**3.** *Ex situ* **conservation strategies**

*Ex situ* conservation strategies ensure the conservation of plant genetic resources outside their natural habitat. The genetic material is conserved either in field, seed, *in vitro* and cryo genebank. The explants for *ex situ* conservation include seed, *in vitro* cultures, shoot tips, dormant buds, pollen, DNA, etc. Germplasm of *Fragaria* is always in demand by breeders and researchers who need germplasm with good quality traits for crop improvement purposes. Thus, conservation of available germplasm is of utmost important. As strawberries are vegetatively propagated, most common method of conservation is in the field genebank, orchards, glass house and net houses as live plants. Seeds are cross-pollinated and heterozygous thus not encouraged for genotype conservation. Germplasm conservation by conventional methods has several limitations, e.g., high inputs of cost and labour (as in field genabank), seed dormancy, seed-borne diseases, etc. The biotechnological tools, like tissue culture and cryopreservation (in liquid nitrogen), help to overcome these problems [19, 20]. Thus, strawberry germplasm is being conserved *ex situ* in the field-, *in vitro*- and cryo-genebanks in many countries [2].

#### **3.1 Seed conservation**

Seed conservation of any species is based on the seed storage behavior of seeds. Orthodox seeds are conserved in the seed genebank while recalcitrant and intermediate seeds are stored by other methods. Seeds of *Fragaria* exhibit orthodox seed storage behavior and are being conserved in the genebank (**Table 2**) [21]. *Fragaria* seeds are heterozygous and can be stored in the seed genebank for gene pool conservation.

#### **3.2 Field genebank**

Conventionally *Fragaria* species are maintained in field genebanks as live plants. For example, in the Field gene bank, Bhowali, India, 80 accessions of *Fragaria* spp. are being maintained. At NCGR, Corvallis, 1500 accessions of *Fragaria* are maintained under screen house, and other global genebanks. Conservation in field genebanks comes with limitations like: large land requirement; establishment and maintenance expenses; risk of loss due to disease and insect attacks, and loss due to natural disasters and climate change impacts.

#### **3.3** *In vitro* **conservation**

*In vitro* conservation refers to maintaining germplasm on a defined nutrient medium under controlled environmental conditions. Three major *in vitro* conservation strategies are in practice. Conservation of germplasm at: (1) normal growth (2) slow growth and (3) cryopreservation. Slow growth techniques are for short- to medium-term-conservation storage of clonal plant material to be stored under *in vitro* conditions with extended shelf life. Slow growth methods aim for reducing the growth of *in vitro* shoots thus prolonging the subculture interval without causing any adverse effect on the plant tissue. Cryopreservation techniques are used for the longterm conservation of plant material. *In vitro* conservation strategies are discussed below:

#### *3.3.1 Normal growth*

Under this method, germplasm cultures are maintained under normal growing conditions by frequent subculturing at regular intervals. The normal growth method is advantageous as the cultures are available for immediate multiplication and distribution and it avoids the requirement of low-temperature facility (thus economical for tropical countries) or the application of stresses. *In vitro* maintenance of germplasm under normal growing conditions is the best method if the subculture interval may be extended up to a year or more [27]. About 1900 accessions of various horticultural crops including *Fragaria* are being conserved *in vitro* under normal growing conditions at National Genebank at ICAR-NBPGR, New Delhi, India. There is *in vitro* back-up of *Fragaria* germplasm conserved in the field genebank at Bhowali. Runners and suckers were collected from field genebank, Bhowali and established *in vitro*. Vegetative explants are taken from the field and established in MS media [28]. Shoot were multiplied on MS media supplemented with BAP (1 mg/l) IAA (1 mg/l) and GA3 (0.1 mg/l). Thirty-five accessions of *F*. *vesca* are conserved in the *In vitro* genebank at 25°C/light on MS + 0.2 mg/l BAP for a conservation period of 6-months [29]. Besides 45 exotic accessions of *Fragaria* are also part of *in vitro* collection, maintained under normal growth conditions. *In vitro* shoots are rooted on IBA (1 mg/l) media. Plantlets were


#### **Table 2.**

*Seed storage behavior and storage conditions of Fragaria species.*

transferred in the sterilized soilrite filled pots for acclimatization. The plants raised through tissue culture exhibited normal growth, flowering and fruit setting [29].

#### *3.3.2 Slow growth*

The main aim of this method is to maintain cultures undergrowth limitation conditions to reduce the requirement of frequent subculture. Some of the various approaches in practice are discussed below:

#### i.Low-temperature incubation

This method applies to wide range of genotypes, especially of temperate nature. Here, the *in vitro* cultures are maintained at low temperature that affects the metabolic activities which in turn restrict the growth of the plant. The storage temperature, generally, is crop-specific. *In vitro* conservation of *Fragaria* spp. at low temperature was successfully reported (**Table 3**).

ii.Use of growth retardants

Growth retardants are used to reduce the overall growth of the *in vitro* plants thereby prolonging their subculture intervals. But, the use of some of the growth retardants may


#### **Table 3.**

*In vitro conservation of* Fragaria *spp. at low temperature.*

cause mutation, owing to their mutagenic properties. These may also pose a physiological problem if used for a longer time. The most commonly used growth retardants are abscisic acid, dimethylamino succinamide, phosphon D, maleic hydrazide [27].

iii.Use of minimal growth media and restrictive growth conditions

Carbon source affects the growth rate of *in vitro* cultures. Alteration of optimum dose could reduce the growth rate of cultures in many species. The inclusion of sugar alcohol like mannitol or sorbitol in culture media is quite effective in restricting the growth of many plant species *in vitro* [27]. The use of minimal media may be more effective at low temperature.

#### *3.3.3 Cryopreservation*

Cryopreservation provides a low-input method for storing base collection (longterm backup) of clonal materials. Cryopreservation techniques are based on the removal of all freeze-able water from tissues by physical or osmotic dehydration, followed by ultra-rapid freezing. Cryopreservation can be achieved through classical and new vitrification-based techniques. Classical techniques involve freeze-induced dehydration, whereas new techniques are based on vitrification. Vitrification can be defined as the transition of water directly from the liquid phase into an amorphous phase or glass, while avoiding the formation of crystalline ice [39]. The main advantages in cryopreservation are simplicity and applicability to a wide range of genotypes [40].

Literature survey showed that, *in vitro* grown shoot tips of *Fragaria* have been cryopreserved using various techniques, encapsulation-vitrification [41], encapsulation-dehydration [42], vitrification [43], droplet-vitrification [44], cold acclimation + vitrification, Encapsulation-dehydration, controlled rate cooling [45] and V-cryoplate [46]. Another cryopreservation procedure using aluminum cryo-plates, termed D-cryoplate, was successfully developed for *in vitro* mat rush (*Juncus decipiens* Nakai) basal stem buds [47]. Encapsulation-dehydration and vitrification-based techniques viz., vitrification, V-cryoplate and D-cryoplate, were applied for cryopreservation of non-cold-acclimated shoot tips of a cultivar of strawberry *F*. x *ananassa* cv. Earliglow

[48]. In comparison, the recently developed new aged technique D-cryoplate, resulted in the best with 40% recovery among the four techniques tested. Both the recently developed new-aged techniques, D-cryoplate and V-cryoplate techniques have been used in many crops due to their high efficiency and operational simplicity. Cultivars of *F*. × *ananassa* were cryopreserved by V-cryoplate method and 81% recovery of cryopreserved shoot tips was obtained [46]. The protocol included cold acclimation at 5°C for 3 weeks before cryopreservation. The importance of cold acclimation for cryopreservation has been emphasized in other studies [45, 49–52]. In case of temperate species, a cold acclimation period, which triggers cold adaptation mechanisms, is often beneficial [53].
