Propagation Strategies and Genetic Fidelity in Strawberries

Abstract

While strawberries have long enjoyed huge popularity among consumers, tremendous progress in plant tissue culture, resulting in great advances in micropropagation, has occurred. The in vitro morphogenesis seems to be highly dependent on plant growth regulators and media used for culture, which is again genotype specific. Although automation of micropropagation in bioreactors has been advanced as a possible way of reducing propagation cost, optimal plant production depends upon a better understanding of physiological and biochemical responses of plants to the signals of culture microenvironment and an optimization of specific physical and chemical culture conditions to control the morphogenesis of strawberry plants in liquid culture systems. Clonal fidelity can be a serious problem and molecular strategies have been developed in order to reduce the variation to manageable levels. The article describes the progress in-depth of various aspects of strawberry propagation in vitro on semi-solid gelled media and in liquid media using bioreactors, for their improvement and for commercial production. The article also focuses on the employment of molecular markers in micropropagated plants for the assessment of genetic fidelity, uniformity, stability, and trueness-to-type among donor plants and tissue culture regenerant.

Keywords:

• bioreactors
• Fragaria × ananassa
• gelled and liquid media
• micropropagation
• molecular marker

INTRODUCTION

The cultivated strawberry (Fragaria × ananassa Duch), a hybrid between the ‘Scarlet’ or ‘Virginia’ strawberry (F. virginiana Duch) and the pistillate South American F. chiloensis (L.) Duch. is a dicotyledonous, perennial low-growing herb grown in most arable regions of the world. Strawberries are enjoyed by millions of people in all climates, including temperate, Mediterranean, sub-tropical, and taiga zones (Hancock et al., 1991) and are predominantly used as fresh fruit. Their use in processed forms as cooked and sweetened preserves, jams, jellies, frozen whole berries, juice extracts or flavorings, and their use in making a variety of other processed products made them one of the most popular berry crops (Childers, 1980). The berry is valued for its low-calorie carbohydrate and high fiber contents. Strawberries are a good source of natural antioxidants, including carotenoids, vitamins, phenols, flavonoids, dietary glutathionine, and endogenous metabolites (Larson, 1988), and exhibit a high level of antioxidant capacity against free radical species: superoxide radicals, hydrogen peroxide, hydroxyl radicals, and singlet oxygen (Wang and Jiao, 2000) that are believed to reduce carcinogens in humans (Chung et al., 2002), protect against tumor development (Kresty et al., 2001), and reverse age-related effects on memory (Bickford et al., 2000).

Micropropagation is the first major and widely accepted practical application of plant biotechnology that has gained the status of a multibillion dollar industry throughout the world. The commercial use of this technology is for clonal mass propagation of specific clone and of parental stocks for hybrid seed production, maintenance of pathogen-free (indexed) germplasm, uses as the initial step in a nuclear stock crop production system, and year-round production of plants. Large-scale propagation of clones from hybrids or specific parental lines through commercial micropropagation holds the promise of alleviating problems of shortage of planting material and lack of disease-resistant clones across the world. Complete new plants can be derived from tissue either from pre-existing buds through shoot proliferation, following shoot morphogenesis through adventitious shoot regeneration, or through the formation of somatic embryos—the production of bipolar structures with a root and a shoot meristem (Steward et al., 1970). Micropropagated strawberry plants can be stored under refrigeration (Mullin and Schlegel, 1976), making it a valuable technique for storage of germplasm. Various culture conditions, basal media, and growth regulators have been investigated for strawberry micropropagation on semi-solid gelled media (for review, please see Debnath, 2003; Graham, 2005; Debnath and Teixeira da Silva, 2007) or in liquid media in a bioreactor system (Debnath, 2008a, 2009).

In Vitro Culture on Gelled Media

Strawberry micropropagation on a gelled medium was introduced in the mid 1970s (Boxus, 1974). Since the most important European nurseries produced several million plants per year, the technique gave a definitive answer to the problems of soil fungi, which caused a lot of damage to the strawberry fields, and in another way, tissue culture plants seemed to produce more runners per mother plant in a short time (Mohan et al., 2005). Micropropagation on gelled media has also been widely used in the USA (Zimmerman, 1981) in commercial propagation of strawberries and in breeding programs to produce many plants rapidly. Conventionally, strawberries are propagated vegetatively by runners, arising from axillary leaf buds on the plant crown, but it produces a limited number of propagules. Micropropagation differs from all other conventional propagation methods in that aseptic conditions are essential to achieve success.

Shoot Proliferation and Virus Elimination

Plants produced by axillary branching normally retain the genetic composition of the mother plant and this method has proven to be the most applied and reliable method for true-to-type in vitro propagation in general. Successful shoot proliferation has been obtained in strawberry from single meristem (Boxus, 1974), meristem callus (Nishi and Oosawa, 1973), and from node culture (Bhatt and Dhar, 2000). Meristem-tip culture alone or in combination with heat treatment (Yoshino and Hashimoto, 1975) is widely used to obtain virus- and fungus-free strawberry plants (Molot et al., 1972). Potted plants are placed in a growth chamber at ambient temperatures, which is then followed by raising the temperature a few degrees per day up to 38°C, and they are then grown for 6 weeks (Lines et al., 2006). The meristem, a dome of actively dividing cells, about 0.1 mm in diameter and 0.25 mm long, with two or three leaf primordials is then removed from the shoot tips and cultured on a nutrient medium containing no or low levels of auxin and higher levels of cytokinins to promote axillary budding while preventing callus formation. Mullin et al. (1974) grew strawberries with strawberry mild yellow edge (SMYE) viruses for 6 weeks in a 36°C growth chamber before excising 0.3 to 0.8 mm of the meristematic tips with leaf primordia. Cultures can be initiated and maintained on Boxus (Boxus, 1974) medium containing Knop's (Knop, 1965) macronutrients and Murashige and Skoog (1962, MS) micronutrients and organic components, or MS medium supplemented with 2.2–4.4 μM 6-benzyladenine (BA), 0.5–2.5 μM indole-3-butyric acid (IBA), and 0.3 μM Gibberellic acid (GA₃; Cerović and Ružić, 1989) at 23–25°C during the light period, and 17°C in the dark; the quantum irradiance is 46 μmol m⁻² s⁻¹ for a 16-hr photoperiod (Sowik et al., 2001). Runners can be initiated from in vitro culture on media containing AgNO₃ (10–20 mg⁻¹ L). GA₃ (28.9–57.7 μM) increased the efficiency of AgNO₃ significantly (Zatykó et al., 1989). Although agar (0.6–0.8%, w/v) is the most commonly used gelling agent for in vitro strawberry culture on semi-solid medium, Lucyszyn et al. (2006) reported that the agar/galactomannan (gour, Indian Gum Industries, Jodhpur, India) mixture in the proportion of 0.3/0.3 (w/v) in MS medium showed better performance and enhanced shoot proliferation compared to the medium containing agar (0.6%, w/v) only. Debnath (2005, 2006) found that 3.5 g⁻¹ L Sigma A 1296 agar and 1.25 g⁻¹ L Gelrite (Sigma Chemical Co., St. Louis, MO, USA) were most effective for in vitro strawberry culture on a gelled medium. Cultures are maintained at 23°C under a photosynthetic photon flux density (PPFD) of 30 μmol m⁻² s⁻¹ from ‘warm-white’ fluorescent lamps and a 16-hr photoperiod. Bhatt and Dhar (2000) cultured nodal segments (2.5–3 cm) of wild strawberry (F. indica Andr.) on MS medium supplemented with 4 μM BA and 0.1 μM α-naphthalene acetic acid (NAA) for shoot initiation and multiplication.

Adventitious Shoot Regeneration

There have been a number of reports for adventitious bud and shoot regeneration from leaves (Nehra et al., 1989; Passey et al., 2003; Qin et al., 2005a, 2005b; Yonghua et al., 2005; Debnath, 2005, 2006), petioles (Passey et al., 2003; Debnath, 2005, 2006), peduncles/peduncular base of the flower bud (Lis, 1993), stems (Graham et al., 1995), stipules (Rugini and Orlando, 1992; Passey et al., 2003), stolons (Lis, 1993), roots (Rugini and Orlando, 1992; Passey et al., 2003), runners (Liu and Sanford, 1988), mesophyll protoplasts (Nyman and Wallin, 1988), anther cultures (Owen and Miller, 1996), and from immature embryos (Wang et al., 1984) of strawberries on gelled media. Shoot regeneration directly from field- (Nehra et al., 1989) or greenhouse-grown strawberry plants (Debnath, 2005, 2006) or from in vitro-grown shoots (Passey et al., 2003, Yonghua et al., 2005) have been reported. Explants taken from field-grown plants are difficult to sterilize to establish in vitro cultures due to a high degree of contamination. It is usually recommended to take explants from plants grown under controlled conditions, such as a growth room or greenhouse, or from buds that flush from dormant shoots stored indoors.

Plant regeneration is a crucial aspect of plant biotechnology methodology and tissue culture that facilitates the production of genetically engineered plants and somaclonal variants (Larkin and Scowcroft, 1981), and the rapid multiplication of difficult-to-propagate species. Adventitious plant regeneration from strawberry explants can be divided into the following steps: (i) formation of viable adventitious buds on the explant, (ii) elongation of the buds into shoots, and (iii) rooting of the shoots to form whole plants. A number of factors, such as genotype, culture medium (including growth regulators and their combinations), physical environment, explant development stage, etc., affect adventitious shoot regeneration.

Thidiazuron (TDZ), a the substituted phenylurea (N-phenyl-N′-1,2,3-thidiazol-5-ylurea) with its cytokinin- and auxin-like effects, alone (Debnath, 2005) or in combination with 2,4-dichlorophenoxy-acetic acid (2,4-D) (Passey et al., 2003) or IBA (Yonghua et al., 2005) was found to be effective for shoot regeneration from strawberry leaves. While leaf explants were used in most of the early studies for bud and shoot regeneration, sepals, a floral leaf or individual segment of the calyx of a flower that forms the outer protective layer in a bud, have been tested for shoot regeneration of in vitro strawberry cultures (Debnath, 2005). Shoot regeneration was obtained from sepal, leaf, and petiole explants by incorporating TDZ (2–4 μM) in the culture medium and a dark treatment for 14 days before incubating the explants under a 16-hr photoperiod. A dark treatment similar to that used for strawberry (Barceló et al., 1998) leaf generation was used to achieve the highest response. Such TDZ-induced shoots were transferred to 2–4 μM zeatin-containing medium for elongation (Debnath, 2005). Callus formation and shoot regeneration depended not only on the explant orientation and polarity, but also on genotype (Passey et al., 2003). Young expanding sepals with the adaxial side touching the culture medium produced the best results. Qin et al. (2005b) reported that ‘Toyonoka’ strawberry leaf explants cultured for 10 days in shoot regeneration medium in the presence of AgNO₃ not only enhanced shoot regeneration efficiency but also expedited the inhibition of adventitious buds. Being an ethylene inhibitor; AgNO₃ can markedly promote organogenesis in strawberries.

Rooting and Acclimatization

Proliferated shoots can be rooted in vitro on Boxus (Borkowska, 2001; Sowik et al., 2001), half-strength MS (Yue et al., 1993), or modified cranberry (Debnath and McRae, 2001a, 2001b) medium without growth regulators, or on half-strength MS with activated charcoal (0.6 g⁻¹ L) and IAA (5.7 μM) (Moore et al., 1991). The in vitro-formed roots are thick, possess no hairy roots, grow horizontally, and are fragile and easily damaged. In vitro-grown plantlets have low photosynthetic activity, poor water balance, and their anatomy and morphology are far from being optimal (Borkowska, 2001). Plantlets that are rooted ex vitro have a larger root system and more runners as compared to those formed by in vitro-rooted strawberry plants (Borkowska, 2001).

Micropropagated plants are acclimatized gradually to ambient conditions to avoid mortality that might otherwise occur under an abrupt change in relative humidity, temperature, or irradiance. For in vitro-rooted plantlets, the standard procedure is to wash the plantlets and transfer to pots containing ProMix BX (Premier Horticulture Limited, Riviére-du-Loup, Québec, Canada) (Debnath, 2005, 2006) or 1 peat:1 vermiculite (Zhou et al., 2005), and maintained in a humidity chamber, and acclimatized gradually by lowering the humidity over 2–3 weeks (temperature 20 ± 2°C, humidity 95%, PPF = 55 μmol m⁻² s⁻¹, 16-hr photoperiod). Hardened-off plants can be maintained in a greenhouse (temperature 20 ± 2°C, humidity 85%, maximum PPF = 90 μmol m⁻² s⁻¹, 16-hr photoperiod) (Debnath, 2005, 2006).

Debnath (2006) developed a protocol that enables strawberry micropropation in one step, i.e., multiplying shoots and having them rooted in the same culture medium. The use of microcuttings, giving both root and shoot growth in a medium containing cytokinin, is emerging as a better choice for micropropagation of strawberries than multiple shoot proliferation, using a cytokinin supplemented medium with subsequent rooting of microshoots. In vitro-derived strawberry shoots can be proliferated, elongated, and rooted on zeatin-containing medium. Zeatin alone at very low levels (1–2 μM) produced two to three shoots per explant, averaging 88% rooting incidence in a single medium in ‘Bounty’ strawberry. Furthermore, the protocol did not use auxin in the culture medium, which lowers the cost and reduces the probability of somaclonal variation among the proliferated plants. The main advantage of this protocol is that all the shoot tips of the in vitro-grown plantlets can be used for shoot proliferation and rooting, whereas basal rooted nodal segments can be transferred to the peat-perlite medium and acclimatized in the greenhouse. The protocol can eliminate stage II of micropropagation and can increase both multiplication rate and rooting rate; this translates into a faster micropropagation of cranberry. The technique is now routinely used at the author's laboratory in Canada.

Somatic Embryogenesis

Somatic embryogenesis involves the development of bipolar embryos from embryogenically-competent somatic cells in vitro. In contrast to organogenesis, where microshoots and roots develop on different media, somatic embryogenesis is apparently a one-step procedure involving the development of embryos having both a shoot and a root pole, as in the zygotic embryos. Wang et al. (1984) reported somatic embryogenesis from strawberry cotyledons on MS medium supplemented with 22.6 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 2.2 μM BA, and 500 mg/l casein hydrolysate where a few of the embryogenic tissues developed into somatic embryos. Morphologically, normal plants were obtained from somatic embryos that were transferred to MS medium containing 2.89 μM GA₃ or 2.22 μM BA + 0.54 μM NAA. Maintenance of the embryogenic cultures was, however, unsuccessful. Donnoli et al. (2001) reported somatic embryogenesis in 8% of the embryogenic calli in the strawberry cultivar Clea on MS medium supplemented with 4.88 μM BA and 4.90 μM IBA. Somatic embryogenesis research with strawberries is still in a preliminary stage and more efforts would be required to develop the technology (Graham, 2005).

Bioreactor Micropropagation

Automated bioreactors for large-scale production of tissue culture plants are important for commercial success of the micropropagation industry. Bioreactors are self-contained, sterile environments that capitalize on liquid nutrient or liquid/air inflow and outflow systems, designed for intensive culture and control over microenvironmental conditions (aeration, agitation, dissolved oxygen, etc.; Paek et al., 2005). The use of large-scale liquid cultures and automation has the potential to resolve the manual handling of the various stages of micropropagation and decreases production costs significantly. Bioreactor systems have been introduced for mass propagation of horticultural plants (Levin and Vasil, 1989) and have proven their potential for large-scale micropropagation. Different types of bioreactors developed for optimal mixing of oxygen, nutrients, and culture without severe shear stress are generally two types: (i) mechanically agitated bioreactors and (ii) pneumatically agitated and non-agitated bioreactors (Paek and Chakrabarty, 2003).

Culture in liquid medium is advantageous for several plant species but often causes asphyxia and hyperhydricity, resulting in malformed plants and loss of material. The malformations are manifested in glossy hyperhydrous leaves with a distorted anatomy. To overcome these problems, two major solutions for malformation control includes: use of growth retardants to control rapid proliferation and temporary immersion bioreactors (TIB, Ziv et al., 2003) in which the explants are alternately exposed to liquid cultivation medium and air.

Limited reports are available on in vitro bioreactor strawberry culture (Takayama and Akita, 1998). Hanhineva et al. (2005) reported shoot regeneration from leaf explants of five strawberry cultivars in a commercially available TIB bioreactor (RITA^®, VITROPIC, Saint-Mathieu-de-Tréviers, France) containing liquid MS medium with 9 μM TDZ and 2.5 μM IBA. The TIB system proved to be well suited for shoot propagation and for subsequent subculture of the developing plantlets. Regeneration frequencies were 70 ± 8 to 94 ± 2% and 83 ± 5 to 92 ± 3% in the TIB system and on semi-solid medium, respectively. The labor time taken by the TIB system was less than half of the time required for handling plant material for cultivation on a semi-solid medium.

Debnath (2008a) developed a protocol for adventitious shoot regeneration, proliferation, and rooting of ‘Bounty’ strawberry using a TIB bioreactor system in a liquid medium combined with in vitro culture on a semi-solid gelled medium. Multiple shoot regeneration is obtained using leaf, sepal, or petiole explants from greenhouse- or field-grown plants by incorporating 2–4 μM TDZ on the semi-solid culture medium for 4 weeks, and including a dark treatment for 14 days before incubating the explants under a 16-hr photoperiod at photosynthetic photon flux density (PPFD) of 30 μmol m⁻² s⁻¹ at the culture level provided by a cool white fluorescent lamp (Debnath, 2005, 2006), followed by culturing in the same liquid medium in a bioreactor system with immersion of explants for 15 min every 4 hr. Shoots are proliferated in the bioreactor containing 0.1 μM TDZ liquid medium for another 8 weeks. Such TDZ-induced shoots do not elongate in the same liquid medium (Debnath, 2008a) or on solid medium even after culturing for another 8 weeks (Debnath, 2005, 2006). In liquid medium, TDZ supports rapid shoot proliferation at a low concentration (0.1 μM) but induces hyperhydricity (Debnath, 2008a). Bioreactor-multiplied hyperhydric shoots when transferred to a medium containing 2–4 μM zeatin, produce normal shoots within 4 weeks of culture. Shoot proliferation and rooting of in vitro strawberry plants can be achieved in the bioreactor system with the same medium with 0.5–1 μM zeatin. In vitro plantlets are propagated by subculture to a fresh medium at 6- to 8- week intervals. Rooted shoots are rinsed free of tissue culture medium and are planted on ProMix BX (Premier Horticulture Limited, Rivière-du-Loup, QC) potting medium. Plantlets are placed in a humidity chamber with a vaporizer and are acclimatized by gradually lowering the humidity over 2 to 3 weeks (temperature 20 ± 2°C, humidity 95%, PPFD 55 μmol m⁻² s⁻¹ with cool white-fluorescent tubes, 16-hr photoperiod). Hardened-off plants are maintained in a greenhouse under natural light conditions (temperature approximately 20 ± 2°C, humidity approximately 85%, maximum PPFD 90 μmol m⁻² s⁻¹, 16-hr photoperiod). The new procedure is expected to be simpler and requires less time to produce plants. After acclimatization, plantlets grow actively in the greenhouse and in the field with an apparently normal leaf and shoot morphology (Debnath, 2008a).

Growth and Development of Micropropagated Strawberries

Tissue culture-derived strawberry plants grow more vigorously producing more crowns and runners and increased petiole length, yield per area, and number of inflorescences per crown than conventionally propagated plants (Boxus et al., 1984; Cameron et al., 1989; López-Aranda et al., 1994). Zebrowska et al. (2003) reported that compared to conventionally propagated plants, ‘Teresa’ strawberry microplants produced more leaves, runners, and inflorescences with better yield and more resistance to leaf scorch. Litwińczuk (2004) compared strawberry plants of ‘Senga Sengana’ obtained in vitro from axillary and adventitious shoots with their runner progeny and with standard runner (control) plants under field conditions. In the planting year, in vitro-obtained plants developed significantly more crowns and runners compared to other groups. Such differences, especially in runners' number were not observed in the next 2 years. In the planting year, all in vitro propagated plants and about 80% of their runner progeny flowered contrary to the control (only 3% plants). Every year tissue culture plants developed significantly more inflorescences than other studied groups. Plants obtained in vitro produced bigger fruits and higher yield than other groups in the first 2 years. However, a reduction of berry yield for tissue culture plants in contrast with the control was observed in the third year only. The primary effects, increased vigor and axillary bud activity, are possibly related to the forced proliferation in vitro through hormonally induced crown branching (Swartz et al., 1981). The micropropagated strawberry ‘Gorella’ showed higher resistance to frost damage than did standard runner plants, when injury was evaluated in the field in the spring (Rancillac and Nourrisseau, 1989). Similarly, Dalman and Malata (1997) found that the micropropagated ‘Senga Sengana’ strawberry plants overwintered better than did the plants produced from runners, although for ‘Mari’ the opposite was observed, and for ‘Jonsok’ no differences between the two types of plants occurred.

Palonen and Lindén (2001) compared cold hardiness and overwintering of three types of strawberry plants of cultivars Senga Sengana and Jonsok: (i) micropropagated virus-free elite plants, (ii) certified plants (runner plants from elite plants), and (iii) ordinary plants (runner plants of conventionally propagated plants from a strawberry farm). No consistent differences in cold hardiness among the three types of plants were detected during the winter. Field evaluation did not reveal any differences in their winter survival either. Micropropagated plants flowered more freely than did the plants produced through runners.

Clonal Fidelity in Strawberry Micropropagation

Although there are advantages for the use of micropropagation, there are concerns about genetic changes resulting from the process (Dale et al., 2008). Somaclonal variation can result in a range of genetically stable variations useful in crop improvement (Jain, 2001). It is unpredictable in nature, and can be both heritable (genetic) and non-heritable (epigenetic).

Somaclonal variation has been reported in berry plants and concern has been expressed about the genetic stability of micropropagated plants. Discrete morphological variants (Swartz et al., 1981) and sporadic occurrences of abnormal fruit setting and a hyper-flowering habit that might be due to DNA methylation, have been reported in micropropagated strawberry plants (Boxus et al., 2000). Strawberry regenerants produced from anther culture have been demonstrated to vary with respect to earliness, calyx separation, rate of ripening, and mildew (Sphaerotheca macularis L.) tolerance (Simon et al., 1987). Somaclonal variants with fungal resistance in strawberry have been reported (Damiano et al., 1997).

A number of molecular markers, including restriction fragment length polymorphism (RFLP), random-amplified polymorphic DNA (RAPD), arbitrary primed polymerase chain reaction, DNA amplified fingerprinting, simple (short) sequence repeat (SSR), short tandem repeat, sequence characterized amplified region, sequence-tagged sites (STSs), amplified fragment length polymorphism (AFLP), inter simple sequence repeat (ISSR), expressed sequence tag (EST)-PCR, and cleaved amplified polymorphic sequences derived from EST-PCR markers are available for genetic analysis of tissue culture-raised plants (Debnath, 2008b, 2010). While reviews of these techniques are plentiful (Varshney et al., 2005) because of the rapidity with which relevant technology is proceeding, these may not remain compressive for long. PCR development has set the stage to overcome many of the shortfalls in the Southern blotting RFLP technique (Saiki et al., 1985). PCR-based DNA marker systems can be divided into two basic classes: those that use primers designed from arbitrary or non-specific sequences, such as RAPD and AFLP, and those that use primers designed from a known sequence for targeting a single specific locus, such as SSRs and STSs.

The introduction of DNA-based markers allows direct comparisons of different genetic material, independent of environmental influences (Weising et al., 1995). The degree of similarity between banding patterns can provide information about genetic similarity and relationships between the samples studied. Each marker system has its own strengths and limitations, making the choice of marker an important decision.

RAPD and ISSR marker analyses have been developed in the author's laboratory to identify genetic diversity in strawberry (Debnath et al., 2008), and can be used to verify trueness-to-type of micropropagated strawberries. ISSR markers (Gupta et al., 1994; Zietkiewicz et al., 1994) are now being used in bioreactor micropropagated berry plants in the author's laboratory to verify clonal fidelity. ISSR primers target microsatellites that are abundant throughout the plant genome (Wang et al., 1994). These markers have proved to be more reproducible than RAPD markers. They cost less and are easier to use than AFLPs and do not require prior knowledge of flanking sequences, like SSRs (Reddy et al., 2002).

Debnath (2009) compared bioreactor-derived tissue culture (BC) ‘Bounty’ strawberry plants obtained from sepal explants grown ex vitro with those propagated by tissue culture on gelled medium (GC) and by conventional runner cuttings (RC), for growth, morphology, anthocyanin content, and antioxidant activity over three growth seasons. The BC and GC plants produced more crowns, runners, leaves, and berries than the RC plants although berry weight per plant did not differ significantly. BC and GC plants produced berries with more anthocyanin contents and antioxidant activities than those produced by the RC plants. But ISSR marker assay produced a homogenous amplification profile in the tissue culture and donor control plants confirming the clonal fidelity of micropropagated plants. However, it is imperative to regularly check the genetic purity of the micropropagated plants in order to produce clonally uniform progeny. In vitro culture on nutrient media apparently induces the juvenile branching characteristics that favored enhanced vegetative growth with more crown, runners, leaf, and berry production. Whether the useful agronomic traits observed in the first three seasons of these plants are stable has to be ascertained in subsequent years and in field trials.

CONCLUSIONS

Significant opportunities exist for the application of in vitro propagation in strawberries. The relative ease by which techniques can be applied depends on the genotype. Automation, using a bioreactor, is one of the most effective ways to reduce the costs of micropropagation (Paek et al., 2001). The system improves the efficiency of in vitro propagation of strawberries; the shoot proliferation is about three times more than those on gelled medium (Debnath, 2009) Less cytokinin concentration (1–2 μM zeatin) is required in liquid culture (Debnath, 2009)compared to gelled medium (2–4 μM) for maximum shoot proliferation. However, the main challenge with a bioreactor system without active oxygen supply is hyperhydricity (vitrification). To overcome this challenge, careful optimization for each species is important. The most important factor here is the immersion time, since it governs nutrient uptake and expression of hyperhydricity. A balance between immersion frequencies and immersion duration to optimize shoot proliferation and reduce hyperhydricity is needed. Vessel volumes also come into this, as larger volumes often give better air exchange and better acclimatization of shoots in subsequent ex vitro conditions. The TIB has been shown to reduce some problems usually encountered in permanent liquid cultures, such as hyperhydricity, poor quality of propagules, and necessity of transplanting on a solid medium in the elongation and/or rooting stage (Debnath, 2008a).

Clonal fidelity is one of the main concerns in berry micropropagation. True-to-type propagules and genetic stability are prerequisites for the application of micropropagation of berry crops. The occurrence of somaclonal variation during micropropagation can be controlled by numerous factors, including genotype, presence of chimeral tissue, explant type and origin, media type, types and concentrations of growth regulators, cultural environment (temperature, light, etc.), and duration of culture (Graham, 2005). Clonal fidelity of micropropagated berry plants can be monitored by their morphological, biochemical, physiological, and genetic characteristics. Molecular markers are powerful tools in genetic identification of somaclonal variation.

ACKNOWLEDGMENTS

I wish to thank S. Khanizadeh, A. R. Jamieson, and C. Kempler of Agriculture and Agri-Food Canada Research Centres at St-Jean-sur-Richelieu, Kentville, and Agassiz, respectively, for supplying strawberry cultivars and advanced lines; and Darryl Martin, Sarah Leonard, Glenn Chubbs, Sarah Halfyard, and Shawn Foley for their excellent technical help. This work is the Atlantic Cool Climate Crop Research Centre Contribution No. 213.