In Vitro Propagation and Conservation of Fragaria Species

Abstract

The genus Fragaria L. belongs to the family Rosaceae. The basic chromosome number is 7 (x = 7). Around 20 species of diploid, tetraploid, hexaploid and octoploid are found in the genus. The species of Fragaria are commonly known as strawberries. The genus is found in the temperate regions of the northern hemisphere as well as South America. The most extensively distributed species in the genus is F. vesca L. and the commonly cultivated strawberry is F. x ananassa Duch. While strawberries are native to temperate climates, some varieties can be grown in subtropical climates. Fragaria is a vegetatively propagated crop. The easiest and most direct method for conservation of the strawberry plants is in a field gene bank. Their germplasm remains at risk of loss due to biotic and abiotic factors including climate change. Besides, this approach does not result in the distribution of healthy, virus-free plants. In vitro techniques are in place to propagate and conserve Fragaria germplasm. In vitro storage may be done in cold conditions, or liquid nitrogen as meristem or shoot tip. In this review chapter, tissue culture propagation technique, various aspects and strategies for conservation of Fragaria species will be discussed to present a holistic view of ex situ conservation of Fragaria genetic resources.

1. Introduction

The genus Fragaria L. belongs to the family Rosaceae. The species of Fragaria are commonly known as strawberries. The fruit is technically known as accessory fruit (Achne) because the fleshy part is not derived from ovaries but from receptacles. The basic chromosome number is 7 (x = 7). There are around 20 species found in the genus of diploid, tetraploid, hexaploid and octoploid in nature. Fragaria is found in the temperate regions of the northern hemisphere as well as South America. Natural hybridization between F. chiloensis (octaploid) with F. virginiana (octaploid) gave rise to the present-day strawberry cultivars, F. × ananassa Duch. [1]. A large number of commercial varieties evolved later. Fragaria has Duchesnea and Potentilla as close relatives [2, 3]. Most extensively distributed species in the genus F. vesca L., is native to northern Eurasia, North and South America [4]. The commonly cultivated strawberry, F. x ananassa Duch., is grown in most of the arable regions of the world [5]. While strawberries are native to temperate climates, some varieties can be grown in subtropical climates. Many countries in Asia, North America, Europe and Africa produce strawberries. In 2018, the highest producer of strawberries was China (2.7 million tons) followed by US (1.2 million tons) and Mexico (0.59 million tons) [6]. Generally, strawberry is used for table and desert purposes; however various value-added products are also prepared like strawberry jam, jelly, candy and canned strawberry. A holistic approach is required to conserve such a valuable economically important species. Tissue culture technology has significantly contributed towards the propagation and conservation of Fragaria germplasm. The sections below will be useful for breeders, researchers, farmers, farm managers to have an overview of Fragaria genetic resource and its ex situ conservation.

1.1 Genetic resources of Fragaria

Genus Fragaria includes 20 species (Table 1) distributed in the Northern temperate and Holarctic zones [7, 8]. In Fragaria, four specific fertility classes are primarily associated with their ploidy level or chromosome number. Most widely distributed species F. vesca is diploid in nature (2n =14) and the most cultivated species is F. × ananassa (2n = 56) is octoploid in nature. Diploid strawberries can be differentiated by their foliage, flower and fruits, inflorescence structure and plant habit. The F. vesca genome size was estimated at 197 Mb [9] and 206 Mb [10] and of F. ananassa 698 Mb [11]. Out of 20 species listed in Table 1, three species of Fragaria occur wild in the Himalayas in India [12, 13, 14]. These are: 1. F. nilgerrensis Schecht. (Nilgiri strawberry): Habitat: A creeping herb found in Nilgiris and higher hills of north-eastern India. Uses: The fruit is pinkish, sub-acidic and juicy; 2. F. nubicola Lindl. (Alpine Strawberry): Habitat: A herb distributed in the temperate Himalayas. Uses: Fruits are edible; and 3. F. vesca L. (perpetual strawberry): Habitat: A herb found in the higher altitudes of temperate Himalayas. Uses: The red delicious fruits are edible.

Species	Ploidy	Distribution
Fragaria vesca L.	2x	Worldwide
F. viridis Duch.	2x	Europe and Asia
F. nilgerrensis Schlect.	2x	Southeastern Asia
F. daltoniana J. Gay	2x	Himalayas
F. nubicola Lindl.	2x	Himalayas
F. iinumae Makino	2x	Japan
F. yesoensis Hara	2x	Japan
F. mandshurica Staudt	2x	North China
F. nipponica Makino	2x	Japan
F. gracilisa A. Los	2x	North China
F. pentaphylla Losinsk	2x	North China
F. corymbosa Losinsk	2x	North China
F. orientalis Losinsk	4x	Russian Far East/ China
F. moupinensis (French.) Card	4x	North China
F. ×bringhurstii Staudt	5x	California
F. moschata Duch.	6x	Euro-Siberia
F. chiloensis (L.) Miller	8x	Western N. America and Chile
F. virginiana Miller	8x	North America
F. iturupensis Staud	8x	Iturup Island
F. × ananassa Duchesne ex Lamarck	8x	Worldwide

Table 1.

Species of Fragaria, ploidy and global distribution.

Source: [3].

2. In vitro propagation

Fragaria species are propagated primarily by stolons, also called runners, and at a lesser extent by seeds [15, 16]. From a Horticulture point of view, micropropagation has been practised for more than 50 years. Micropropagated strawberry plant has been introduced to prevent most of the plant and soil transmissible diseases. Over the years various protocols have been established for in vitro propagation of strawberry. Adventitious shoot regeneration in strawberry has been widely done using different explants such as a leaf, leaf disk, sepals, petiole and root mainly for transformation studies [17]. The in vitro culture has been successful in the mass propagation of true-to-type strawberry plants. A protocol that enabled strawberry micropropagation in one step, i.e., shoots multiplication and rooting in the same culture medium, emerged as a better choice for micropropagation of strawberries than shoot proliferation, (on a cytokinin supplemented medium) with subsequent rooting of shoots. In ‘Bounty’ strawberry, zeatin at very low levels (1–2 μM) produced 2–3 shoots per explant, averaging 88% rooting incidence on a single culture medium [18]. An efficient micropropagation protocol is a prerequisite for an in vitro conservation programme.

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.

Species	Seed storage Behavior	Seed Storage Conditions
Fragaria chiloensis (L.) Mill.	Orthodox	100% viability following drying to mc’s (moisture content) in equilibrium with 15% RH and freezing for 1 month at −20°C at RBG Kew, WP
F. moschata Duchesne ex Weston	Orthodox	92% viability following drying to mc’s in equilibrium with 15% RH and freezing for 34days at −20°C at RBG Kew, WP
Fragaria spp. (strawberry)	Orthodox	No problem for long-term storage under IPGRI preferred conditions (SSLR); no loss in viability following 23 years hermetic air-dry storage at 4.5°C [22]
F. vesca L.	Orthodox	Viability is halved after 3 years open storage [23]; viability maintained after 8 years hermetic storage at −18°C [24]; long-term storage under IPGRI preferred conditions at RBG Kew, WP. Oldest collection 16 years; germination change 78 to 91.2%, 14years, 1 collection
F. virginiana Mill.	Orthodox	75% viability following drying to mc’s in equilibrium with 15% RH and freezing for 107 days at -20C at RBG Kew, WP. Longevity: When stored in sealed containers the seeds can remain viable for up to 20 years [25].
F. viridis Weston	Uncertain	The species has been shown to form a transient soil seed bank, with seeds persisting in the soil for <1 year [26]. Although, this may suggest that seeds of the species are short-lived under ambient conditions, and perhaps recalcitrant or intermediate, several factors may have resulted in the inclusion of orthodox seeds within this category (see [26] for further detail). Further research is necessary before the storage behavior of the taxon can be reliably classified.

Table 2.

Seed storage behavior and storage conditions of Fragaria species.

Compiled from: [21].

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 long-term 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 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:

1. 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).

1. Use of growth retardants

Species	In vitro conservation strategy	Conservation duration	References
Fragaria spp. and cultivars (50)	4°C, dark, add liquid med. Every 3 m	6 years	[30]
Fragaria cultivars (21)	2 °C	9–27 m, 10–100% survival	[31]
Fragaria spp. and cultivars (96)	4°C, dark, tissue culture bags	9–24months; 15 months mean storage time	[32, 33, 34]
Fragaria spp. and Cultivars (9)	4°C, 12 h photoperiod or dark, 4 BA conc., CH or not	More than 12 months, best with CH, photoperiod, low BA	[35]
Fragaria spp.	4 °C	Up to 7 months	[36]
Fragaria (wild species)	4 °C, five chamber bags, Jungnickel medium	6 to 9 months	[37]
Fragaria vesca	4 °C, dark, culture tubes	10 months	[29]

Table 3.

In vitro conservation of Fragaria spp. at low temperature.

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 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].

1. 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 (long-term 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, 50, 51, 52]. In case of temperate species, a cold acclimation period, which triggers cold adaptation mechanisms, is often beneficial [53].

4. Genetic stability

Maintenance of genetic fidelity is essential for a successful cryopreservation strategy [54] and requires tools for evaluating the genetic stability of conserved plants. Theoretically, during cryopreservation all metabolic activities stop at the ultralow temperature of LN, consequently, after rewarming from cryopreservation recovery of true-to-type plants is expected [55]. Cryopreservation protocols involve cooling in and rewarming from LN, in vitro culture and regeneration processes, phenotypic and genomic changes can occur due to somaclonal variation. Hence, it is necessary to verify true-to-type plants after cryopreservation [56]. The literature is overall ‘positive’ regarding the outcome of stability assessments from cryopreservation with studies. The various types of DNA markers detect different levels of polymorphism and different amounts of DNA change. Genetic stability is the norm in most studies of possible plant genetic variation following cryopreservation [56].

The development of molecular techniques in the recent year provides additional means for assessing genetic fidelity in plants. Single sequence repeats (SSRs) are tandemly repeated motifs of one to six bases present in coding and non-coding regions and are highly polymorphic [57]. F. x ananassa shoot tips were cryopreserved using encapsulation-dehydration and vitrification along with that new modified method cryoplate (V and D cryoplate) [48]. Plants raised through these techniques were subjected to genetic stability analysis using SSR markers. No differences were observed between Fragaria in vitro mother plants and in vitro cryopreserved plants using eight SSR primers [58]. This lack of variation suggests that there were no changes in the genetic fidelity of the plants due to cryopreservation. This was also the case in Solanum, in which the microsatellite sequences of plants regrown from cryopreserved apices were identical to the profiles of the parent plants and their progeny [59]. No structural changes were observed in the in vitro control or the Solanum plants grown from the cryopreserved germplasm, indicating stable inheritance of SSR sequences in the somatic progeny [59]. The low coverage of the genome is one criticism of molecular techniques. Despite being highly polymorphic and co-dominant, SSRs may be clustered and distributed unevenly in certain chromosome locations.

5. Cryotherapy and virus elimination

An important pre-requisite for the conservation of germplasm either in vitro or under cryopreservation is the availability of ‘virus-free’ starting material. The presence of viruses hinders international and national exchange and conservation of germplasm and also becomes a hurdle for utilization of germplasm in its crop improvement. The viruses cause the most serious diseases in Fragaria. Viruses causing serious diseases are arabis mosaic virus (ArMV), raspberry bushy dwarf virus (RBDV), strawberry mild yellow edge virus (SMYEV) and raspberry ringspot virus (RpRSV). These viruses are transmitted during traditional vegetative propagation through runners or by modern methods of in vitro multiplication. Thus, periodic screening for in vitro conserved Fragaria germplasm is important for viruses’ status in an in vitro genebank [60].

It was established for the first time that cryopreservation was not only useful for germplasm conservation, but also for virus eradication in in vitro shoots of plum infected with plum pox virus (PPV) [61]. Cryotherapy is a novel method for virus eradication in economically important plant species [62]. In Cryotherapy, more hydrated, infected cells die because of freezing, and only small compact cells which are close to the meristem and generally virus-free survive cryopreservation [63]. Cryotherapy has been attempted for virus eradication in many important crops such as banana, grapevine, Prunus, raspberry, citrus and potato. In F. vesca post-cryotherapy 96.67% of cultures were found free from Raspberry ringspot virus (RpRSV) [64].

6. Conclusions

The development of new elite cultivars with high yield, quality, and biotic and abiotic resistance or tolerance, will improve the efficiency of Fragaria production. In addition, the use of virus-free plant propagules may also increase yields and limit the spread of viral diseases within and between countries. Therefore, Fragaria germplasm conservation and its back up is of utmost important.

For a cryopreservation protocol to be successful or to provide optimal results, experimental conditions for each of its successive steps must be optimized. The protocols stated in this study need to be optimized for better regrowth. There is scope for improvement at each step of the tested techniques, to obtain higher recovery of cryopreserved shoot tips. The modified protocols can be tested on other Fragaria cultivars and further can be used for cryopreservation of genetic resources of Fragaria.

Acknowledgments

The author gratefully acknowledges Former and Present Directors, ICAR-NBPGR for providing research facilities, Dr. Barbara M. Reed and Dr. Kim E. Hummer for providing Fragaria accessions and Dr. T. Niino for providing cryo-plates to facilitate cryo-research.