There are many possible reasons. Among others, a poorly developed root system, the lack of well-developed leaves that can sustain ex vitro growth or acclimatization conditions that don't allow plants to adapt to the new conditions, or the plants obtained from in vitro culture are too soft or even vitrified. In these papers they seem to have successfully propagated strawberry plants and stablished them in soil... maybe you can check if there is any difference with your conditions. Good luck!

http://www.idosi.org/aejsr/2%282%2907/15.pdf

http://www.ijat-aatsea.com/pdf/JUNE_v4_n1_08/IJAT2008_14_Manosh.pdf

I recommend the following protocol:

STRAWBERRY MICROPROPAGATION PROTOCOL

This protocol for in vitro multiplication of strawberry includes all the four stages.

Used cultivars

Premial, Real, Magic, Redgauntlet, Benton, Floral, Elsanta, Marmolada, Idea, Everest, Record, Senga Sengana

Culture media

The culture media for strawberry micropropagation is currently completely under control. The medium for shoot induction and multiplication is MS nutritive medium (Murashige and Skoog, 1962) containing kinetin - N6-furfuryl adenine (K) and indole-3-acetic acid (IAA) for initiation of culture, and N6 - benzyladeniene (BAP) in combination with IAA for buds proliferation. For in vitro rooting of plants medium is composed of ½ MS macroelements, LF (Lee and de Fossard, 1977) microelements, MS vitamins with gibberellic acid (GA3) and 3 – indolylbutiric acid (IBA) (Coman and Neculae, 1981; Teodorescu and Neculae, 1994; Neculae, 1996), (Table 1).

Initiation of culture – explant excision and sterilization

The original strawberry plant material is provided by strawberry breeder responsible with genetic conformity of a cultivar. Quality micropropagation is related to the availability of homogenous plant material at the initial step of the process. Runner tips about 2 cm were collected in early June from a field strawberry plants before runners develop new leaves and roots. It is easier to excise from young runner tips than from well developed plants as there are hairs in the meristem zone. Runner tips from each cultivar must be keeping in a separate bag and labeled.

Procedure for surface sterilization involves the following steps:

1. washing samples under running tap water;

2. dipping samples for 4 min in an 94% ethanol;

3. dipping samples for 8 min in a calcium hypochlorite solution;

4. washing with sterile distilled water.

Meristems are further excised one by one using fine forceps and pieces of razor blade.The explant size should be < 0.3 mm so as to ensure sanitary status of the plant production line (Boxus, 1974). Apical tips (larger than 0.3 mm) or axillary buds can also be cultured by the same methods but will not exclude foliage nematodes and so must be taken from nuclear stock plants tested for foliage nematodes (OEPP/EPPO, 1998).They are asepticaly placed in test tubes containing 2 ml culture medium. About a month later the excised explant tips develop into a rosette of leaflets. They need to be transferred to the multiplication medium and now explants need to be carefully cleaned by removing all necrotic parts, callus, and roots. Independently of the variety more than 80 % of well - excised meristems develop into shoots (Redgauntlet 95%, Benton 96%, Premial 93%, Real 91%, Magic 86%, etc.).

Shoots proliferation / multiplication

The new rosette of leaflets it is transferred to culture vessel containing 20 ml multiplication medium, sealed with caps. Within the first 3 to 4 weeks, 2 to 3 new buds appear one by one at the base of the oldest leaves. These young axillary buds grow very quickly and, in turn, produce secondary axillary buds that cover the entire surface of a culture vessel. The initial rosette becomes a cluster consisting of 12 to 37 small buds. Each bud shows several short petioles with a small unifoliate leaves at the edges. At this stage, to progress in the clonal propagation buds can be aseptically separated and transferred onto a fresh multiplication medium. Every 4-6 weeks, each isolated bud will produce averagely 20 to 35 new axillary buds. Clusters are divided roughly into 4 to 5 small tufts (clusters) of buds that are transferred onto fresh proliferation medium. About 4 weeks later large clusters of new buds are formed and can be divided. Up to 10 multiplication steps can be achieved, but this figure should not be exceeded (OEPP/EPPO, 1998). Monitoring the transfers prevents the vitroculturist from overlaying the tenth subculture and from the risks of multiplying stipular buds. A continuous high proliferation rate depends on the transfers that must be done after 3 - 4 weeks of in vitro culture. If it is not possible to transfer the plants either due to lack of media or manpower unavailaibility, culture vessels must be stored at +2°C. At this stage, it is possible to create a stock of plantlets that could then be used for the initiation of new propagation lines.

Rooting

Rooting phase is an important step in micropropagation proces. Plantlets are separate from clusters and transferred onto a BAP free medium containing indole butyric acid to favour rooting (Table 1). Roots will appear 10 -15 days later in parallel with first true leaves. Depending on variety the rooting rate is between 72 - 96 %. Within 5 – 8 weeks, well rooted plantlets with 2 to 4 cm height leaves and 1,5 to 3 cm long roots are ready for acclimatization.

Acclimatization and adaptation rate

Strawberries are one of the species with a great capacity to support stress of plantlets transfer to the septic life conditions. Acclimatization can be achieved with a perlite substrate and covered with a plastic sheet maintaining a critical 100% humidity in the atmosphere. After 4- 5 weeks, plants developed new leaves and the plastic sheet can progressively be lifted up. The average percentage of acclimatized plants is ranging between 80 and 95 %.