IN VITRO PRESERVATYION OF STRAWBERRY GENETIC RESOURCES Aim of the work is the development of efficient protocols for the in vitro proliferation and conservation of strawberry germplasm. With this aim have been tested different methods for: in vivo buds sterilization In vitro auxiliary shoot proliferation In vitro auxiliary shoot frigo conservation In vitro auxiliary shoot rooting Shoot acclimatization Evaluation of the in vivo true to type quality of the in vitro proliferated shoots have been also carried out.
STRAWBERRY IN VITRO PROLIFERATION PROTOCOL Type of explants and size New runners from initial material virus tested (nuclear stock), with 1 to 3 emerged leaves. It is also possible with smaller runners. The terms/time of explants taking Explant collection is possible all year round, depending on the physiological status of the mother plant (initial material). Whenever there are some runners, the in vitro introduction is a success. Due to the normal cycle of the plant it is best to do the in vitro introduction between March and November. The way of surface sterilization of explants First of all, the most advanced leaves are removed with a scalpel and the runners are washed under tap water in order to remove earth, sand or substrate remains. Surface sterilization protocole: Five min in Mercryl: combination of chlorhexidine gluconate (CHG) and benzalkonium chloride; 5 min in Alcohol 70%; 20 25 min in Calcium hypochlorite: Ca(ClO) 2 solution at 45 g/l (0.31 mol/liter) and 3 rinses with sterile water. Meristem extraction is done under binocular magnifying glass in a clean room, one meristem by test tube. Laminar flow not necessary. The best media for shoot induction The shoot induction is done with a modified DKW medium (Driver and Kuniyuki, 1984) (Fig. 2). The way of media sterilization The medium is sterilized for 25 to 30 minutes at 118 114 C in an autoclave. The best media for multiplication The proliferation medium used is the Murashige and Skoog MS (1962) composition supplemented with BA (Benzyl Adenine 1,1 mm 0,25 mg/l), sugar 30 g/l and agar 7,5 g/l. The best media for frigo conservation The frigo conservation medium used is the Murashige and Skoog MS (1962) composition without the addition of plant growth regulators and supplemented sugar 40 g/l and agar 7,5 g/l.
STRAWBERRY IN VITRO PROLIFERATION PROTOCOL Duration of subculture The multiplication subculture takes 3 weeks. Multiplication parameters The multiplication is done by division of tufts. The upper part of the leaves are first removed and then the tuft is divided into 2 to 3 small new tufts. The multiplication is possible due to the pre existing buds at the laef basis. For total conformity of the plants there should not be neo formation. Multiplication index Depending on the cultivars, the multiplication index is from 2 to 4. Generally most of the varieties have an index between 2.5 and 3. That means 1 jar give 2.5 to 3 jars when subcultured (after 3 weeks). The best media for rooting The rooting medium is the Murashige and Skoog MS (1962) composition without the addition of plant growth regulators and supplemented sugar 40 g/l and agar 7,5 g/l. Growth conditions in vitro Temperature For proliferation the temperature ranges from 21 24 C. For conservation the temperature ranges from 2 4 C. Photoperiod The photoperiod is 16 hours day, 8 hours night. Light intensity on culture surface Light is provided by neon tubes 58 W White industry. Two neon tubes per shelf. Acclimatization percentage of acclimatized and adopted plants Before acclimatization, the plants are washed under tap water in order to remove agar and old leaves. The plants are then packed and labelled with the official certification label if necessary. The percentage of acclimatized plants is close to 100 %.
STRAWBERRY GENOTYPES IN VITRO P1 AN has developed protocols for the in vitro conservation of the varieties expected for the in vivo preservation program of this research unit and study on their fruit quality and content of bioactive compounds: Varieties: Adria, Ciflorette, Irma, Patty, Candonga, Fragaria virginiana glauca x F. x ananassa selections: AN 94.414.52, AN 94.470.51, AN 94.472.52 F. x ananassa selections: AN 01.211.51, AN 94.159.59, AN 00.235.52, AN 99.78.51, AN 01.188.53, AN 93.231.53.
Strawberry (fragaria x ananassa Duch.) shoots cv. ADRIA, 2 weeks old
Strawberry (fragaria x ananassa Duch.) shoots cv. ADRIA, 2 weeks old
Strawberry (fragaria x ananassa Duch.) shoots cv. ADRIA, 2 weeks old