variations. Trees cultivated under field conditions reached 1 m in height at the end of growth season. Trees are now 4-years old and are grafted with different varieties for studying their field performance.
Field performance of the micropropagated plants was studied where these acclimatized plants were healthy, vigorously growing reaching around 60-70 cm height within one-season and free of any abnormalities or morphological variations. Trees cultivated under field conditions reached 1 m in height at the end of growth season. Trees are now 4-years old and are grafted with different varieties for studying their field performance.
The described method has potential to produce thousands of healthy and virus-free plantlets of these rootstocks over a short time. It has the potential to be commercialized to produce these rootstocks in a large-scale making it available to cherry growers to take advantages of these rootstocks to improve stone fruit production in the country.
Cold storage experiments showed that shoots excised from in vitro cold storage did not lose their ability to resume growth after 8-12 month period storage at 4 C which makes it available to resubculture every 8-12 month instead of every 4 weeks intervals. This also permits the establishment of in vitro gene bank to preserve the valuable genetic resources within a small place and resume its propagation when required.
A simplified method for in vitro micrografting of cherry rootstock Maxma-14 and Weiroot with the cvs P. serrulata and Bing was also developed.
The use of grafted plants is expanding. An in vitro propagation system was established for the rootstocks and the cultivars to combine micropropagation with micrografting. Micropropagated shoots were micrografted onto 3 week-old rootstocks with a union rate of over 80%. After acclimatisation, micropropagated plants were established ex vivo.
The aim of this study was to test and adapt existing regeneration and micrografting protocols for routine and reliable application to grafted plant production in the studied rootstocks and scions
Three week-old rootstock seedlings were decapitated; shots of the cvs were cut longitudinally and were inserted in the rootstock. The lower part of the inserted shoot had a similar diameter to the rootstock. The wedge was inserted into the rootstock slit. Micrografted plants were transferred to MS medium without growth regulators and, 3 weeks later, were potted in plastic pots in a 2:1 peat:perlite mixture, watered and covered individually with plastic bags to decrease transpiration and transplantation shock. Pots were placed in a growth chamber. For the next 2 weeks, the bags were removed for 15 minutes each day and sprayed with 0.03% (w/v) benomyl (Benlate 50 WP). After acclimatization, the plants were transferred to a greenhouse.
Elisa tests which done in Germany approved that the propagated plants were virus-free from the fellow:
1. Plum pox virus (PPV).
2. Pruns necrotic Ringspot Virus (PNRsV).
3. Apple mosaic virus.
4. Apple chlorotic leaf spot virus (ACLSV).
5. Tomato Ring Spot Virus (ToRSV).
6. Prune dwarf virus (PDV).

In vitro regeneration system has been established using leaf explants for three cherry rootstocks, i.e. Maxma Delbard 14 Brokforest®, P. mahaleb and Weiroot cherry rootstocks as a prerequisite for later genetic transformation studies and also for the aim of studying somatic variations for breeding purposes with rooting and establishment in the field under natural conditions. Adventitious shoot organogenesis from three explants was monitored on MS media supplemented with different concentrations of BA at high concentration and NAA. The highest percentage of shoot formation was observed on MS medium supplemented with 5 mg l, 1 BA and 0.3 mg l, 1 NAA of explants formed adventitious shoots was placed onto MS-Basal medium. On MS containing BA (1 mg/l) in combination with IBA at 0.3 mg/l and GA3 0.2 mg/l, multiplication rate of about 7 fold every 4 weeks was obtained. For root initiation, 2 to 3 cm- long shoot tips were excised from proliferating cultures and grown on full or half strength of 1/2MS macroelements with 0.5 IBA gelled with 0.7% agar then incubated for one week in darkness then transferred to the light 16 hour each day, with 95-100% rooting efficiency within 2-4 weeks.

Rooted plantlets were transplanted into pots with a mixture of 2:1 (v/v) peat : Perlite and acclimatized gradually to filed condition where they were covered with transparent polyethylene bags and gradually hardened off by cutting increasingly large holes in the bags until the bags were removed completely after about 4 weeks.
On the other hand, cloning of DNA is one of the key methods in biotechnology, in which specific segments of DNA are recombined to plasmid vectors or other cloning vehicles, which will then replicate and generate numerous copies of introduced DNA in bacteria.
CP gene of the Prune dwarf virus (PDV) was isolated and sequenced. RT-PCR amplified PDV cDNA fragments were detected by electrophoresis on agarose gel, followed by gel staining. Cloning of CP gene into the pGreen vector. E.coli transformation was followed. Confirmation of the transformants was done, where transformants could be identified and stored at -80 C until to be used.
The overall goal of this part of the the study was the generation of PDV-resistant cherry plants.
Cloning involved the following steps:
1. Isolation of the RNA from the PDV,
2. Selection and preparation of a vector,
3. Ligation of a DNA-fragment or a cDNA fragment generated by RT-PCR into a vector,
4. Transformation of the recombinant molecule into a suitable host strain (E. coli), and
5. Selection of E . coli cells carrying the recombinant molecule.
The Future Perspective includes: Transformation and regeneration of cherry with the PDV- CP gene
Production of transgenics resistant to the virus and growing it under containment conditions
Confirmation of gene expression by molecular biology techniques.
In-containment evaluation of transgenics for PDV resistance