Micropropagation of apple — A review

Biotechnology Advances 28 (2010) 462–488

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Biotechnology Advances j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b i o t e c h a d v

Research review paper

Micropropagation of apple — A review Judit Dobránszki a,⁎, Jaime A. Teixeira da Silva b a b

Research Institute of Nyíregyháza, Research and Innovation Centre, Centre of Agricultural Sciences and Engineering, University of Debrecen, H-4400 Nyíregyháza, P.O. Box 12, Hungary Faculty of Agriculture and Graduate School of Agriculture, Kagawa University, Miki-cho, Ikenobe 2393, Kagawa-ken, 761-0795, Japan

a r t i c l e

i n f o

Article history: Received 13 September 2009 Received in revised form 16 February 2010 Accepted 17 February 2010 Available online 25 February 2010 Keywords: In vitro Malus sp. Shoot multiplication Rooting Production of propagation materials

a b s t r a c t Micropropagation of apple has played an important role in the production of healthy, disease-free plants and in the rapid multiplication of scions and rootstocks with desirable traits. During the last few decades, in apple, many reliable methods have been developed for both rootstocks and scions from a practical, commercial point of view. Successful micropropagation of apple using pre-existing meristems (culture of apical buds or nodal segments) is influenced by several internal and external factors including ex vitro (e.g. genotype and physiological state) and in vitro conditions (e.g., media constituents and light). Specific requirements during stages of micropropagation, such as the establishment of in vitro cultures, shoot multiplication, rooting of microshoots and acclimatization are summarized in this review. New approaches for increasing shoot multiplication and rooting for apple and current use of micropropagated plantlets as tools in basic and applied research are also discussed. © 2010 Elsevier Inc. All rights reserved.

Contents 1. 2. 3.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . Techniques and stages of micropropagation in theory . . . Micropropagation of apple in practice . . . . . . . . . . 3.1. Establishment of in vitro culture . . . . . . . . . . 3.1.1. Explant choice . . . . . . . . . . . . . . 3.1.2. Sterilization . . . . . . . . . . . . . . . . 3.1.3. Phenolic browning . . . . . . . . . . . . 3.2. Shoot multiplication . . . . . . . . . . . . . . . 3.2.1. Cultivar/genotype . . . . . . . . . . . . 3.2.2. Explants and subculture . . . . . . . . . 3.2.3. Composition of medium . . . . . . . . . 3.2.4. Physical factors . . . . . . . . . . . . . 3.3. Rooting . . . . . . . . . . . . . . . . . . . . . 3.3.1. In vitro rooting of microshoots . . . . . . 3.3.2. Ex vitro vs. in vitro rooting . . . . . . . . 3.3.3. Adventitious rooting . . . . . . . . . . . 3.4. Acclimatization . . . . . . . . . . . . . . . . . . Current applications . . . . . . . . . . . . . . . . . . . 4.1. Long-term cultures, automation and culture stability . 4.2. Synthetic seeds . . . . . . . . . . . . . . . . . . 4.3. Disease control, biotic and abiotic stress . . . . . .

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Abbreviations: AA, ascorbic acid; ABA, abscisic acid; AC, activated charcoal; BA, 6-benzylaminopurine or benzyladenine; BAR, benzyladenine-9-riboside; CA, citric acid; GA3, gibberellic acid; IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; KIN, kinetin; LPM, last proliferation medium; NAA, α-naphthaleneacetic acid; PA, polyamine; PG, phloroglucinol; PGR, plant growth regulator; PPO, polyphenol oxidase; REM, root elongation medium; RIM, root induction medium; TDZ, thidiazuron; TOP, N6-meta-hydroxy-benzyladenin or meta-topolin. ⁎ Corresponding author. Tel.: + 36 42 594 300; fax: + 36 42 430 009. E-mail address: [email protected] (J. Dobránszki). 0734-9750/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2010.02.008

J. Dobránszki, J.A. Teixeira da Silva / Biotechnology Advances 28 (2010) 462–488

4.4. Micrografting . . . . . . . . 4.5. Physiological, biochemical and 4.6. Genetic transformation . . . 5. Conclusions and future prospects . . Acknowledgements . . . . . . . . . . . References . . . . . . . . . . . . . . .

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1. Introduction Apple is one of the most important fruits in temperate zones and it is the third most important fruit crop (64.3 million t/year) in the world following bananas (81.3 million t/year) and grapes (66.3 million t/year) (FAO, 2009). Apple is conventionally propagated by vegetative methods, such as budding or grafting. Although these traditional propagation methods do not ensure disease-free and healthy plants, they depend on the season; moreover, they typically result in low multiplication rates. Micropropagation of apple rootstocks has opened up new areas of research and fruit tree propagation allowing the problems of conventional methods to be overcome and enabling rapid multiplication of disease-free fruit plants at a commercial scale (Zimmerman and Debergh, 1991). Furthermore, micropropagation allows quick propagation of new varieties or breeding lines or variants for apple breeders. It is an essential step in the success of regeneration of transgenic lines and determines the effectiveness of a transformation protocol (Aldwinckle and Malnoy, 2009). The history of apple tissue culture dates back to the late 1960s and the early 1970s when apple shoots had been cultured in vitro and their axenic growth was first reported (Jones, 1967; Elliott, 1972; Walkey, 1972). Since then many genotypes have been successfully cultured in vitro, and a number of papers have been published on different aspects of apple micropropagation. In addition, reliable methods have been developed for both rootstocks and scions from research and commercial points of view. The results of this research on apple micropropagation are detailed in this review paper, which is mainly based on the literature obtained from the past two decades. 2. Techniques and stages of micropropagation in theory Generally in vitro tissue culture or micropropagation is defined as the culture of different somatic cells, tissues or organs of plants under controlled in vitro conditions with the aim of producing a large number of progeny plants, which are genetically identical to the stock plant, in a relatively short time compared to conventional propagation. This implies in vitro cloning based on the fact that different plant parts, buds, meristems, tissues and cells are capable of regeneration into whole plants under adequate in vitro conditions. A special characteristic of plant cells and meristems in which they retain a latent capacity to produce a whole plant is called totipotency (Reinert and Backs, 1968; Vasil and Vasil, 1972; Verdeil et al., 2007; George, 2008). The main advantages of micropropagation of apple are a) enormous capacity to multiply target plant material compared to conventional cloning methods, b) the ability to produce progeny all-year round, c) the production of disease-free plant material and d) the possibility of multiplying genotypes which produce seeds uneconomically or which are sterile. Various methods and uses of micropropagation are presented in detail by George and Debergh (2008). In the present review only the main points are briefly summarized with a view to better understand its application in apple. Two main methods of in vitro propagation can be distinguished: (1) Propagation from axillary or terminal buds, in which propagation is based on pre-existing meristems. The following techniques are included: (i) meristem-tip culture, (ii) shoot or shoot tip culture and (iii) node (single or multiple) culture.

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(2) Propagation by the formation of adventitious shoots or adventitious somatic embryos, based on explants originating from somatic tissues. Adventitious shoot or embryo formation can occur (i) directly from tissues of the excised explants without previously formed callus (direct organogenesis or direct embryogenesis) or (ii) indirectly, when shoots or embryos regenerate on previously formed callus or in cell culture (indirect organogenesis or indirect embryogenesis). In many plant species direct organogenesis is rarely observed or unknown. The likelihood of the genetic identity of progenies decreases when propagation occurs via an indirect, callus phase. Inheritable genetic variability induced during in vitro propagation is defined as somaclonal variation which can be a problem in commercial micropropagation since it can negatively affect the uniformity of progenies (Hammerschlag, 1996). When propagation occurs via adventitious regeneration (by the formation of adventitious shoots or somatic embryos) and mainly when regeneration occurs on previously formed callus (indirectly), the frequency of somaclonal variation can increase (Son et al., 1993; Karp, 1995; Abeyaratne et al., 2004; Bordallo et al., 2004; George and Debergh, 2008). If regeneration occurs after a long-term period of callus growth, or shoot masses with basal callus are cut up in order to provide explants for subculture, the relative incidence of somaclonal variation increases (George, 1996a; Pontaroli and Camadro, 2005; George and Debergh, 2008). Moreover, somaclonal variation is also affected by the origin of callus; the rate of somaclonal variation was very low in regenerants in which the regeneration system involved a callus phase than when calli originated from vegetative apices in apple (Caboni et al., 2000). This finding may be due to the fact that a stricter genetic control acts in meristematic cells than in the cells of other tissues (De Klerk, 1990; Caboni et al., 2000). Genetic stability of progenies during micropropagation via meristem-tip culture was reported in other plant species, such as strawberry (Mohamed, 2007). However, somaclonal variation can be a useful and important tool for crop improvement in plant breeding by increasing genetic variability which contributes to the selection of traits, such as resistance to diseases, herbicides or tolerance to different abiotic stresses in different plant species (rice, tomato, asparagus, strawberry) including apple (Skirvin et al., 1993; Karp, 1995; Abeyaratne et al., 2004; Pontaroli and Camadro, 2005; Biswas et al., 2009). In vitro propagation can also be achieved by the formation of adventitious (storage) organs. In general, secondary in vitro cultures based on the propagation of primary in vitro shoot cultures includes the differentiation and propagation of adventitious organs, such as bulbs or tubers. Micropropagated plants are produced mainly by method (1) in most plant species because it can ensure the highest genetic stability during in vitro propagation since regeneration is based upon a preexisting meristems and new shoots form without an intervening callus phase. This method is defined as micropropagation as a part of an in vitro propagation method, sensu stricto. Generally, four stages are necessary for successful micropropagation to proceed (George and Debergh, 2008): Stage 1 — establishment of in vitro culture; Stage 2 — shoot multiplication; Stage 3 — rooting of microshoots; and Stage 4 — acclimatization. During Stage 1 explants are transferred to in vitro culture, which means that they should be surface sterilized so that they can survive and grow under artificial conditions. For successful initiation of an

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aseptic in vitro culture, stock plants should be selected and these or their parts often have to be pre-treated. Physical (e.g., light, temperature, etc.) or chemical pre-treatments can be necessary either for reducing the contamination of the stock plant in order to be able to have successful surface sterilization of explants or for enabling or improving the growth of explants in subsequent in vitro conditions. The process of stock plant selection and pre-treatments is defined as Stage 0 (George and Debergh, 2008). After successful initiation of an aseptic culture an efficient and reliable method for shoot multiplication should be achieved during Stage 2. Its success depends on various factors, such as plant species, cultivar, or genotype; organic and inorganic compounds, plant growth regulator (PGR) content or consistency of the medium, and other physical culture conditions such as light, temperature, vessel humidity, etc. The aim of this stage involves inducing the development of in vitro shoots (microshoots) capable for further cycles of shoot multiplication (subcultures) or for introducing them into Stage 3 (rooting). During Stage 3 microshoots originating from Stage 2 should be rooted either under in vitro or under ex vitro conditions. Successful rooting depends on similar factors listed in Stage 2 (plant species, media and environment). For rooting, suitable sized (high) microshoots are necessary, which often requires insertion of a microshoot-elongationstep because the size of in vitro shoots can decrease during Stage 2 due to the intensive multiplication. This inserted stage is defined as Stage 3a and the subsequent rooting as Stage 3b according to Debergh and Maene (1981). The transfer of rooted shoots to the natural environment occurs during Stage 4. This process is a crucial step for commercial use of micropropagation. Micropropagated plants are mixotrophic, i.e. they are not fully dependent on photosynthesis because of the low light intensity in vitro and on the carbohydrate content of the medium. In culture vessels there is frequently high humidity resulting in morphological and physiological modifications which cause rapid water loss of plants when moved to external conditions. During acclimatization the most important tasks are the step-by-step decrease of high humidity and concomitant increase of light intensity in order to avoid significant loss in propagated material. The present review summarizes the results obtained from apple micropropagation sensu stricto. Results are reviewed according to the above-mentioned stages of micropropagation. 3. Micropropagation of apple in practice 3.1. Establishment of in vitro culture The success of the initial stage of micropropagation is influenced by different parameters. The critical points for the effective in vitro culture establishment from apple, like other woody fruits or nuts (PérezTornero and Burgos, 2007; Ríoz Leal et al., 2007), are associated with the efficacy of sterilization of collected explants and the inhibition of phenol-induced browning of explants. These are dependent on choice of explant, the sterilization method, the in vitro physical and chemical conditions, etc. It has been reported that different genotypes do not respond in the same way to in vitro manipulations, such as establishment, proliferation or rooting (Abdul-Kader et al., 1991; Karhu and Zimmerman, 1993). Hence, a micropropagation procedure developed for an apple genotype could not always be extrapolated with the same success for another genotype. During establishment differences are commonly found between genotypes, mainly in the rate of explant browning or in the demand for the composition of medium (Modgil et al., 1999; Dobránszki et al., 2005). 3.1.1. Explant choice From the point of view of true-to-type propagation, explant choice is very important. Meristems or shoot tips (dormant or actively growing buds) can be used as explants due to their genetic stability. In

apple, both of these explants have been used depending on the aim of in vitro culture establishment (Abbott and Whiteley, 1976; Jones, 1976; Abbott, 1978; Lane and Looney, 1982; Kausal et al., 2005). Meristem culture was utilized to obtain virus-free plants by culturing 0.2 mm-long sections from terminal and axillary buds of ‘Jonathan’. Only 60% of the excised buds survived and about half of them sprouted successfully, while the other half of surviving buds formed calli (Huth, 1978). Kausal et al. (2005) studied the effect of explant size and position on tissue browning intensity and survival of explants. They concluded that axillary buds ranging from 0.2 to 0.6 cm in size showed less browning intensity and higher survival (60%) compared to larger (0.6–1 and 1–2 cm) explants; however, only a few buds survived. When actively growing buds were used as explants, establishment was successful independent of their position on the mother plant (distal or basal) but when dormant buds were used as explants, distal buds were most suitable as the source of explants. The survival of explants depends on their rate of microbial contamination and of explant browning, which pertain not only to the explants used for culture initiation but also to the physiological stage of mother plants and to the season when explants are collected (Table 1) (Werner and Boe, 1980; Webster and Jones, 1989; Marin et al., 1993; Yepes and Aldwinckle, 1994; Modgil et al., 1999; Dobránszki et al., 2000a). 3.1.2. Sterilization Even though in vitro cultures can be established at any time of the year, success depends on the season for collection of explants. Contamination of explants was found to be less if they were collected during spring or summer compared to explants collected in autumn or winter (Hutchinson, 1984; Webster and Jones, 1991; Modgil et al., 1999). A contamination rate of 7.75 and 11.5% was observed when the explants were collected during summer and spring, respectively (Modgil et al., 1999) whereas explants collected during autumn or winter showed a contamination rate of 26 or 50%, respectively. In our experiments, explants collected during summer had a higher level of bacterial contamination (up to 80%) compared to explants collected in spring (b20%), independent of genotype (Dobránszki et al., 2000a,b,c). To reduce microbial contamination, instead of field-grown plants, the explants could be collected from stock plants grown in a controlled environment, such as a greenhouse (Webster and Jones, 1989; Yepes and Aldwinckle, 1994; Preece, 2001; Preece and Read, 2003). Different methods have been described for surface sterilization of explants (mainly shoot tips). Webster and Jones (1989, 1991) compared the efficacy of sodium hypochlorite (NaOCl) solution (15%) or a solution of mercuric chloride (HgCl2) in sodium hypochlorite (150 mg HgCl2 in 12% Domestos) and found them equally effective in surface sterilization of four cold-hardy dwarfing apple rootstocks (‘B.9’, ‘Ottawa 3’, ‘P.2’ and ‘P.22’). NaOCl (10%) alone for 10 min in the case of ‘M9’ (Grant and Hammatt, 1999) or 1% NaOCl together with 2–3 drops Tween-20 for 20 min in the case of ‘MM 111’ was also effective (Kaushal et al., 2005). Wang et al. (1994) surface sterilized ‘Fuji’ nodal explants by applying 75% alcohol for 30 s followed by 0.5% HgCl2 plus 0.05% Tween-20 for 10 min. The disinfection procedure for 10 scions (‘Empire’, ‘Freedom’, ‘Golden Delicious’, ‘Idared’, ‘Jonathan’, ‘Liberty’, ‘McIntosh’, ‘Mutsu’, ‘Summerland McIntosh’, and‘Rome Beauty’) and two rootstocks (‘Malling 7A’ and ‘Malling 26’) originating from greenhouse stocks resulted in 70–90% survival of explants (Yepes and Aldwinckle, 1994). The explants were washed under running tap water for 2 h then agitated for 5 min in a solution containing 2 drops of Tween-20 in 100 ml of water and finally rinsed three times with distilled water followed by surface disinfection in 3% solution of calcium hypochlorite [Ca(OCl)2] for 10–20 min (Yepes and Aldwinckle, 1994). They concluded that young, newly expanded leaves were very sensitive to Ca(OCl)2; however, a reduction of surface sterilization time from 20 to 10 min increased (to 70–90%) the survival of the tissues. The procedure used in our laboratory was efficient for surface sterilization of explants from field-grown plants even when they

J. Dobránszki, J.A. Teixeira da Silva / Biotechnology Advances 28 (2010) 462–488

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Table 1 Summary of work on explant choice and on inhibition of phenol-induced browning of apple explants (PG: phloroglucinol; AA: ascorbic acid; AC: activated charcoal; CA: citric acid; PVP: polyvinyl-pyrrolidine; C: cysteine; BA: 6-benzyladenine; and BAR: benzyladenine-9-riboside). Cultivar

Explant (type, size, origin)

Arrangements for prevention of tissue browning

M.9

0.5 cm long shoot tips from PG (162 mg/l) glasshouse-grown mother plants

+

Fuji

Shoot tips from fieldgrown plants

++ ++ ++

Collecting explants at right time of the year Darkening of mother plants for 4 weeks Combination of cold (+5 °C) and dark treatment of explants for 6–8 days AA (100–150 mg/l) in the initiation medium for 6 days AC (2.5 g/l) in the initiation medium for 6 days Empire, Freedom, Golden Delicious, Idared, Jonathan, 1–2 cm long growing shoot Liquid medium, dark treatment and daily transfer of explants to fresh medium during Liberty, McIntosh, Mutsu, Summerland McIntosh, tips from greenhousethe 1st week Rome Beauty, Malling 7A, Malling 26, and Malus grown plants (previously Solidified initiation medium with AC (10 g/l) cold-treated) prunifolia xanthocarpa for 1 week CA (0.1 g/l) and AA (0.15 g/l) in the medium M.9 Shoot tips AA (0.1% for 3 days) in the medium Sodium metabisulfite (0.1–1% for 2–18 h) in the medium Chlororesorcinol (0.018% for 2–18 h) in the medium Glucono-lactone (1–2% for 2–18 h) in the medium Darkening of mother plants Heat treatment of mother plants (39 °C for 6 weeks) Pre-treatment of explants in shaked liquid medium (100 rpm) Cold treatment of explants (+ 4 °C for 18 h) End-product inhibition by adding oxidised phenolic compounds to the solidified medium Tydeman's Early Worcester Axillary buds Collecting explants at right time of the year AA (100 mg/l) and C (50 mg/l) in the medium PVP (500 mg/l) and AC (1000 mg/l) in the medium PVP (500 mg/l) and AA (150 mg/l) in the medium AA (50 mg/l), C (10 mg/l), PVP (500 mg/l) and AC (1000 mg/l) in the medium MM.106 Actively growing buds Pre-treatment of explants in shaked liquid medium (100 rpm) with PVP (0.1%) for 1–2 days PG M.26, MM.106, JTE-H, Prima, Galaxy, Jonagold, Growing shoot tips from Collecting explants at right time of the year Liquid medium contained perlite, CA (0.15 g/l), and Húsvéti rozmaring field-grown mother AA (0.1 g/l), PVP (1 g/l) AC (2.5 g/l), and plants substitution of BA with BAR (0.5 mg/l) in the initiation medium JTE-F, Remo, Rewena, Reanda Growing shoot tips from In vitro micrografting field-grown mother plants MM.111 Growing buds Smaller sized explants (0.2–0.6 cm long)

Efficacy for controlling References browning* James and Thurbon (1981), Jones (1985), Webster and Jones (1989) Wang et al. (1994)

++ −/+ ++

Yepes and Aldwinckle (1994)

++ ++ – –

Block and Lankes (1996)

– – ++ ++ −/+ + ++ ++ – ++

Modgil et al. (1999)

+ + ++

Sharma et al. (2000)

– ++ ++

Dobránszki et al. (2000a,b,c)

++

Dobránszki et al. (2005)

++

Kaushal et al. (2005)

*: Not effective for the inhibition of tissue browning, +: low efficiency, and ++: high efficiency.

were collected from strongly contaminated mother plants in a number of scions (‘Prima’, ‘Jonagold’, ‘Idared’, ‘Húsvéti rozmaring’, ‘McIntosh’, ‘Freedom’, ‘Remo’, ‘Reanda’, ‘Rewena’, ‘Royal Gala’, and ‘Galaxy’) and rootstocks (‘JTE-F’, ‘JTE-H’, ‘M.26’, and ‘MM.106’). Explants were first washed in tap water with 0.05% Tween-20 for 1 h in a shaker at 150 rpm and at 26 °C. After washing they were surface sterilized in 70% ethanol for 0.5–3 min followed by 0.1% HgCl2 solution for 5 min and washed three times with sterile distilled water. If necessary, because of extremely high contamination of stock plants, a second round of sterilization was done on the next day with 25% Clorox for 5 min (Dobránszki et al., 2000a,b,c, 2005). 3.1.3. Phenolic browning Explant browning and eventual death of the tissue during the initial stage of apple culture is a frequent problem. It occurs through

the action of polyphenol oxidase (PPO) and peroxidases (POX) by triggering defence reactions induced by wounding (Pan and van Staden, 1998). PPO catalyzes the reaction between different phenolic compounds and molecular oxygen producing quinones which are highly reactive and non-specifically polymerize proteins and produce dark pigments, melanin (Onay and Jeffree, 2000; Leng et al., 2009). In intact cells the enzymes and their substrates do not meet, because polyphenols, the substrates of PPO, are in the vacuoles, while the enzyme is in plastids or chloroplasts. When cells are wounded during explant excision, the browning reaction starts (Murata et al., 1997). The existence of a positive correlation between PPO activity and the level of phenolic compounds and explant browning has described in different lignaceous tree species (Laukkanen et al., 1999; Huang et al., 2002; Sharma and Singh, 2002; Leng et al., 2009). The important role of different antioxidant enzymes, such as POX and superoxide

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dismutase, has also been reported in explant browning (Laukkanen et al., 1999; Leng et al., 2009). The activity of enzymes associated with phenol oxidation is also affected by environmental factors. Light was suggested to increase enzyme activity (Linington, 1991; Wang et al., 1994; Pan and van Staden, 1998), while lowering temperature decreased phenolic biosynthesis by decreasing enzyme activity (Hildebrandt and Harney, 1988; Wang et al., 1994). The rate of tissue browning is influenced by several factors prior to and after explant isolation, as mentioned above (Werner and Boe, 1980; Webster and Jones, 1989; Marin et al., 1993; Yepes and Aldwinckle, 1994; Modgil et al., 1999; Dobránszki et al., 2000a; Kausal et al., 2005) and depends on the season when explants are collected. Browning was less when explants were collected in spring or summer (Modgil et al., 1999), although we (Dobránszki et al., 2000a) found that the majority of explants died because of strong browning of buds if they were collected in winter or spring, when dormancy ended and/or shoot growth was very intensive. Our results match earlier observations in which stock plants had a high phenolic content in the growing season and a high PPO activity which caused a high rate of browning, i.e., browning showed seasonal changes (Biedermann, 1987; Wang et al., 1994). Thus, explants should be collected after stock plants enter dormancy and before bud break (Wang et al., 1994). Several methods have been developed for preventing or controlling explant browning. Two groups can be distinguished. The first group of methods includes treatments of the mother plant such as darkening or heat treatment in order to bring it to optimal physiological conditions (Wang et al., 1994; Block and Lankes, 1996); the second group includes the direct treatment of explants. Explant treatments can include on one hand their pre-treatment during the first phase of culture initiation, such as pre-treatment in liquid culture, cold treatment, or dark treatment (Wang et al., 1994; Yepes and Aldwinckle, 1994; Block and Lankes, 1996; Modgil et al., 1999; Dobránszki et al., 2000a,b,c). On the other hand, supplementing the initial medium with different additives that can prevent the production of phenolics or can remove inhibitory phenolic substances from the media, such as antioxidants, chelate-forming materials or adsorbents (James and Thurbon, 1981; Jones, 1985; Webster and Jones, 1989; Wang et al., 1994; Yepes and Aldwinckle, 1994; Block and Lankes, 1996; Pan and van Staden, 1998; Dobránszki et al., 2000a,b,c; Sharma et al., 2000; Thomas, 2008) (Fig. 1a,b). The type of cytokinin applied in the initiation medium can also have an effect on the rate of tissue browning in apple (Dobránszki et al., 2000a) as reported earlier for other woody species such as Prunus davidopersica and Ailanthus altissima (Jámbor-Benczúr et al., 1995, 1997). When choosing from different methods it should be kept in mind that methods which prevent explant browning can simultaneously alter the metabolism of explants. Wang et al. (1994) reported that although the extension of dark treatment of stock plants successfully increased the prevention of browning, it also reduced the vigour of stock plants and thus explants became too weak to survive. Similarly, the optimal length of dark treatment in the first phase of culture initiation depends on plant species (Ziv and Halevy, 1983; Wang et al., 1994) and in the case of apple, also on the genotype (Table 1). Supplementing the initiation medium with activated charcoal (AC) effectively prevented explant browning in many plant species by adsorbing phenolics, by providing a dark environment and by inactivating PPO and POX, resulting in increased explant survival and organogenesis (Tisserat, 1979; Pan and van Staden, 1998; Nguyen et al., 2007; Thomas, 2008). Many materials, such as inhibitory substances, minerals, different organic compounds and PGRs can be adsorbed or desorped to/from AC and these materials become or cease to be available to explants altering explant metabolism, survival, growth and morphogenesis (Tisserat, 1979; Jaiswal and Amin, 1987; M'Kada et al., 1991; Pan and van Staden, 1998; Thomas, 2008). Hence, AC may either promote or inhibit growth and development in vitro. Application of other inhibitory substances, such as antioxidants or chelate-forming

Fig. 1. (a–h). Micropropagation of apple. In vitro culture establishment (a) on liquid medium contained perlite and activated charcoal and (b) on gelled medium. Shoot proliferation of (c) ‘Royal Gala’ and (d) ‘Húsvéti rozmaring’. In vitro root development of (e) ‘Royal Gala’ and (f) ‘McIntosh’. (g–h) Hardened plants of ‘Royal Gala’ in pots.

materials, in culture initiation medium have been widely used but their applications were only partly successful, possibly due to their influences on the metabolism of explants (Block and Lankes, 1996). Furthermore, the efficiency of treatments is highly genotype dependent in apple and in most cases successful control of explant browning could be achieved by different combinations of these methods (Table 1). 3.2. Shoot multiplication The success and commercial usefulness of an apple micropropagation protocol largely depends on the mode and rate of shoot multiplication. The efficacy of shoot multiplication is influenced by several factors, such as genotype, media composition, in vitro environmental factors, etc., whose roles are discussed next. 3.2.1. Cultivar/genotype The genotype-dependence of shoot multiplication of apple has been described by several authors (Lane and Looney, 1982; Lane and McDougald, 1982; Yepes and Aldwinckle, 1994; Karhu, 1995; Dobránszki et al., 2000a,b; Kovalchuk et al., 2009). Genotypic differences in growth

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and developmental responses were already demonstrated in the 1980s (Lane and Looney, 1982; Lane and McDougald, 1982). Lane and McDougald (1982) studied the shoot multiplication of four apple cultivars (‘M.27’, ‘M.9’, ‘M.26’, and ‘Macspur’) and found that cultivars differed in their response to the concentration of a cytokinin, 6-benzylaminopurine (BA) in the medium: optimum levels for the development of a maximum number of shoots varied between cultivars (5 µM for ‘M.26’ and ‘Macspur’ and 10 µM for ‘M.27’ and ‘M.9’). These different responses of apple cultivars to BA, namely different requirements for exogenous BA, might be associated with differences in the endogenous PGR levels (cytokinins and/ or other PGRs), with subsequent differences in the uptake of cytokinin from the medium, in the efficiency of transport via cultures or in the metabolism of BA (Lane and McDougald, 1982). Lane and Looney (1982), studying the in vitro growth of three strains of ‘McIntosh’ with different in vivo growth habits (standard, compact and extremely compact), found that all strains had similar optima (3–6 µM BA) for maximal shoot proliferation but their tolerances to a supra-optimal concentration (10 µM) of BA were different and were related to the growth habit in vivo; the production rate of new shoots as a percent of the rate at optimal concentration of BA were 90, 20 and 0% for extreme compact, moderate compact and standard strains, respectively. Webster and Jones (1991) noted differences in shoot production of four apple rootstocks. Shoot production was readily achieved with ‘P.22’ and ‘Ottawa 3’, although it was more difficult with ‘P.2’ and ‘B.9’. In the case of these latter rootstocks some improvement was achieved in shoot production on medium containing phloroglucinol (PG) (162 mg/l) and when BA was increased from 1 to 2 mg/l. Comparing shoot multiplication of 10 scions, two rootstocks and Malus prunifolia xanthocarpa, Yepes and Aldwinckle (1994) also found high variation in the number and length of shoots. Similarly, the multiplication rate and length of newly developed shoots were also strongly influenced by the genotype in our experiments (Dobránszki et al., 2000a,b) (Fig. 1c,d): the response of different scions (‘Prima’, ‘Galaxy’, and ‘Jonagold’) and rootstocks (‘M.26’, ‘MM.106’, and ‘JTE-H’) to PGRs in the media was significantly different. Studying the effect of carbohydrate quality on shoot multiplication of four apple cultivars (‘Gala’, ‘McIntosh’, ‘Make’, and ‘Jaspi’), Karhu (1995) observed that shoot growth and proliferation of ‘Gala’ was only hardly affected by the carbohydrate quality whereas significant differences in the shoot development of three other cultivars occurred. Highest shoot number was observed on medium containing sorbitol in ‘McIntosh’ (4.8±0.3) and in ‘Jaspi’ (1.7±0.2); however, shoots were longest on medium containing sucrose or glucose in ‘Make’ (11.7±1.5, 9.3±1.6) and ‘McIntosh’ (10.3±1.2, 10.2±1.2). Kovalchuk et al. (2009) studied the role of 16 wild and cultivated apple genotypes (‘Agat’, ‘Aport’, ‘Damira’, ‘Golden Delicious’, ‘Grushovka Verneskaya’, ‘Makpal’, ‘N 36’, ‘N 43’, ‘Naggit’, ‘Nurgul’, ‘Starkrimson’, ‘Royal Red delicious’, ‘Syislepper’, ‘TM-6’, ‘Ulan’, and ‘Voskhod’) on the in vitro storage of germplasm on PGR-free medium and three different responses of genotypes (capacity for short-, moderate-, or long-term storage) to cold (4 °C) storage were observed. 3.2.2. Explants and subculture Generally, shoot tips or shoots with multiple nodes are used as explants during the shoot multiplication of apple cultivars. Modgil et al. (1999) studied the effect of explant type of ‘Tydeman's Early Worcester’ on shoot proliferation in detail, comparing three different explant types: shoot tips, nodal cuttings and basal mass. They concluded that the number of new shoots was highest (8.4) when nodal cuttings were used as explants and that these shoots were mainly axillary shoots but shorter (3.16 cm) compared to shoots originating from shoot tip explants (4.4 new shoots per explant with a mean length of 5.16 cm). Shoot tips produced relatively few shoots (4.4) but their mean length was highest (5.16 cm) therefore they were suitable for root initiation. When the basal mass was used as the explant both the mean number (2.4) and mean length (1.22 cm) of shoots were lowest; moreover, the risk of formation of adventitious shoots increased. If 2–3 mm of shoot tips were removed causing the cessation of apical dominance and if they

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were placed horizontally in the medium, the growth of axillary shoots could be stimulated. The position of shoot cuttings can affect not only shoot proliferation but subsequent rooting as well, as was shown for ‘Húsvéti rozmaring’ (Dobránszki et al., 2000c). The vertical position of shoots on different proliferation media for a week prior to root induction inhibited rooting (0–6% rooting percentage), although shoots cultured horizontally prior to root induction rooted in 88.6% of all cases. Subcultures during micropropagation are reported to lead to rejuvenation, namely to a gradual physiological change which cause an increase in shoot production (Webster and Jones, 1989; Noiton et al., 1992). Webster and Jones (1989) noted an increase in the production of ‘M.9’ shoots over 21 months of subculture. Grant and Hammatt (1999) increased shoot production by increasing culture time: mean number of shoots was five-fold higher after culturing the apple lines subcultured at 28–42-day intervals in vitro for 430 days. Although shoot production depended on the time spent in in vitro culture it was not influenced by subculture frequency (28, 35 or 42 days). Kaushal et al. (2005) observed that the multiplication rate of ‘MM 111’ was lower after the first subculture than after the second and third. 3.2.3. Composition of medium 3.2.3.1. Salts and organic compounds. The most commonly used medium for apple micropropagation is Murashige and Skoog (MS) medium (1962). However, the application of other media has also been reported. Welander (1985), studying the effect of different media such as MS, Lepoivre (Quoirin et al., 1977) and C (Cheng, 1978) on shoot formation, found MS medium to be the most efficient for the production of in vitro shoots N1 cm from ‘Åkerö’. Van Nieuwkerk et al. (1986) used LS (Linsmayer and Skoog, 1965) medium for shoot proliferation on shoot tip explants of ‘Gala’. DKW (Driver and Kuniyuki, 1984) medium was applied by Muleo and Morini (2006, 2008) for ‘MM.106’ and ‘M.9’. Webster and Jones (1991) used modified MS medium where NaEDTA and FeSO4 were replaced by [CH2N(CH2COO2]2FeNa (20 mg/l). Modified MS medium with nitrogen at a third of the strength was used in the case of ‘M.9’ for studying the functionality of the photosynthetic apparatus in vitro (Zanandrea et al., 2006). Mouhtaridou et al. (2004) studied the effects of boron at a concentration range of 0.1 to 6.0 mM on the fresh mass, chlorophyll and mineral content of in vitro shoots of ‘MM.106’, and found that increasing boron concentration significantly decreased the fresh mass, shoot length and chlorophyll content of in vitro shoots. Moreover, the boron, phosphorous, calcium and magnesium content of shoots increased in the shoot while the potassium, iron, manganese and zinc content decreased, probably because boron might affect either the uptake or the transport of these mineral elements within plants. Ciccoti et al. (2008, 2009) compared the effect of MS, QL (Quorin and Lepoivre, 1977), WPM (Lloyd and McCown, 1981) and DKW (Driver and Kuniyuki, 1984) media on the shoot proliferation in M. sieboldii and 10 M. sieboldii-derived hybrid genotypes. A very strong genotype dependence was detected regarding the salt composition of the best propagation media; MS medium caused the best propagation in 3.3 (M. sieboldii) to 5.7 (‘C1907’). However, in the case of other two genotypes (‘D2212’ and ‘H0801’) QL salts led to significantly better proliferation rates (3.3, 3.9) and in two other genotypes (‘H0909’ and ‘Gi 477/4’) statistically similar proliferation was observed on MS medium (4.4 compared to 3.9 and 3.4 compared to 3.0, respectively). Using DKW medium the best proliferation (4.3) was achieved in one genotype (‘4608’). Reduced shoot height and chloroses were observed on WPM medium, possibly due to its low nitrogen content. In general, the highest proliferation rate was observed on MS medium and shoot growth was favoured when Fe-EDTA was replaced by Fe-EDDHA as the iron source. Organic compounds, especially vitamins of MS medium have also been modified. Karhu (1995, 1997) used MS medium without glycine but with 3 µM thiamine-HCl for ‘Gala’, ‘McIntosh’, ‘Make’ and ‘Jaspi’. Sriskandarajah et al. (1990a) used Staba (1969) vitamins for ‘Gala’, ‘Royal Gala’ and ‘Jonagold’, Baraldi et al. (1991) used LS vitamins for

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‘Golden Delicious’, while Liu et al. (2009) and Nabeela et al. (2009) used B5 vitamins (Gamborg et al., 1968) for ‘Orin’ and ‘Golden Delicious’ and ‘MM111’, respectively instead of standard MS vitamins. Liu et al. (2009) studied the effects of D-arginine, a potential inhibitor of arginine decarboxylase alone and in combination with exogenous putrescine (Put) on in vitro shoot growth of ‘Orin’. D-arginine increased growth retardation (fresh weight and shoot length) with an increase in its concentration from 1 to 5 mM. Moreover, its retarding effect was paralleled by a decrease in endogenous Put level. Growth of shoots treated by D-arginine could be recovered by adding exogenous Put. The effect of exogenous Put was proportional to its concentration added and to the increase of endogenous Put level. They concluded that modification of the endogenous polyamine (PA) pool could modify the growth of in vitro apple shoots, which could be associated both with its action as a nitrogen provider for shoot growth (Arias et al., 2005; Liu et al., 2009) and with the hormone-mediator effect of PAs at cellular levels (Hanzawa et al., 2000; Kuznetsov and Shevyakova, 2007; Pang et al., 2007). The stimulating effect of PG (1,3,5-trihydrixylbenzene, C6H3 (OH)3·2H2O) on the growth and proliferation of in vitro ‘M.26’ apple shoots was reported by Jones et al. (1977). However, James and Thurbon (1981) found that PG could not increase the number of ‘M.9’ shoots at any of 12 combinations of PGRs they studied while in one combination it had a decreasing effect. Webster and Jones (1991) reported that PG (162 mg/l) caused some improvement in the shoot production of apple rootstocks reported to be difficult to micropropagate (‘P.2’ and ‘B.9’), mainly when it was applied in combination with increased BA content and with a long subculture period. Maximum shoot number (6.66) and shoot length (2.73 cm) of ‘MM.106’ was observed on shoot proliferation media that was supplemented with 100 mg/l PG. Taken together, it seems that the usefulness of PG for improvement of shoot proliferation depends on genotype, PGR content of the medium and presumably on the number of subcultures. A fluoro derivate of 28-homoethylcastasterone (5F-HCTS), a brassinosteroid derivate, was reported to enhance the in vitro shoot multiplication of M. prunifolia in the concentration range of 100 to 10,000 ng per shoot through inhibition of stem elongation but mainly through increasing lateral branching. The stimulation of lateral branching caused by 5F-HCTS may be due to the effect of 5F-HCTS itself or to its stimulatory effect on cytokinin biosynthesis. Moreover, its inhibitory effect on stem elongation might be due to its stimulatory effect on ethylene biosynthesis (Schaefer et al., 2002). 3.2.3.2. Carbohydrates. Carbohydrates in the medium serve as energy, osmotic regulator and source of carbon. They can influence the growth and differentiation of tissues by regulating gene expression (Borges de Paiva Neto and Campos Otoni, 2003; Gibson, 2004; Zhou et al., 2006; Thorpe et al., 2008). In general, sucrose (3%) has been used as a carbohydrate source during shoot multiplication of apple. It is wellknown that carbon metabolism in apple is special because sorbitol and sucrose are the main photosynthetic products and more than 60% of carbon export occurs from source leaves as sorbitol (Loescher and Everard, 1996; Noiraud et al., 2001). High production of axillary shoots and biomass of apple in the presence of sorbitol has been reported (Pua and Chong, 1984; Welander, 1989). In ‘Macspur’, sorbitol enhanced axillary branching but did not increase the production of biomass (Pua and Chong, 1985). Karhu (1995) found that different carbohydrates affected the growth and proliferation of shoots in a different way in different cultivars: ‘Gala’ was little affected, but the presence of sorbitol at 165 mM enhanced axillary branching in ‘McIntosh’ and ‘Jaspi’ and longest shoots developed in ‘McIntosh’ and ‘Make’ compared to the effects of media containing glucose (165 mM) or sucrose (82.5 mM). Moreover, biomass production was lowest on medium containing sorbitol compared to media with sucrose or glucose. In experiments with ‘McIntosh’, Karhu (1997) proved that both the quality and the concentration of carbohydrates affected the production of axillary shoots. When the medium contained 4.4 µM BA, sorbitol-containing

medium (165 mM sorbitol) stimulated axillary branching of shoots compared to sucrose-containing medium (88 mM sucrose) but to a lesser extent than on sucrose medium (88 mM) with a high level of BA (13.3 µM), although this level of BA increased biomass production. Similar observations were made by Bahmani et al. (2009a) who found that shoot regeneration and proliferation of ‘MM.106’ rootstock improved on medium supplemented with 90 mM sorbitol whereas maltose was found to be the most detrimental. The different findings concerning the effectiveness of different carbohydrates may be either due to genotype dependence or to the adaptation of in vitro shoots to the carbon source of the medium on which they were previously maintained (Pua and Chong, 1985; Karhu, 1995). Sotiropoulos et al. (2006) also found genotype dependence in the use of carbohydrate when they studied the effect of different sucrose (44, 88, and 176 mM) and sorbitol (82, 164, and 329 mM) concentrations on the shoot growth and in vitro proliferation of ‘M.9’ and ‘MM.106’. While the optimal sucrose concentration was the same (88 mM) for both rootstocks in terms of shoot proliferation, there were differences in the optimum between the rootstocks regarding shoot length. Genotype dependence was more pronounced when sorbitol was used; optimal sorbitol concentration for shoot proliferation and growth was 164 mM in ‘M.9’, but ‘MM.106’ produced most shoots with the highest shoot length when 329 mM sorbitol was applied to the medium. Zhou et al. (2006) carried out growth analysis of transformed ‘Greensleeves’ with decreased sorbitol synthesis; moreover, they studied the activity and transcript level of the key enzymes [sorbitol dehydrogenase (SDH), sucrose synthase (SUSY)] in sorbitol and sucrose metabolism in shoot tips. They proved that the expression of SDH and SUSY are modulated by sorbitol and sucrose because these carbohydrates act as signal molecules. Therefore both enzymes are important in the regulation of plant growth and development by determining the sink strength of apple shoot tips. The effects of different carbohydrates were studied on the activity of different enzymes (POX and catalase) in the in vitro culture of ‘M.9’ and ‘MM.106’ (Sotiropoulos et al., 2006): sorbitol increased the activity of POX and catalase and that of POX was influenced by the addition of sucrose. 3.2.3.3. Plant growth regulators. Shoot branching depends on the initiation and activity of axillary meristems, which are hormonally controlled mainly by cytokinins; however, they act in interaction with auxins even though the auxin-effect is indirect (Ward and Leyser, 2004). Shoot multiplication of apple is based on media containing cytokinins as the major PGR, in lower concentration also auxins and in several cases gibberellin. The effect of different PGRs is highly genotype dependent (Table 2). In most work on shoot multiplication of apple, BA was used as the cytokinin source mainly in a concentration range between 0.5 and 2 mg/l (Baraldi et al., 1991; Marin et al., 1993; Yepes and Aldwinckle, 1994; Dobránszki et al., 2000a; Sharma et al., 2000; Kaushal et al., 2005). Lane and McDougald (1982) compared the response of apple shoot cultures to different concentrations of BA ranging from 1 to 10 µM for different cultivars (‘M.27’, ‘M.9’, ‘M.26’, and ‘Macspur’). They observed that a low concentration of BA (1 µM) resulted in fewer shoots (1.22 in ‘M.27’, 0.50 in ‘M.9’, 0.50 in ‘M.26’ and 1.47 in ‘Macspur’). Supra-optimal BA concentrations (10 µM) decreased the number of shoots in ‘M.26’ (2.72) and in ‘Macspur’ (3.10) but to a lesser extent than when applied at a low (1 µM) concentration. Optimal concentration of BA (5 µM for ‘M.26’ and ‘Macspur’ and 10 µM for ‘M.27’ and ‘M.9’) for the production of maximal number of shoots differed with the cultivar (7.54 in ‘M.27’, 3.91 in ‘M.9’, 3.41 in ‘M.26’ and 4.56 in ‘Macspur’). A similar effect of increasing BA concentration (1.1–15.4 µM) was observed in the case of ‘Åkerö’ (Welander, 1985). The optimum level was around 8.8 µM, above which no more increase could be observed in shoot number; however, the rate of hyperhydrated shoots increased. A strong negative correlation was reported between shoot length and shoot number; therefore, BA at 4.4 µM was used for shoot proliferation. Even though shoot number was

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Table 2 Efficient combination of plant growth regulators and their effects on shoot multiplication of apple. Cultivar (SC)a

M.27 (SC:1) M.9 (SC:1) M.26 (SC:1) Macspur (SC:1) Åkeröb (SC:6) Gala

Ougnoe Chernomorskoe letneey Golden Delicious Granny Smith Mutsu Spur Golden Delicious Julyred Greensleeves Tuscan (SC:7-10) Empire Freedom Golden Delicious Idared Jonathan Liberty Malling 7A Malling 26 McIntosh Malus prunifolia xanthocarpa Mutsu Rome Beauty Summerland McIntosh McIntosh Tydeman's Early Worcester M.26 MM.106 JTE-H Jonagold Prima Galaxy Húsvéti rozmaring MM.106c MM.106 EM7 M.26 JTE-H Jonagold MM.111 M.27 Malus sieboldii 4551 (Laxton's Superb × M. sieboldii) 4556 (Laxton's Superb × M. sieboldii) D2118 (open pollinated) D2212 (Laxton's Superb×M. sieboldii open poll.) H0801 (4556 × M9) H0901 (4556 × M9) H0909 (4556 × M9) 4608 (M. purpurea cv. Eleyi×M. sieboldii) Gi 477/4 (4608 open poll.) C 1907 (4608 open poll.) Golden Delicious Sukari Golden Delicious Golden Delicious MM.111 a b c

Plant growth regulators Cytokinin

Auxin

Gibberellin

10 µM BA 10 µM BA 5 µM BA 5 µM BA 4.4 µM BA 10 µM TDZ 1 µM TDZ 0.1 µM TDZ 4.4 µM BA 0.5–5.0 mg/l BA 0.5–5.0 mg/l BA 1 mg/l BA 0.7 mg/l BA 1 mg/l BA 0.5 mg/l BA 0.7 mg/l BA 4.4 or 8.8 µM BA 8.8 µM BA 1 mg/l BA 1 mg/l BA 1 mg/l BA 1 mg/l BA 1 mg/l BA 1 mg/l BA 1 mg/l BA 1 mg/l BA 1 mg/l BA 1 mg/l BA 1 mg/l BA 1 mg/l BA 1 mg/l BA 13.3 µM BA 2.2 µM BA + 7.5 µM KIN 0.5 mg/l BA 1.0 mg/l BAR 1.0 mg/l BAR 1.0 mg/l BAR 1.0 mg/l BAR 0.5 mg/l BA+1.5 mg/l KIN 1.0 mg/l BA+1.0 mg/l KIN 1.0 mg/l BA+1.0 mg/l KIN 0.5 mg/l BA+1.5 mg/l KIN 1.0 mg/l BA+1.5 mg/l KIN 0.5 mg/l BA 0.5 or 1.0 mg/l BA 0.5 or 1.0 mg/l BA 0.5 or 1.0 mg/l BA 3.5 mg/l TOP 5.0 mg/l TOP 2.0 mg/l BAR 1.0 mg/l BA 4.44 µM BA 4.44 µM BA 4.44 µM BA 4.44 µM BA 4.44 µM BA 4.44 µM BA 4.44 µM BA 4.44 µM BA 4.44 µM BA 4.44 µM BA 4.44 µM BA 4.44 µM BA 4.44 µM BA 4.44 µM BA 4.44 µM BA 2 mg/l TDZ

– – – – – – – – – – 0.5 µM IBA 1.4 µM GA3 0.5 µM IBA 1.4 µM GA3 0.5 µM IBA 1.4 µM GA3 0.5 µM IBA 1.4 µM GA3 0.1% IBA/IAA – 0.1% IBA/IAA – – 0.1 mg/l IBA – 0.1 mg/l IBA – 0.1 mg/l IBA – 0.1 mg/l IBA – 0.5 µM IBA – 0.5 µM IBA – 0.1 mg/l IBA 0.5 mg/l GA3 0.1 mg/l IBA 0.5 mg/l GA3 0.1 mg/l IBA 0.5 mg/l GA3 1.0 mg/l IBA 0.1 mg/l GA3 0.1 mg/l IBA 0.5 mg/l GA3 0.1 mg/l IBA 0.5 mg/l GA3 0.1 mg/l IBA 0.5 mg/l GA3 0.1 mg/l IBA 0.5 mg/l GA3 0.1 mg/l IBA – 1.0 mg/l IBA 0.1 mg/l GA3 0.1 mg/l IBA 0.5 mg/l GA3 0.1 mg/l IBA 0.5 mg/l GA3 0.1 mg/l IBA – 0.5 µM IBA – – – 0.1 mg/l IBA 0.2 mg/l GA3 0.1 mg/l IBA 0.2 mg/l GA3 0.1 mg/l IBA 0.2 mg/l GA3 0.3 mg/l IBA 0.2 mg/l GA3 0.3 mg/l IBA 0.2 mg/l GA3 0.3 mg/l IBA 0.2 mg/l GA3 0.3 mg/l IBA 0.2 mg/l GA3 0.3 mg/l IBA 0.2 mg/l GA3 0.3 mg/l IBA 0.2 mg/l GA3 0.3 mg/l IBA 0.2 mg/l GA3 0.1 mg/l IBA 1.0 mg/l GA3 0.1 or 0.3 mg/l IBA 0.2 mg/l GA3 0.1 or 0.3 mg/l IBA 0.2 mg/l GA3 0.1 or 0.3 mg/l IBA 0.2 mg/l GA3 0.3 mg/l IBA 0.2 mg/l GA3 0.1 mg/l IBA 0.2 mg/l GA3 0.1 mg/l IBA 0.2 mg/l GA3 – 0.5 mg/l GA3 1.47 µM NAA 0.58 µM GA3 0.25 µM IBA 0.28 µM GA3 0.25 µM IBA 0.28 µM GA3 0.25 µM IBA 0.28 µM GA3 0.25 µM IBA 0.28 µM GA3 0.25 µM IBA 0.28 µM GA3 0.25 µM IBA 0.28 µM GA3 0.25 µM IBA 0.28 µM GA3 0.25 µM IBA 0.28 µM GA3 0.25 µM IBA 0.28 µM GA3 0.25 µM IBA 0.28 µM GA3 0.25 µM IBA 0.28 µM GA3 0.25 µM IBA 0.28 µM GA3 1.47 µM NAA 0.58 µM GA3 1.47 µM NAA 0.58 µM GA3 0.2 mg/l NAA –

Multiplication rate

Shoot length

Reference

7.54 3.91 3.41 4.56 4.9 ± 0.3 10.6 ± 3.2 10.0 ± 2.4 8.5 ± 2.3 8.0 ± 2.4 – – 4.8 7.5 10.4 7.7 9–11 6.8 or 6.9 9.8 6.7 ± 2.8 6.6 ± 1.4 9.0 ± 1.0 5.0 ± 1.4 2.5 ± 0.7 7.0 ± 2.6 11.6 ± 2.5 5.3 ± 1.5 6.6 ± 1.5 7.8 ± 3.7 8.2 ± 1.2 6.3 ± 1.5 7.8 ± 0.7 4.1 ± 0.3 5–10 7.7 6.9 9.5 9.9 6.5 6.3 8.1 10.4 10.9 6.2 6.66 7.0 5.0 10.0 13.3 6.9 5.9 5.0 5.0 3.3 ± 0.4 3.7 ± 0.4 4.6 ± 0.4 3.5 ± 0.4 3.3 ± 0.1 3.9 ± 0.4 4.9 ± 0.4 4.4 ± 0.4 4.3 ± 0.4 3.4 ± 0.1 5.7 ± 0.3 4.8 ± 0.6 6.0 3.5 4.0 4.1

– – – – 1.6 ± 0.4 cm 2.3 ± 0.4 2.4 ± 0.3 2.8 ± 0.6 5.1 ± 0.7 – – – 2.7 3.6 4.9 – 10.8 or 9.8 mm 15.8 mm 2.0 ± 0.8 cm 2.0 ± 0.8 cm 2.0 ± 0.2 cm 2.3 ± 0.8 cm 2.8 ± 1.8 cm 2.3 ± 0.5 cm 1.5 ± 0.8 cm 1.3 ± 0.5 cm 2.1 ± 0.2 cm 2.0 ± 0.8 cm 2.1 ± 0.2 cm 1.2 ± 0.4 cm 1.5 ± 0.1 cm – – 31.9 mm 39.9 mm 22.1 mm 19.6 mm – – – – – 44.8 mm 2.73 cm – – – 12.6 mm 20.9 mm 21.9 mm 1.0–4.0 cm – 2.8 ± 0.1 cm 3.3 ± 0.2 cm 2.4 ± 0.2 cm 2.2 ± 0.1 cm 2.3 ± 0.2 cm 2.6 ± 0.3 cm 2.2 ± 0.1 cm 2.7 ± 0.1 cm 2.3 ± 0.1 cm 1.3 ± 0.1 cm 2.0 ± 0.1 cm 2.4 ± 0.2 cm

Lane and McDougald (1982)

2.0 cm –

Welander (1985) Van Nieuwkerk et al. (1986)

Kataeva and Butenko (1987) Baraldi et al. (1991) Abdul-Kader et al. (1992)

Lászlóffy et al. (1992) Marin et al. (1993) Yepes and Aldwinckle (1994)

Karhu (1997) Modgil et al. (1999) Dobránszki et al. (2000a)

Dobránszki et al. (2000b)

Dobránszki et al. (2000c) Sharma et al. (2000) Zaid et al. (2000)

Magyar-Tábori et al. (2001a) Magyar-Tábori et al. (2002a) Kaushal et al. (2005) Al-Rihani et al. (2005) Ciccoti et al. (2008, 2009)

Ali Bacha et al. (2009b) Al-Tiawni et al. (2009) Nabeela et al. (2009)

(SC) indicates the number of subcultures after which multiplication rate was determined, only in cases where this information was clearly available. Number and length of shoots N 1 cm and total number of shoots was 5.2 ± 0.5. PG was added to the medium (100 mg/l).

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suboptimal, this level was used to promote longer shoots. Marin et al. (1993) also reported the shortening and thickening of shoots at higher concentrations of BA: at 8.8 µM and 17.6 µM BA the shoots were swollen and distorted in ‘Greensleeves’. Yepes and Aldwinckle (1994) found that at a constant BA level (1 mg/l) the balance between gibberellic acid (GA3) and indole-3-butyric acid (IBA) affected shoot elongation: when the level of IBA was increased from 0.01 up to 1.0 mg/l, shoot elongation was stimulated but only in the presence of GA3 (0.5 mg/l). Effects of different combinations and concentrations of PGRs (BA, kinetin (KIN), GA3, and IBA) were tested on the shoot multiplication of ‘MM.111’ (Kaushal et al., 2005). The best multiplication (4–5 shoots per explant) and growth were observed with 0.5–1.0 mg/l BA and 0.5 mg/l GA3 without auxin. The effects of other types of cytokinins were also studied on shoot multiplication of different apple cultivars. Thidiazuron (TDZ) was tested during shoot proliferation of ‘Gala’ (Van Nieuwkerk et al., 1986). It stimulated shoot multiplication in a concentration range of 0.1–10 µM, although its stimulating effect was not significantly different from that of 4.4 µM BA (Table 2). However, shoots and leaves were smaller on medium with TDZ than on medium with BA. Moreover, by using TDZ, the formation of large shoot clumps was observed and some of these shoots seemed to be adventitious, which is unfavourable in true-to-type micropropagation protocols. The effects of different BA-derivates were also studied during shoot multiplication of apple in order to increase the efficacy of shoot proliferation and avoid the side effects of BA, such as difficulties in subsequent rooting (Webster and Jones, 1991), or toxicity after several subcultures (Werner and Boe, 1980). Better multiplication could be achieved in ‘MM.106’ and in ‘JTE-H’ when benzyladenine-9-riboside (BAR) was used instead of BA. When BA was used at 0.5–1.0 mg/l, the number of shoots obtained was between 4.5 and 5.4 while the use of BAR at 1.0 mg/l increased the rate of multiplication to 6.2–6.9 shoots in ‘MM.106’. In ‘JTEH’, the use of BAR showed a 1.5–2.5-fold increase in the number of shoots obtained when compared to the use of BA (Dobránszki et al., 2000a). The effects of BA, BAR and N6-meta-hydroxy-benzyladenin (TOP) applied between 0.5–5.0 mg/l were tested during shoot multiplication of ‘Jonagold’ and ‘JTE-H’ (Magyar-Tábori et al., 2001a, 2002a). The number of newly developed shoots (3.5–4.1) was not affected by the levels of BA for ‘Jonagold’; optimal concentration was 2.0 mg/l using BAR (5.9 shoots/ explant) and 5.0 mg/l using TOP (6.9 shoots/explant). A negative relationship was detected between the number and length of shoots as reported by others (Welander, 1985; Marin et al., 1993) but at concentrations optimal for shoot multiplication, the suppression of shoot length was 33.5–35.2% using TOP or BAR, while, with an increase of BA concentration from 0.5 up to 5.0 mg/l, shoot length was decreased by 41– 52%. Most ‘JTE-H’ shoots were produced at 2.0 mg/l BAR (11.7 shoots/ explant) and at 3.5 mg/l TOP (13.3 shoots/explant). BA was the least effective cytokinin, optimum at 3.5 mg/l (8.0 shoots/explant). Lower shoot growth suppression (31.2%) was observed using TOP, while the highest (66%) stunting effect was caused by BA. These results suggested that the BA derivate conjugated at the N9-position by a ribose (i.e., BAR) or the hydroxylated BA analogue (TOP) could increase the multiplication rate during shoot proliferation of apple with lower decreasing effects on shoot length compared to BA. Several authors studied dual cytokinin effect during shoot proliferation. A 5–10 multiplication rate was detected on medium containing 2.2 µM BA combined with 7.5 µM KIN while shoot multiplication rate was only 3–6 on medium containing 4.4 µM BA as the single cytokinin in ‘Tydeman's Early Worcester’ (Modgil et al., 1999). Shoot proliferation was enhanced by using 1.0 mg/l BA combined with 1.0 mg/l KIN in ‘Prima’ (8.1 shoots/explant) and in ‘Galaxy’ (10.4 shoots/explant) or 0.5 mg/l BA combined with 1.5 mg/l KIN in ‘Galaxy’ (10.9 shoots/explant) compared to the use of a single cytokinin (1.0 mg/l BA) (5.4 and 9.5 shoots/explant in ‘Prima’ and ‘Galaxy’, respectively). However, the application of two cytokinins did not favor the shoot multiplication of ‘Jonagold’: the multiplication rate caused by 0.5 mg/l BA (5.6) was not significantly lower than when 0.5 mg/l BA was combined with 1.5 mg/l KIN (6.3). When KIN

was combined with BAR the multiplication rate of ‘Jonagold’ decreased from 6.5 to 4.0 (Dobránszki et al., 2000b). In other experiments with ‘Húsvéti rozmaring’, the application of 1.0 mg/l BA combined with 1.0 or 1.5 mg/l KIN induced significantly more shoots (5.3 or 6.2) than media containing 0.5 or 1.0 mg/l BA, BAR or TOP alone (shoot number=2.3 to 4.7). Shoots were significantly longer on medium containing two cytokinins (44.4–44.8 mm) than on medium containing 1.0 mg/l BA alone (39.5 mm) (Dobránszki et al., 2000c). The addition of KIN (0.25– 1.0 mg/l) to different combinations of BA, GA3 and IBA during shoot proliferation of ‘MM.111’ affected neither the multiplication nor the growth of shoots (Kaushal et al., 2005). Shoot multiplication of apple rootstocks (‘M.9’ and ‘MM.111’) was affected by ethylene concentration (Lambardi et al., 1997). They found that stimulation of ethylene biosynthesis significantly decreased axillary shoot development in both rootstocks. ‘MM.111’ seemed to be more sensitive to ethylene than ‘M.9’. Application of aminoethoxyvinylglycine (AVG), an inhibitor of ethylene biosynthesis, together with the use of gas traps inside the culture flasks enhanced both axillary shoot formation and shoot elongation. 3.2.3.4. Other chemicals. The effect of potassium humate (KH) extracted from peat was tested at a concentration range of 0 to 500 mg/l in combination with different concentrations of BA on shoot proliferation of ‘Golden Delicious’ (Baraldi et al., 1991). When applied alone, KH had no effect on shoot proliferation, although when applied in combination with a cytokinin (1 mg/l BA), it had a negative impact. Nabeela et al. (2009) also found 1.0 g/l morpholino ethane sulfonic acid to be important for shoot proliferation when used in conjuction with other PGRs. Han et al. (2009) maximized the multiplication of shoot cultures of Malus hupehensis Rehd. var. pinyiensis Jiang by adding 20–30 µM sodium nitroprusside. Triacontanol at 20 µg/l (Tantos et al., 2001) increased shoot length and numbers more than the control. 3.2.3.5. Gelling agents. Most of the culture media used for micropropagation of apple are solidified with agar (0.6–0.8%). Agar is a natural polysaccharide obtained from mainly Graciliaceae and Gelidiaceae red algae. It is rather expensive, can affect the growth and development of in vitro cultures and can cause hyperhydricity and necrosis of apple cultures (Pasqualetto et al., 1988). Pereira-Netto et al. (2007) studied the effects of five agar brands on the proliferation and physiological state of ‘Marubakaido’ (M. prunifolia Borkh). They found differences in the rate of multiplication and hyperhydricity. The proliferation and performance of in vitro shoots were related to the degree of polydispersity and to the amount of higher molecular weight fractions in the agar brands and they were not connected to agar gel strength. Other natural polymers, such as galactomannans (Babbar et al., 2005), have also been used during apple shoot proliferation (Lucyszyn et al., 2005). Commercial galactomannans, guar gum or cassia, were mixed with agar (3–3 g/l) and used for gelling the medium during micropropagation of ‘Marubakaido’ (Lucyszyn et al., 2005). The use of these mixtures showed comparative shoot proliferation rates (7.60±1.45 for agar/guar mixture and 6.19±1.76 for agar/cassia mixture) compared to multiplication on standard agar-containing (6 g/l) medium (7.16±1.24). The occurrence of hyperhydric shoots was also lower (2.50±0.88% for agar/cassia mixture, 6.65±2.24% for agar/guar mixture and 19.35±1.90% for agar) (Lucyszyn et al., 2005). Similar enhancement of multiplication rate and decreased hyperhydricity occurred for both ‘Marubakaido’ and ‘Jonagored’ when gels created from a blend of 0.4% agar and 0.2% xyloglucan extracted from seeds of Hymenaea courbaril were used (Lima-Nishimura et al., 2002). Proliferation of six apple cultivars was equal or significantly better on medium gelled with a blend of corn starch and Gelrite compared to the same medium gelled with agar (Zimmerman et al., 1995). Bommineni et al. (2001) obtained more shoots from ‘Gale Gala’ on phytagel (0.25%)solidified shooting medium (21.0–28.4 shoots) than on agar (11.0–13.1 shoots). Substitution or partial substitution of agar with these different mixtures or with phytagel resulted in not only better efficiency of shoot

J. Dobránszki, J.A. Teixeira da Silva / Biotechnology Advances 28 (2010) 462–488

multiplication but was able to decrease the cost of micropropagation since the cost of the gelling agent per litre can be cheaper by as much as 90% (Babbar et al., 2005). Mohan et al. (2004) used sugarcane bagasse to increase rooting of ‘Marubakaido’ by 22% when particle sizes were b18 mm. Increased in vitro culture productivity and better medium utilization were reported by the application of a double-phase medium technique in three semi-dwarf apple rootstocks (‘M.26’, ‘MM.106’, and ‘P 14’) (Litwińczuk, 2000). The double-phase medium was obtained by pouring liquid MS solution onto an agar-solidified (6.4 g/l) medium at the beginning of subculture. Shoot fresh weight of ‘M.26’ and ‘P 14’ was higher than on agar-solidified (6.4 g/l) control medium partly due to the development of larger leaves. In the case of ‘P 14’ and ‘MM.106’ shoot proliferation and elongation were also enhanced. No undesirable changes such as hyperhydricity, leaf or shoot tip necrosis, or shoot fasciation, were observed during three successive subcultures of ‘M.26’ and ‘P 14’ and even after six subcultures for ‘MM.106’. Moreover, double-phase medium favored sucrose hydrolysis and the uptake of sugar and mineral components by cultures due to the liquid layer of the medium. They concluded that this technique is easy-to-use and its application could reduce the costs of micropropagation.

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dent on the exposure time to different light qualities. The results from these two experiments may be helpful to improve the setting of light conditions during the multiplication phase. 3.2.4.2. Temperature. Little attention has been paid to the effect of temperature during apple shoot multiplication in vitro. The applied temperature regimes have depended mostly on the facilities of different laboratories and no direct investigations were conducted regarding the effects of culture temperature on apple shoot multiplication. The temperature regimes under which different apple rootstocks and scions were cultured are summarized in Table 3. In most cases 24–26 °C was applied (Jones et al., 1977; Snir and Erez, 1980; Zimmerman and Fordham, 1985; Van Nieuwkerk et al., 1986; Noiton et al., 1992; Yepes and Aldwinckle, 1994; Wang et al., 1994; Karhu, 1995, 1997; De Klerk et al., 1997; Grant and Hammatt, 1999; Modgil et al., 1999; Litwińczuk, 2000; Bommineni et al., 2001; Lucyszyn et al., 2005; Liu et al., 2009), however, there are several reports when lower (22 °C) (Werner and Boe, 1980; Webster and Jones, 1989; Marin et al., 1993; Dobránszki et al., 2000a,b,c; Magyar-Tábori et al., 2001a, 2002a; Mouhtaridou et al., 2004) or higher (27 °C) (Schaefer et al., 2002) temperature was used. Some authors reported the application a day–night cycle with 28 and 22 °C (Lane and Looney, 1982; Lane and McDougald, 1982) or 24 and 22 °C (Welander, 1985).

3.2.4. Physical factors 3.2.4.1. Light. Light, one of the most important environmental signals, can affect morphogenesis through photoperiod, light intensity, or spectral wavelength. Usually in vitro cultures of apple during shoot multiplication were provided with a 16-h photoperiod from cool- or warm-white fluorescent lamps (400–700 nm). Several authors applied continuous illumination (Noiton et al., 1992; Schaefer et al., 2002). The applied light intensity varied between 38 and 105 µmol m− 2 s− 1. Marin et al. (1993) compared the effect of 8- and 16-h photoperiods on the shoot multiplication of columnar apple rootstocks (‘Tuscan’, ‘Trajan’, ‘Telemon’, and ‘Greensleeves’) and concluded that shoot production was significantly higher under an 8-h photoperiod. Irrespective of the irradiance level and quality of light during the proliferation phase, both of which can affect development, very little work has been done on the regulatory role of light on axillary branching in in vitro culture of apple. Zanandrea et al. (2006) evaluated chlorophyll fluorescence in ‘M.9’. During the multiplication phase a 16-h photoperiod was applied and in vitro shoots were irradiated with 15 µmol m− 2 s− 1 for 55 or 90 days and additional higher light intensity (15 µmol m− 2 s− 1) was added to plants for 55 days at 2 h/day before determining fluorescence. They concluded that in vitro shoots had a functional photosynthetic apparatus but the efficiency of the radiant energy use was low presumably due to low irradiance since the supply of additional light increased the photosynthetic activity. The effect of light quality was studied during shoot multiplication of apple rootstocks (‘MM.106’ and ‘M.9’) (Muleo and Morini, 2006, 2008). These studies showed that light quality regulated axillary branching and two independent actions were determined, one blue light- and the other red light-dependent. Blue light reduced branching and increased apical dominance while red light enhanced branching and inhibited apical dominance. The interaction between the two systems controlled by phytochromes and UV–A/blue light receptors regulates the development of the shoot system. In these studies with ‘MM.106’ bud differentiation and apical dominance could be regulated proving that shoot proliferation in apple is the response of an interaction between lateral bud differentiation and new shoot development, both of which are regulated by light quality (Muleo and Morini, 2006). The later study (Muleo and Morini, 2008) on the physiological analysis of blue and red light regulation of stem growth, apical dominance and branching in in vitro shoot culture of ‘M.9’ showed the effect of phytochrome photoequilibrium on shoot growth and the interaction between phytochrome and blue light receptors. Photomorphogenesis in shoot clusters was depen-

3.2.4.3. Others. Other physical factors, such as relative humidity in culture vessels, ethylene and carbon dioxide content (CO2) of gas phase in vessels can have an important influence on shoot growth and multiplication. Ethylene and CO2 accumulate in the gas phase of culture vessels during in vitro culture; the rate of accumulation depends on the volume and sealing of vessels, the tissue type being grown, the tissue weight and culture conditions, such as temperature or light (George and Davies, 2008). Predieri et al. (1991) examined the effect of CO2 enrichment (daily injecting of 100 and 200 ml pure CO2) on shoot development of ‘M.9’ and ‘Gala’ under different irradiance (30 and 70 µM s− 1 m2) and in the case of ‘Gala’ on medium with different sucrose concentrations (1 or 2%). In ‘M.9’, CO2 enrichment caused an increase in shoot proliferation, and fresh and dry weight after 21 days in culture on medium with 1% sucrose. In ‘Gala’, only a high CO2 concentration increased proliferation when the sucrose content of medium was low (1%) and the irradiation of cultures was high (70 µM s− 1 m2). When a low sucrose content (1%) was used in the medium any degree of CO2 enrichment increased the plant mass. In contrast, a higher sucrose concentration (2%) and lower CO2 enrichment (100 ml per day) increased the weight only under high irradiance. Li et al. (2001) observed that CO2enrichment (1000 µmol mol− 1) increased the fresh weight of shoots of ‘M.9’ from 46.2 to 101.3 mg under 40 µmol m− 2 s− 1 and from 70.4 to 147.4 mg under 100 µmol m− 2 s− 1. It also increased the number of leaves per plantlet (from 5.3–5.5 to 7.5–7.7) Albeit physiological effects of accumulated ethylene or relative humidity in vessels are proved in other plant species (reviewed by George and Davies, 2008; Moshkov et al., 2008), systematic studies regarding the role of relative humidity and ethylene in apple shoot cultures is not available. 3.3. Rooting Successful rooting of in vitro shoots prior to their establishment in soil is a prerequisite for any propagation method. Rooting of microshoots can be performed both under in vitro (Fig. 1e,f) and ex vitro conditions. 3.3.1. In vitro rooting of microshoots Adventitious rooting of plants depends on a series of interdependent phases (Gaspar et al., 1992) and in apple (‘Jork 9’) microshoots De Klerk et al. (1995) distinguished three main phases: dedifferentiation, induction and morphological differentiation. They concluded

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Table 3 Summary of temperature regimes applied during the shoot multiplication phase of micropropagation of different apple cultivars. Applied temperature regimes

Cultivar

References

22(± 2)°C

Malling 7 M.9 Golden Delicious Greensleeves, Tuscan M.26, MM.106, JTE-H, Prima, Galaxy, Jonagold, and Húsvéti rozmaring JTE-H Jonagold MM.106 Gala, Royal Gala, and Jonagold Åkerö

Werner and Boe (1980) Webster and Jones (1989) Baraldi et al. (1991) Marin et al. (1993) Dobránszki et al. (2000a,b,c) Magyar-Tábori et al. (2001a) Magyar-Tábori et al. (2002a) Mouhtaridou et al. (2004) Sriskandarajah et al. (1990a) Welander (1985)

Fuji M.9 Gale Gala M.26 Delicious, Redspur Delicious, Redchief Delicious, Vermont Spur Delicious, Royal Red Delicious, Gala, Golden Delicious, McIntosh, Spur McIntosh, Mutsu, Spartan, and York Imperial Gala Jork M9 Empire, Freedom, Golden Delicious, Idared, Jonathan, Liberty, McIntosh, Mutsu, Summerland McIntosh, Rome Beauty, Malling 7A, Malling 26, and Malus prunifolia xanthocarpa Jork 9 Gala, McIntosh, Make, Jaspi Tydeman's Early Worcester Marubakaido (Malus prunifolia Borkh) Orin MM.104, MM.106, and MM.109 Jonathan M.26, MM.106, and P 14 Marubakaido (Malus prunifolia (Willd) Borkh) KSC-3 M.9, M.26, and M.27, Macspur

Wang et al. (1994) Grant and Hammatt (1999) Bommineni et al. (2001) Jones et al. (1977) Zimmerman and Fordham (1985)

Summerland Red McIntosh, Macspur, and McIntosh Wijcik

Lane and Looney (1982)

22–24 °C 24/22 °C day/night temperature 24 °C 24.6 ± 2 °C 24 ± 2 °C 25(± 2)°C

26(± 2)°C

27 ± 1 °C 27–29 °C 28/22 °C day/night temperature

that dedifferentiation occurred in the first 24 h, induction from 24 to 72–96 h, and differentiation after 3–4 days. An in vitro rooting procedure of apple shoots in practice involves a root induction phase followed by a root elongation phase with specific requirements at each phase. The knowledge of anatomical, biochemical and physiological processes during the different phases of adventitious rooting is indispensable and can provide tools for direct manipulation of in vitro rooting even in cultivars known to be recalcitrant to rooting. Hicks (1987) followed the time course of adventitious rooting in an apple rootstock (‘KSC-3’) microcuttings (1.5–2.5 cm) cultured on media with IBA (0.3 mg/l) for 0, 1.5, 3, 5, 7 and 10 days. Emergent root primordia were visible from the 7th day and the frequency of rooted shoots reached 59–78% by the 21st–27th day. Anatomical changes occurred within a few mm of the cut surface in shoots. Activation response was detected over the first 3 days such as densely stained cells in a variety of tissues of the cambial zone with enlarged nuclei and nucleoli, from which root meristemoids developed outside the xylem by the 5th day. These meristemoids developed into root primordia by the 10th day and a vascular connection between root and stem had been initiated. Comparing individual stems of the same chronological age asynchrony was detected in the timing of developmental events, which may be due to unidentified variation between stems used for microcuttings. Anatomical and ultrastructural observations in ‘Jork 9’ showed a very similar pattern (De Klerk et al., 1995; Jásik and De Klerk, 1997). Harbage et al. (1993) examined the development of adventitious rooting in ‘Gala’ and detected changes during induction (medium contained 1.5 µM IBA) and post-induction (medium without IBA) stages. A time-course analysis of root induction showed that root initiation was detectable within 1 day (cytoplasm of phloem parenchyma cells became dense and cells had prominent nuclei) and root

Van Nieuwkerk et al. (1986) Welander and Pawlicki (1993) Yepes and Aldwinckle (1994)

De Klerk et al. (1997) Karhu (1995), Karhu (1997) Modgil et al. (1999) Lucyszyn et al. (2005) Liu et al. (2009) Snir and Erez (1980) Noiton et al. (1992) Litwińczuk (2000) Schaefer et al. (2002) Hicks (1987) Lane and McDougald (1982)

primordia developed by the 4th day. Meristem activity was detected only in the basal part (1 mm) of microcuttings. Only phloem parenchyma cells took part in the formation of meristematic regions. Time-course analyses of the post-induction phase rapidly shifted (within 1 day) root formation from primordial initiation to the development of a root tissue system, which indicates that transfer of microcuttings to auxin-free medium caused differentiation and organization of the root system. A tracheid connection between root and stem was continuous. Welander and Pawlicki (1993) developed a model system for rooting stem discs originating from the basal part of the stem from axillary in vitro shoots (‘Jork M9’) and performed anatomical studies using light, transmission and scanning electron microscopy for tracing histological events leading to root development. Histological events detected were similar those reported by Hicks (1987); on the 2nd day cambial activity increased, on the 4th and 5th day meristemoids developed, and by the 7th day root primordia started to emerge from the stem cortex. Variation in rooting capacity between shoots within the same shoot cluster was also detected. They concluded that 1 day of IBA treatment (24.6 µM) was sufficient to induce rooting despite no anatomical changes being detected after one day (Welander and Pawlicki, 1993). Naija et al. (2008) studied the anatomical and biochemical aspects of adventitious rooting of ‘MM.106’ and described the same histological events, such as increasing cambial activity and first cell divisions at day 3, development of meristemoids by day 5 followed by development of an organized root system by day 10, as reported earlier in other apple cultivars (Hicks, 1987; Welander and Pawlicki, 1993). POX activity of induced shoots after placing them on rooting medium decreased and reached a minimum at days 1 and 2 corresponding to the induction phase

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and then it increased until day 5 corresponding to the end of the initiation phase, which indicates a correlation between the ontogenic stages and biochemical events during in vitro rooting as was suggested earlier (Gaspar et al., 1992; Auderset et al., 1994; Gaspar et al., 1994). Ali Bacha et al. (2009a) easily rooted ‘Golden Delicious’ and ‘MM.111’ rootstock in vitro by transferring 2–3 cm long shoot tips to half-strength MS basal rooting medium supplemented with 1.0 mg/l IBA followed by auxin-free half-strength MS medium in the case of ‘Sukari’ (Ali Bacha et al., 2009b). In vitro rooting of microshoots is affected by the interaction of several internal and external factors which are discussed next. 3.3.1.1. Cultivars/genotypes. Differences in rooting ability between rootstocks and scions are not surprising considering that rootstocks need to have high rooting frequencies in the field; therefore, this trait is selected for during breeding. However, it is well-documented that genotype dependence exists both in rootstock varieties and scions regarding in vitro rooting ability, or rather, their requirement for optimal rooting conditions. Lane and McDougald (1982) compared the response of ‘M.27’, ‘M.9’, ‘M.26’, ‘MM.111’ and ‘Macspur’ to different concentrations (0.1, 0.33, 1.0, 3.3, 10 and 33 µM) of α-naphthaleneacetic acid (NAA) and found that the optimal level of NAA for rooting differed between cultivars. ‘M.27’, ‘M.26’ and ‘Macspur’ rooted best at 1 µM while ‘MM.111’ at 3.3 µM NAA. They also observed that rootstocks (84–85%) rooted better than scion ‘Macspur’ (58%) at the same NAA optimum. However, ‘M.9’ required a special treatment for rooting. Excised ‘M.9’ shoots were dipped into a solution containing 1.61 µM NAA followed by culture on auxin-free medium and 52.2% rooting could be achieved. The effects of subculture for increasing rooting percent were reported to be different in different scions (Sriskandarajah et al., 1982). They observed that in ‘Jonathan’ 95% rooting was obtained after 9 subcultures whereas 31 subcultures were necessary for a maximum of 79% rooting for ‘Delicious’. Similarly, Zimmerman and Fordham (1985) observed that in ‘Mutsu’ and ‘McIntosh’ 4 subcultures were enough for increasing rooting above 90%. Other cultivars were not able to root better even after many more subcultures as observed in the case of ‘Redspur Delicious’, which rooted to a maximum of 74% after 38 subcultures. PG treatment (100 mg/l) during rooting caused different rooting responses in 12 apple scions (Zimmerman and Fordham, 1985) (Table 4). James (1983) examined indole-3-acetic acid (IAA) uptake and distribution in a difficult-to-root rootstock (‘M.9’) and in an easy-toroot rootstock (‘M.26’). He found that differences in rooting reflect the metabolism of exogenous IAA rather than differences in its uptake or distribution. Later, Alvarez et al. (1989) found that conjugated IAA levels were significantly higher in ‘M.9’ than in ‘M.26’ while free IAA levels in apical shoot sections of both rootstocks were comparable (298 and 263.7 ng/FW) but in basal sections the free level of IAA was 2.8-times higher in ‘M.26’ than in ‘M.9’. These differences can explain the differences which were found in rooting responses of these rootstocks. Yepes and Aldwinckle (1994) studied the rooting of 10 scions and two rootstocks and reported that the optimal IBA concentration was cultivar-dependent (Table 5). Isutsa et al. (1998) compared the in vitro rooting of three rootstocks (‘G.65’, ‘G.30’, and ‘G.11’) at 10 levels of IBA (up to 5 mg/l) for determining optimal conditions of rooting and concluded that the rooting response of different rootstocks was different and ‘G.65’, ‘G.30’ and ‘G.11’ were found to be difficult-, moderate- and easy-to-root in vitro (Table 5). Litwińczuk (2000) also found genotype dependence in the in vitro rooting of three rootstocks. ‘P 14’ and ‘MM.106’ showed a high percentage of rooting (98.5 and 89.9%, respectively) at 1.0 mg/l IBA after using double-phase shoot multiplication medium whereas ‘M.26’ showed only 66.4% rooting. Other rootstocks (‘JTE-H’, ‘M.26’ and ‘MM.106’) also demonstrated genotype dependence in the rooting responses to IBA level (1, 2, or 3 mg/l IBA). Rooting in ‘JTE-H’ was high (91–100%) at all levels of IBA, rooting in ‘MM.106’ was low (33–46%) while rooting in ‘M.26’

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decreased as IBA concentration increased (from 94.3 to 62.9%) (MagyarTábori et al., 2002b). 3.3.1.2. Explant position and subculture. The position of microshoots and number of subcultures prior to rooting was reported to affect the efficacy of in vitro rooting. Isutsa et al. (1998) investigated the effects of two orientations of shoots on the in vitro rooting in three apple rootstocks. Cut ends of microshoots were inserted into rooting medium. In half of the Petri dishes shoots were placed in an upright position in the medium while in the remaining Petri dishes they were inserted in an inverted position to reverse polarity and after 2 weeks inverted microshoots were placed upright. Rooting percentage significantly depended on the orientation of microshoots and a strong interaction was found between microshoot orientation, genotype and auxin level regarding rooting percentage. ‘G.30’ rooted (2–65%) over a wide range of IBA concentrations (from 1.0 to 5.0 mg/l) when they were in an inverted orientation but they rooted only at 2 or 3.5 mg/l IBA when they were in an upright orientation with a rooting percentage of 20 and 100%, respectively. 100% rooting was achieved with ‘G.11’ at 1.0 or 2.0 mg/l IBA in the inverted orientation but at 3 mg/l IBA in the upright orientation. ‘G.65’ rooted only in an inverted orientation with maximum 30% rooting at 2.0 mg/l IBA. Shoot orientation on the last shoot multiplication medium prior to rooting was reported to influence the efficacy of the subsequent rooting process (Dobránszki et al., 2000c). Explants were placed horizontally on shoot multiplication media during shoot multiplication. Newly developed 3-week-old shoots from the last multiplication media immediately prior to exposing shoots to root induction were cut and placed either vertically or horizontally on the same proliferation media for a week. When shoots of ‘Húsvéti rozmaring’ were placed in a vertical orientation for a week prior to transfer onto rooting medium, rooting percentage decreased considerably (0–6%) compared to shoots (88.6%) which were placed horizontally on the last proliferation medium. The vertical position stimulated callus formation at the base of shoots (Dobránszki et al., 2000c). Increasing the number of subcultures could increase the rooting of different apple cultures due to rejuvenization (Webster and Jones, 1989). Sriskandarajah et al. (1982) studied the in vitro rooting of usually difficult-to-root scions (‘Jonathan’ and ‘Delicious’) after initial culture of shoots (subculture-0) and after a maximum of 32 subcultures and concluded that there was a progressive improvement of rooting as subculture increased; after subculture-0 5–8% of shoots rooted but rooting percentage reached 95% in ‘Jonathan’ after 9 subcultures and 79% in ‘Delicious’ after 31 subcultures. In ‘Jonathan’, the rooting percentage did not increase further as the number of subcultures increased while the mean number of roots per shoot increased from 9.4 (after 9 subcultures) to 12.1 after 25 subcultures. Repeated subcultures alone could not improve adventitious rooting, only if they were combined with optimisation of some physical factors (i.e., continuous lighting, 26 °C). Noiton et al. (1992) studied the effects of subcultures on the endogeneous levels of IAA and abscisic acid (ABA) and their effects on the rootability of ‘Jonathan’. They concluded that the transition between difficult-to-root (0% rooting) and easy-to-root (N70% rooting) conditions occurred after 4 subcultures corresponding to a decrease in endogenous ABA content (from 1328.6 to 186.7 ng/g dry weight) and with an increase in the ratio of IAA/ABA from 0.2 to 0.6. The IAA/ABA ratio increased up to 0.7 after 26 subcultures when 100% rooting was detected. Improvement in rooting ability was not correlated with the level of endogenous IAA. Similarly, Zimmerman and Fordham (1985) observed that the rooting ability increased in several cultivars by increasing the number of subcultures; however, they did not specifically compare the changes in rooting percentage with culture time. ‘M.9’ gradually showed enhanced adventitious root production, from a few percent up to 70–90%, as the number of subcultures increased over 21 months (Webster and Jones, 1989). According to Grant and Hammatt (1999) rooting ability of ‘M.9’ was mainly affected by total time spent in culture rather than subculture frequency, similar to their observation

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Table 4 Summary of work on the effects of phloroglucinol (PG) application during in vitro rooting of apple. Cultivar

Treatment

M.9

PG (162 mg/l) IBA (2 mg/l) PG (162 mg/l)+IBA (2 mg/l) PG (162 mg/l) IBA (2 mg/l) PG (162 mg/l) + IBA (2 mg/l) PG + (1 mM) PG − PG + (1 mM) PG − PG + (1 mM) PG − PG + (1 mM) PG − PG + (1 mM) PG − PG + (1 mM) PG – PG + (1 mM) PG – PG + (1 mM) PG − PG + (1 mM) PG − PG + (1 mM) PG − PG + (1 mM) PG − PG + (1 mM) PG − PG (162 mg/l)

Delicious Royal Red Delicious Vermont Spur Delicious Spartan Redchief Delicious Redspur Delicious Gala Golden Delicious McIntosh Spur McIntosh Mutsu York Imperial M.9

a

Tydeman Early Worcester b

MM.106

PG (100 mg/l)

PG (100 mg/l)

Rooting percentage

Root number per shoot

References

For 38 days

0% 8% 62%

0 2.2 3.6

James and Thurbon (1979)

For 4 days prior to culture on hormonefree medium

5.5% 30% 69%

1.8 4.4 12.0

31% 74% 15% 45% 20% 60% 66% 80% 0% 12% 90% 98% 97% 100% 92% 95% 68% 85% 82% 87% 70% 50% 15% 15%

− − − − − − − − − − − − − − − − − − − − − − − −

in shooting medium − − + + in liquid medium − − + in shooting medium − + − +

in rooting medium − + − + in solid medium − + + in rooting medium − − + +

Zimmerman and Fordham (1985)

Webster and Jones (1989) 77% 93% 69% 93%

− − − − Modgil et al. (1999)

50% 68.4% 64%

− − − Sharma et al. (2000)

65.75% 80.5% 53.25% 60.25%

− − − −

+: Presence and −: absence. a After 21 subcultures. b Shoots was rooted in liquid medium for 9 days followed by the agar-solidified medium.

regarding shoot multiplication, which also increased as culture time increased. Webster and Jones (1991) reported that subculturing increased the rooting of four cold-hardy dwarfing rootstocks: ‘P.22’ and ‘Ottawa 3’ reached 85–100% rooting after 9 months of subculture, after 27 months of subculture for ‘P.2’ to achieve 70% rooting and a maximum of 50% rooting even after 39 subcultures in ‘B.9’. 3.3.1.3. Composition of medium 3.3.1.3.1. Salts and organic compounds. In general MS medium is employed in which the salt concentration is reduced mainly by half or a third (Werner and Boe, 1980) during in vitro rooting of apple. Besides MS medium also the use of Lepoivre medium (Quoirin et al., 1977) with 1/2-strength macroelements (Welander, 1985; Welander and Pawlicki, 1993; Druart, 1997; Puente and Marin, 1997) and Linsmayer and Skoog (1965) medium with full (James and Thurbon,

1979; Karhu and Zimmerman, 1993) or 1/2-strength macroelements (James and Thurbon, 1979, 1981) is common. Sriskandarajah et al. (1990b) observed that the reduction of ammonium nitrate (NH4NO3) to 1/4-strength in MS medium increased the rooting percentage of ‘Gala’ and ‘Royal Gala’. Omitting NH4NO3 from the rooting medium increased the rooting of ‘Jonagold’. 100% rooting was achieved on NH4NO3-free medium in all three cultivars when potassium nitrate (KNO3) was provided at full strength. Druart (1997) examined the effects of different macroelements during root initiation and elongation in ‘Compact Spartan’. The presence of NH4NO3 inhibited root emergence and growth in root elongation medium but in root induction medium it only had a delaying effect on root emergence but did not affect final rooting. KNO3 promoted rooting when it was applied in root induction medium, which contained auxin but it reduced root growth and the final length of the root system in root

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Table 5 Commonly used auxins and their effects on in vitro rooting in apple. Cultivars (SC)a

IBA

IAA

NAA

Rooting percentage

References

M.9 MM.104 MM.106 MM.109 Granny Smith M.27 (SC:1) M.26 (SC:1) MM.111 (SC:1) Macspur (SC:1) Jonathan (SC:9)

2 mg/l 1 mg/l 1 mg/l 1 mg/l 10 µM – – – – –

– – – – – 1 µM 1 µM 3.3 µM 1 µM 10 µM

2.5 µM 0.3 mg/l – – 0.3 mg/l – – 0.3 mg/l – – 0.3 mg/l – – 0.3 mg/l – – 0.3 mg/l – – 0.3 mg/l – – 0.3 mg/l – – 0.3 mg/l – – 12 µM 4 µM 0.2 mg/l 1.0 mg/l 0.3 mg/l 0.2 mg/l 0 mg/l 0.3 mg/l 1.0 mg/l 2 mg/l 3 mg/l 2 mg/l 1.5 µM – – 0.5 mg/l 1 mg/l – 1 mg/l 2 mg/l 1 mg/l 0.49–14.70 µM 0.5 mg/l 25 µM 25 µM 25 µM 25 µM 25 µM 25 µM 25 µM 25 µM 25 µM 25 µM 25 µM 25 µM 4.9 µM

95 100 100 100 78–82 85 85 84 58 95 95 90 56 36 66 45 20 18 0 2 2 90 87 93 82 70 90 64 31 64 100 97 97 87 60 63 80 40 73 80 100 100 71 100 100 100 100 100 30 100 100 50–68.4 0–7 10.67–11 20.33–25 20.33–22.67 91 94.3 46 100 85 90 100 67 55 82 100 60 95 100 75 73 85–100 100 95

James and Thurbon (1979) Snir and Erez (1980)

Åkerö (SC:11) Delicious (SC:26,27)

– – – – – – – – – – 100 µM – – 0.3 mg/l – – 0.3 mg/l – – 0.3 mg/l – – 0.3 mg/l – – 0.3 mg/l – – 0.3 mg/l – – 0.3 mg/l – – 0.3 mg/l – – 0.3 mg/l – – – – – – – – – 0.1 mg/l – – – – 0.5 mg/l 1 mg/l – – 17.1 µM – – – – – – – – – – – – – – – – – –

Redspur Delicious (SC:38)

Royal Red Delicious (SC:29)

Gala (SC:33)

Golden Delicious (SC:23)

McIntosh (SC:4,6)

Spur McIntosh (SC:22)

Mutsu (SC:4)

Spartan (SC:22)

M.9 M.26 Empire Freedom Liberty Malling 7A McIntosh Malus prunifolia xanthocarpa Compact Spartan G.65 (difficult-to-root) G.30 (moderate-to-root) G.11 (easy-to-root) Tydeman Early Worcester MM.106

Gale Gala M.26 MM.106 JTE-H M.27 MM.111 Malus sieboldii 4551 (Laxton's Superb × M. sieboldii) 4556 (Laxton's Superb × M. sieboldii) D2118 (open pollinated) D2212 (Laxton's Superb × M. sieboldii open poll.) H0801 (4556 × M9) H0901 (4556 × M9) H0909 (4556 x M9) 4608 (M. purpurea cv. Eleyi × M. sieboldii) Gi 477/4 (4608 open poll.) C 1907 (4608 open poll.) Golden Delicious Golden Delicious

– – – 0.3 mg/l – – 0.3 mg/l – – 0.3 mg/l – – 0.3 mg/l – – 0.3 mg/l – – 0.3 mg/l – – 0.3 mg/l – – 0.3 mg/l – – 0.3 mg/l – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –

Sriskandarajah and Mullins (1981) Lane and McDougald (1982)

Sriskandarajah et al. (1982) Welander (1985) Zimmerman and Fordham (1985)

Alvarez et al. (1989) Yepes and Aldwinckle (1994)

Druart, 1997 Isutsa et al. (1998)

Modgil et al. (1999) Sharma et al. (2000)

Bommineni et al. (2001) Magyar-Tábori et al. (2002b)

Al-Rihani et al. (2005) Kaushal et al. (2005) Ciccoti et al. (2008, 2009)

Al-Tiawni et al. (2009) (continued on next page)

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Table 5 (continued) Cultivars (SC)a

IBA

IAA

NAA

Rooting percentage

References

McIntosh Red Fuji Prima Royal Gala

1 mg/l 1 mg/l 3 mg/l 2 mg/l

– – – –

– – – –

100 82.9 94.3 94.3

Magyar-Tábori et al. (2009)

a

(SC) indicates the number of subcultures after which rooting was induced, only in cases where this information was clearly available.

elongation medium. Root emergence occurred fastest, and root growth and viability of roots were stimulated in the presence of Ca (NO3)2; MgSO4 (0.27 g/l MgSO4·7H2O) favoured root elongation. Longest roots were detected in the presence of KH2PO4 (0.14 g/l) and Ca(NO3)2 (0.6 g/l Ca(NO3)2·4H2O). There are great differences regarding the application of different combinations of vitamins during the in vitro rooting phase. In general MS vitamins have been used but application of Linsmayer-Skoog (James and Thurbon, 1979, 1981; Baraldi et al., 1991) or Lepoivre (Welander and Pawlicki, 1993; Puente and Marin, 1997) vitamins is also common. However, in some reports (Webster and Jones, 1991; Druart, 1997; Bolar et al., 1998), the medium was supplemented only by thiamine (0.4–1 mg/l) and myo-inositol (100 mg/l). The vitamin composition of root induction and elongation media differed in some experiments. Myo-inositol (100 mg/l) and vitamin B1 (0.5 mg/l) was applied in root induction medium and only myo-inositol at 1/2strength (50 mg/l) in root elongation medium (Dobránszki et al., 2000c; Magyar-Tábori et al., 2001b,c, 2002b). The effect of different concentrations of KH (50, 250, 500 mg/l) in combination with different concentrations of IBA (0.06, 0.3 mg/l) was studied in ‘Golden Delicious’ (Baraldi et al., 1991). An interaction between IBA and KH was observed. At 0 or 0.06 mg/l IBA with KH at 500 mg/l increased the rooting up to 96% and the combination of 0 or 0.06 mg/l IBA with KH at 250 and 500 mg/l increased the number of roots as well. However, root length decreased by increasing the concentration of KH. When KH was increased from 50 to 500 mg/l, the root length decreased from 28 to 11 mm and from 26 to 7 mm in the presence of 0.06 and 0.3 mg/l IBA, respectively. An auxin-like effect of KH was apparently related to the activity of carboxylic and oxidrillic groups, which protect auxin from oxidation (Mato and Mendez, 1970). The interaction of phenolic metabolism with auxin metabolism controls root formation, mainly at an early stage. Coumarin (1,2benzopyrone) at 30–150 µM has been used to enhance rooting in apple scions (‘Gala’, ‘Delicious’, ‘Redchief Delicious’, and ‘Redspur Delicious’) (Karhu and Zimmerman, 1993). Coumarin significantly affected the rooting percentage in all cultivars. Coumarin (90 µM) increased the rooting of ‘Delicious’ even without IBA from 23 to 63% and it improved the rooting of ‘Gala’ up to 100% in the presence of IBA (1.5 µM). Coumarin significantly increased the number of roots of ‘Delicious’, whose increase was quadratically related to the increase in concentration of coumarin. Systematic studies were conducted regarding the effect of PG on in vitro rooting to enhance the rooting process. PG was able to increase the rooting percentage (James and Thurbon, 1979, 1981; Webster and Jones, 1989; Modgil et al., 1999) and also the number of roots per shoot but not in all cultivars (Zimmerman and Fordham, 1985; Sharma et al., 2000). There was an interaction between the PG and IBA content of the medium (James and Thurbon, 1979). Moreover, Sharma et al. (2000) showed the preconditioning effect of PG on shoots for subsequent rooting. Results of studies on the effects of PG are summarized in Table 4. Naija et al. (2009) proved the involvement of PAs in the rooting process during the root induction phase and their interaction with auxin in ‘MM.106’. Put, spermidine (Spd), and inhibitors of PA biosynthesis, such as cyclohexylamine (Cha) and aminoguanidine (AG) were applied during the 1st day of culture in the absence of IBA. They enhanced rooting from 0 to 42.22% (Put), 26.66% (Spd), 53.33% (Cha) and 26.66% (AG). However, another inhibitor of PA metabolism, α-difluoromethylornithine (DFMO) inhibited rooting when it was applied together with IBA (from 96.67 to 63.33%). On

IBA-free medium Put and Cha increased the endogenous levels of IAA and indole-3-acetylaspartic acid (IAAsp) but Spd, AG and DFMO had no effect. The endogenous level of Put was increased to a maximum by the 2nd day under rooting conditions and was slightly affected by the exogeneous application of Put or Cha, but was reduced by Spd, AG and DFMO. 3.3.1.3.2. Carbohydrates. Sugar is essential for root growth; no roots developed on sugar-free medim in ‘Merton Malling’ apple rootstocks but rooting could be restored by transferring shoots to sucrosecontaining (2%) medium (Snir and Erez, 1980). The effects of sucrose between concentration ranges of 0 to 2% on rooting of ‘Granny Smith’ showed that 1% sucrose concentration was optimal at 10 µM IBA in agitated liquid culture (Sriskandarajah and Mullins, 1981). Karhu and Ulvinen (1995) examined the effects of different carbohydrates (88 mM sucrose, 175 mM sorbitol, glucose, fructose and xylitol) on the rooting of ‘Make’ and ‘McIntosh’ and on the development of plantlets transplanted into soil. The rooting percentage of ‘McIntosh’ was highest on medium containing sucrose and no rooting occurred on medium containing xylitol in both scions. Root number was significantly highest (5.7 compared to other carbohydrates with values of 3.7–3.9) on medium containing sucrose in ‘Make’ and a similar tendency was observed in ‘McIntosh’ (3.0 roots per shoot on medium containing sucrose and 1.8– 2.5 roots on other media). Reduced chlorophyll fluorescence values after transplanting may indicate increased sensitivity to environmental stress. Plantlets rooted on sorbitol, glucose or fructose showed a decline in chlorophyll fluorescence values one week after transplanting but plantlets rooted on sucrose presented no decline (Karhu and Ulvinen, 1995). Pawlicki and Welander (1995) used stem discs from in vitro ‘Jork 9’ shoots to study the effects of carbohydrate quality and concentration on the rooting process and proved that the type of sugar influenced root regeneration. 29–59 mM sucrose promoted root development but also increased callus formation while sorbitol reduced callus formation. 100% rooting with 6 roots per disc was achieved using a combination of glucose/sorbitol (117/59 mM) or of sucrose/mannitol (59/29 mM). The latter combination reduced the time required for rooting and the rate of callus formation remained low (4.4%). Sucrose concentration influenced the number of adventitious roots and there was an interaction between sucrose and auxin: by increasing the sucrose concentration the optimum auxin concentration shifted (Calamar and De Klerk, 2002): The absence of sucrose in the initial rooting period (0-48 h) reduced rooting but in the latter phase it was not necessary. Bahmani et al. (2009b) studied the influence of fructose, glucose, sucrose, sorbitol and maltose at 30, 60, 90, and 120 mM on the rooting and hyperhydricity of ‘MM.106’ and observed that 90 mM sucrose was the most effective. Rooting only occurred when medium contained sucrose, glucose or sorbitol. Rooting percentage was highest (90%) in the presence of sucrose (90 mM= 3%, w/v) with the number (10) and length (7.2 cm) of roots reaching a maximum at this sucrose level. However, an increase in sucrose concentration to 120 mM decreased all parameters (85%, 5 and 3.8, respectively). Hyperhydricity of shoots was highest at 60 mM maltose and 30 mM sorbitol but it was less at 90–120 mM sorbitol, 90 mM sucrose or fructose and 120 nM maltose. 3.3.1.3.3. Activated charcoal. AC increased the root length (from 16.7 to 24.3) in ‘Malling Merton’ apple rootstocks when it was applied at 0.25% (Snir and Erez, 1980). It also reduced basal callusing and improved rooting in ‘Tydeman Early Worcester’ when it was applied at 0.1% (Modgil et al., 1999). The effect of AC added to root elongation medium (REM) at 2.5 g/l was studied on the rooting of ‘Royal Gala’ in

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the presence (0.5 mg/l) and in the absence of NAA (Magyar-Tábori et al., 2001c). Though the rooting percent remained the same (96–100%), AC increased the root number (from 4.5 to 12.1) in the presence of NAA and decreased it (from 14.5 to 8.2) on auxin-free medium whereas NAA decreased both the number and length of roots in the absence of AC. The favourable effect of AC in this study may be due to the adsorption of excess NAA (Pan and van Staden, 1998) since auxin is required only during the induction phase of rooting (De Klerk et al., 1995). Plantlets had several large, dark green leaves and grew most rapidly after the combined application of AC and NAA both in REM and during the first phase of acclimatization. The effect of AC applied in REM was studied on the rooting responses of three rootstocks (‘M.26’, ‘MM.106’, and ‘JTE-H’) after application of root induction medium (RIM) with different IBA concentrations (1, 2, 3 mg/l) (Magyar-Tábori et al., 2002b). An interaction was detected between the IBA content of RIM and the AC content of REM. When RIM contained 1 or 2 mg/l IBA, AC applied in REM decreased the rooting percentage of ‘JTE-H’ (from 100 to 91–96%). The same was true for ‘M.26’ at each IBA level: rooting percentage decreased by 23–36% when AC was present in REM. In contrast, root length increased significantly in the presence of AC in ‘JTE-H’ and ‘M.26’. Root number was not affected by any treatment in ‘MM.106’ but it was significantly lower in the presence of AC in ‘JTE-H’ and ‘M.26’, especially at a low level (1 mg/l) of IBA (in ‘JTE-H’ from 11.5 to 6.1 and in ‘M.26’ from 4.5 to 2.0). A negative effect of AC applied in REM was observed in ‘Red Fuji’ where the rooting percentage decreased from 51.4–82.9 to 45–60% depending on the IBA level applied in RIM (Magyar-Tábori et al., 2009). 3.3.1.3.4. Plant growth regulators. Auxin added exogenously to in vitro shoots or cuttings has the ability to promote adventitious root formation. However, auxin is required only at an early stage of the rooting process for the induction of adventitious roots (Harbage et al., 1993; Abel and Theologis, 1996). In the latter phases of root development, it can modify or even inhibit the development of the root system (Lane, 1978; James and Thurbon, 1979; Druart, 1997; De Klerk et al., 1999). Therefore, most published in vitro rooting methods used two-phase rooting in which root initiation was induced in the first phase (on RIM) and root elongation was supported in the second phase (on REM). Rooting of ‘Granny Smith’ was investigated by Sriskandarajah and Mullins (1981) using different auxins, such as IBA, γ-naphthoxy-acetic acid (NOA), and 2,4-dichlorophenoxy-acetic acid (2,4-D) at 0–40 µM. 2,4-D inhibited rooting even at low concentrations but 59–69% rooting was achieved by the application of 10 µM NOA and IBA. Roots were

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concentrated on the cut ends of stems when NOA was applied but they were produced along the stems and on the cut end when IBA was used. Using 10 µM IBA with an optimized sucrose concentration (1%) 78–82% of stems rooted. The effect of NAA was studied at a concentration range between 0.1 and 33 µM for 21 days in ‘M.27’, ‘M.26’, ‘MM.111’, ‘M.9’ and ‘Macspur’ (Lane and McDougald, 1982). Rootstocks (‘M.27’, ‘M.26’, and ‘MM.111’) rooted better than the scion (‘Macspur’) (Table 5) and under these conditions ‘M.9’ did not root at all. Welander (1985) examined the rooting potential of ‘Åkerö’ under different IBA concentrations (0.5–10.0 µM) and found 2.5 µM IBA to be optimal (up to 100% rooting) but only after shoots reached an ‘easy-to-root condition’, which indicates a sufficient number of subcultures (9–11). The efficacy of different types of auxin (IBA, IAA or NAA at 0.3 mg/l) was investigated on rooting of nine apple cultivars by Zimmerman and Fordham (1985). In ‘Royal Red Delicious’, ‘Gala’, ‘Golden Delicious’ and ‘Spur McIntosh’ there were no differences in the effects of different auxins. IBA significantly induced the most rooting in ‘Delicious’, ‘Redspur Delicious’, ‘McIntosh’, ‘Mutsu’ and ‘Spartan’. In three cultivars (‘Delicious’, ‘McIntosh’, and ‘Spartan’) NAA resulted in better rooting than IAA (Table 5). When root development was induced in ‘Liberty’ by the addition of IBA or NAA (0.1 mg/l to 1.0 mg/l), NAA induced callus formation at the base of shoots even at a low concentration (0.3 mg/l) and poor root development was observed (Yepes and Aldwinckle, 1994). However, minimal callus only formed, and poorly so, when the concentration of IBA was higher than 1.0 mg/l. The optimal concentration of IBA necessary for rooting depended on genotype (Table 5) and it was lower in liquid medium compared to solid medium (Table 6) possibly due to a greater adsorptive area. The effect of NAA or IBA on the rooting of ‘MM.106’ was studied by Sharma et al. (2000) who concluded that IBA was more effective than NAA (Table 5). The effects of different IBA concentrations (1, 2, 3 mg/l) in RIM applied for a week on the rooting of ‘M.26’, ‘MM.106’ and ‘JTE-H’ were studied (Magyar-Tábori et al., 2002b). High (100%) rooting of ‘JTE-H’ was detected at each IBA level. Rooting was similarly high in ‘M.26’ (94.3%) at 1.0 mg/l IBA but with increasing IBA levels rooting percentage decreased to 40–60%. ‘MM.106’ rooted poorly: 33–46% at 2.0 or 3.0 mg/l IBA. Raising IBA level in RIM decreased root length: longest roots (9.0 cm) developed at the lowest IBA concentration compared to the two other IBA concentrations (6–7 cm). The number of roots was not affected by IBA treatment in ‘MM.106’. Snir and Erez (1980) achieved 100% rooting of ‘MM.104’, ‘MM.106’ and ‘MM.109’ when RIM contained 1 mg/l IBA and was applied for 6–8

Table 6 Effect of the status and gelling agents of the rooting medium on the rooting percentage in apple cultivars. Cultivar

Root induction medium Auxin content

Status

Tydeman Early Worcester

0.5 mg/ml IBA 0.2 mg/ml IBA 1.0 mg/ml IBA 0.2 mg/ml IBA 0.5 mg/ml IBA 0.3 mg/ml IBA 0.2 mg/ml IBA 0.3 mg/ml IBA 0.3 mg/ml IBA 0 0.5 mg/ml IBA 0.3 mg/ml IBA 1.5 µM IBA

Gala

24.6 µM IBA

Marubakaido

0 0 0 0

Solid (0.7% agar) Liquid Solid (0.7% agar) Liquid Solid (0.7% agar) Liquid Solid (0.7% agar) Liquid Solid (0.7% agar) Liquid Solid (0.7% agar) Liquid Solid (0.8% agar) Liquid Solid (0.8% agar) Solid (0.25% Phytagel) Solid (agar/xyloglucan 0.4%/0.2%) Solid (0.6% agar) Solid (agar/xyloglucan 0.4%/0.2%) Solid (0.6% agar)

Empire Freedom Liberty Malling 7A McIntosh Malus prunifolia xanthocarpa

Jonagored

Rooting percentage

References

73 100 71 67 78 100 100 88 80 100 92 100 8–10 50–60 91 80 70 6.7 66 10.4

Yepes and Aldwinckle (1994)

Modgil et al. (1999) Bommineni et al. (2001) Lima-Nishimura et al. (2002)

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days. Druart (1997) studied the effect of different periods of auxin treatment on the rooting of ‘Compact Spartan’ and found that a 3-daylong culture period to be sufficient for rooting and 5 days to ensure a reproducibly high percentage of rooting (100%). Works on the effects of auxin in RIM during in vitro rooting are summarized in Table 5. Several authors reported that the dipping method, which is a short-term treatment with a high concentration of auxin prior to placing shoots onto auxin-free REM was more effective than the application of RIM for days prior to cultivation on REM. Sriskandarajah et al. (1982) reported that rooting of ‘Delicious’ could be increased from 60 to 79% by dipping freshly cut shoots into 750 µM IBA solution. In the experiments of Lane and McDougald (1982), ‘M.9’ needed a special requirement for rooting because no root initials developed when shoots were placed on NAAcontaining medium for 21 days. However, exposure to acute levels of NAA, namely dipping shoots into 1.61 mM NAA solution for 2 min then placing them on NAA-free medium, stimulated rooting (52.5%) and extensive callus development could be avoided. Pawlicki and Welander (1995) observed that exposure of root discs of ‘Jork 9’ to 49.2 µM IBA for 540 min was enough for 100% rooting while decreasing the rate of callus formation. A short (3 h) pre-treatment with a high concentration of IBA (30 mg/l) improved rooting of ‘MM.106’ from 20–25 to 80.25% (Sharma et al., 2000). Several authors reported that the survival of rooted plantlets was very low (b10%) when NAA was used for rooting of apple because NAA strongly induced callus formation at the base of shoots and roots that emerged from this callus had no vascular connection to the stem. However, IBA induced abundant root development with minimal callus formation and there was vascular connection between the root and stem (Hicks, 1987; Harbage et al., 1993; Yepes and Aldwinckle, 1994; Bommineni et al., 2001). Rooting of ‘Gala’, ‘Golden Delicious’, ‘Jonathan’, ‘McIntosh’, ‘Vermont Spur Delicious’, and ‘Triple Red Delicious’ was affected by the pH of the RIM (Harbage and Stimart, 1996a). The pH of unbuffered medium raised approximately two units (from 5.6 to 7.0–7.5 depending on genotype) within 4 days, which resulted in a 50% decrease in root number (from 14.5 to 7.8) compared to buffered medium. Loss of IBA from RIM was highest (7%) at pH 5.5 compared to the loss (b2%) at pH 7.0 and was associated with a change in root number; with increasing pH the number of roots (average of cultivars examined) decreased from 10.5 to 5.8. Root formation and uptake of IBA were inversely related to the pH of RIM (Harbage et al., 1998). Maximal IBA uptake (1.7% for ‘Gala’ and 0.75% for ‘Triple Red Delicious’) and highest root number (13.5 for ‘Gala’ and 4 for ‘Triple Red Delicious’) were detected at pH 4.0. Metabolism of IBA was not affected by the pH of the RIM, which suggested that pH regulated root formation by affecting the uptake of IBA. Although the number of root primordia is determined by induction treatment (Harbage et al., 1993), the composition of the REM, such as auxin content or the presence of AC, could influence rooting. When REM contained NAA (0.5 mg/l) both the root number and length of ‘Royal Gala’ decreased (from 14.5 to 4.5 and from 12.7 mm to 2.5 mm, respectively). When the REM contained AC as well, the effect of NAA was eliminated possibly due to the adsorption of excess NAA by AC (Magyar-Tábori et al., 2001c). After inducing rooting of ‘M.26’ and ‘JTE-H’ on RIM with 1 mg/l IBA the number of roots decreased when REM contained AC (from 4.5 to 2.0 and from 11.5 to 6.1, respectively) (Magyar-Tábori et al., 2002b). Rooting ability of in vitro shoots can also be influenced by the hormone-content of the last proliferation medium (LPM). Post-effects of different cytokinins applied in LPM were detected on the rooting of ‘Húsvéti rozmaring’ (Dobránszki et al., 2000c). Rooting percentage was not modified by the cytokinin content of LPM. However, shoots originating from LPM containing 1.0 mg/l BA and 1.5 mg/l KIN formed more roots (6.4) than from LPM containing 1.0 mg/l TOP (4.5). In contrast, roots were longer on shoots originating from LPM containing TOP (22 mm) compared to other LPM (15.3 mm). Rooting ability of ‘Red Fuji’ decreased by up to 55% when the IBA content of RIM was increased from 1 to 3 mg/l (Magyar-Tábori et al., 2001b). Moreover, LPM also

modified its rooting ability, which was lowest (51%) when LPM contained 1 mg/l BA and 0.1 mg/l IBA and highest (95%) after shoots were proliferated on media with 1.0 mg/l BA, 1.5 mg/l KIN and 0.3 mg/l IBA or 1.0 mg/l TOP and 0.3 mg/l IBA. Some carry-over effect of IBA applied in LPM was also detected. A higher IBA level (0.3 mg/l) of LPM tended to increase root number independently of IBA concentration in RIM when it was applied in combination with BA or BAR in LPM. When a higher concentration of IBA was applied together with TOP or BA+KIN in LPM, fewer roots developed on RIM containing 1 or 2 mg/l IBA than after LPM with a lower (0.1 mg/l) level of IBA. Ricci et al. (2001) enhanced the rooting of ‘M.26’ by using two diphenylurea (DPU) derivates, N,N′-bis-(2,3-methylenedioxyphenyl)urea (2,3-DPU) and N,N′-bis-(3,4-methylenedioxyphenyl)urea (3,4-DPU) at 1 µM. While IBA at the same concentration resulted in 75% rooting, 2,3-DPU and 3,4-DPU caused 70 and 91% rooting, respectively. Moreover, roots developed directly from the base of shoots without callus formation using both DPUs while IBA caused callus formation. Rooting activity of these non-hormonal compounds was related to the symmetrical presence of a methylenedioxy benzo-condensed group. Other DPU derivates with weak cytokinin-activity, such as N-(2,3-dimethylphenyl)N′-phenylurea and N-(1-naphtyl)-N′-phenylurea, at 1 µM enhanced the rooting of ‘M.26’ (80–90% rooting percentage, 3.5–3.8 roots per cutting) similar to 1 µM IBA (90% rooting, 3.4 roots per cutting) (Ricci et al., 2003). 3.3.1.3.5. Gelling agents. Use of liquid medium during in vitro rooting of apple is more widespread than during the shoot multiplication phase. Abbott and Whiteley (1976) were the first to report the favourable effect of liquid medium on Malus root growth. Sriskandarajah and Mullins (1981) examined and compared four different methods for root induction of ‘Granny Smith’; rooting was induced on agar-based media, on filterpaper bridges with liquid media, in stationary and in agitated liquid culture. A low frequency (b0.01%) of adventitious root formation was detected on agar-solidified medium. More shoots rooted (up to 4%) by using filter-paper bridges. In stationary liquid media cuttings turned brown and died within a few days. However, in continuously agitated culture 60–80% of cuttings rooted. Comparing the effect of solid and shaken liquid media applied at the first week of the rooting process, Yepes and Aldwinckle (1994) observed that liquid medium was superior to agar-solidified (0.7%) medium in terms of rooting percent, number and length of roots, and a lower IBA concentration was required for root induction (Table 6). However, after 1 week plantlets needed to be transferred either to solid (PGR-free) medium or to soil in order to avoid hyperhydricity. Similarly, culturing in vitro ‘Tydeman Early Worcester’ shoots in liquid culture for 9 days resulted in a higher rooting rate (50–60%) compared to shoots cultured on agar-solidified (0.8%) medium (8–10%) (Modgil et al., 1999) (Table 6). Agarsolidified (0.8%) medium showed a higher percentage of rooting in ‘Gala’ compared to Phytagel-solidified (0.25%) medium (Bommineni et al., 2001) (Table 6). Lima-Nishimura et al. (2002) reported that rooting was higher on medium solidified by agar/xyloglucan (0.4%/ 0.2%) compared to agar-solidified (0.6%) medium in two cultivars (Table 6). A comparison of root elongation on agar-solidified medium and on watered vermiculite after root induction on solid or liquid media was investigated by Druart (1997) in ‘Compact Spartan’. Two traits were superior in vermiculite than in solid medium during root elongation: rooting percent (90–97.5 vs. 40.5–67.5%) and root number (4.4–5.1 vs. 2.0–2.5). When solidified medium was used the hormonal compounds, NH+ 4 ion and the agar gel were identified as inhibitors of root elongation. Rooting rate was lowest (40.5%) when both root induction and elongation occurred on agar-solidified media. Souza et al. (2007) found better rooting of ‘M.9’ on agar-containing medium (67.8% rooting and 7.85 roots) than on vermiculite-containing medium (37.4% rooting and 1.69 roots) at 5 µM IBA and 3% sucrose. Post-effects of solid and liquid RIM were studied during the root elongation phase (Magyar-Tábori et al., 2001b) and interactions between the different media components and conditions were detected. Both rooting percentage (from 66.7 to 96%) and

J. Dobránszki, J.A. Teixeira da Silva / Biotechnology Advances 28 (2010) 462–488

root number (from 6.1 to 25.9) increased after using liquid RIM when REM did not contain AC at 26 °C. AC in REM increased both the rooting percent (from 66.7 to 100% at 26 °C and from 40 to 94.3% at 22 °C) and root number (from 6.1 to 13.1 at 26 °C and from 2.9 to 11.5 at 22 °C) when roots were induced on solid medium. After using liquid RIM root length decreased in the presence of AC (from 37.8 to 27.3) but increased slightly in the absence of AC (from 4.0 to 9.4). Modgil et al. (2009) compared the effects of different substrates (sand at 10 g/l, perlite at 1 g/l and agar at 3 g/l) applied in liquid REM, on the root elongation of ‘M.7’ and ‘M.26’. Best rooting (76.67%) and the highest root number (5.33) were achieved on liquid REM without any substrate. 3.3.1.4. Physical factors 3.3.1.4.1. Light. The role of light in root emergence and growth was reported in the study of Sriskandarajah et al. (1982). They found a progressive improvement in the rooting of ‘Delicious’ and ‘Jonathan’ by the application of continuous (24 h) illumination with 90– 100 µE m− 2 s− 1; however, in these experiments the light treatment was combined with increasing temperature and the number of subcultures; therefore, the specific effects of light were not distinguishable. Zimmerman and Fordham (1985) were able to significantly increase the rooting percentage in four out of nine scions (‘Delicious’, ‘Golden Delicious’, ‘Spur McIntosh’, and ‘Mutsu’) when shoots were cultured in the dark at 25 °C during the first week of the rooting stage. There was an interaction between the effect of temperature applied in the dark and the effect of light: an increase of the dark temperature to 30 °C further increased rooting percentage of ‘Delicious’, ‘Vermont Spur Delicious’ and ‘Gala’ (Table 7). The minimum ‘incubation’ time necessary in the dark for increasing rooting percentage depended on the cultivar and varied between 3–9 days (Table 7). Etiolation of ‘M.9’ shoot cultures for two weeks before rooting led to improved rooting, and rooting percentage increased from 14 to 23% (Webster and Jones, 1989). Rooting percentage was decreased by light in ‘Oregon Spur Delicious’, ‘Triple Red Delicious’, and ‘Golden Delicious’ compared to a 7-day-long dark treatment (Karhu and Zimmerman, 1993). When only the basal part of shoots was kept in the dark for 7 days it was enough for improving the rooting of ‘Oregon Spur Delicious’, but not the other two scions. Darkening of the distal part of shoots significantly reduced the rooting of ‘Golden Delicious’. Rooting percentage was reduced in all cultivars when the basal part of shoots was exposed to light (Table 7). In ‘Jork M9’, 1 day of dark treatment at 25 °C was necessary to induce rooting on medium containing 24.6 µM IBA (Welander and Pawlicki, 1993). Kaushal et al. (2005) found that 90% of shoots rooted when they were cultured in the dark for 9 days. 3.3.1.4.2. Temperature. The effect of temperature on the in vitro rooting process was studied in detail by Zimmerman and Fordham (1985). Increasing temperature from 25 to 30 °C during the first week of rooting affected the rooting of apple scions (Table 7); moreover, rooting increased from 10 to 60% in ‘Vermont Spur Delicious’ and from 57 to 87% in ‘Spur McIntosh’ but it decreased from 87 to 53% in ‘Golden Delicious’. However, a further increase in temperature to 35 °C decreased rooting to 0–9%. Magyar-Tábori et al. (2001c) observed that increasing temperature from 22 to 26 °C during the root induction phase increased the rooting percentage of ‘Royal Gala’ from 40 to 66.7% but did not affect the number or length of roots. 3.3.1.4.3. Others. Relative humidity or the composition of the gas phase in culture vessels can influence rooting. Souza et al. (2007) studied the effect of different flask coverings on the rooting of ‘M.9’. When flasks were covered with cotton–wool both rooting percentage (88.88%) and root number per plantlet (3.3) increased compared to rooting of plantlets grown in flasks covered by aluminium foil (76.72% rooting, 1.4 roots). These results may be due to the reduction of relative humidity and the concentration of ethylene in flasks covered by cotton–wool. Li et al. (2001) investigated the effects of CO2-enrichment under low (40 µmol m− 2 s− 1) and high (100 µmol m− 2 s− 1) photosyn-

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Table 7 Light effects on rooting of different apple cultivars at different temperature. Cultivar

Light treatment

Delicious

7 days in dark 7 days in dark 3 days in dark Vermont 7 days in dark Delicious 7 days in dark 6 days in dark Gala 7 days in dark 7 days in dark 3 days in dark Golden 7 days in dark Delicious 7 days in dark 9 days in dark McIntosh 7 days in dark 7 days in dark 6 days in dark York 7 days in dark Imperial 7 days in dark 6 days in dark Oregon Spur 7 days in dark Delicious 7 days in light Basal darkening for 7 days Distal darkening for 7 days Triple Red 7 days in dark Delicious 7 days in light Basal darkening for 7 days Distal darkening for 7 days Golden 7 days in dark Delicious 7 days in light Basal darkening for 7 days Distal darkening for 7 days Jork M9 1 day in dark MM.111

4 days in dark 7 days in dark 9 days in dark

Temperature in Rooting References the dark (°C) percentage 25 30 30 25 30 30 25 30 30 25 30 30 25 30 30 25 30 30 25

3 30 48 5 10 80 57 67 100 50 87 73 57 66 72 53 67 77 64 36 57

Zimmerman and Fordham (1985)

Karhu and Zimmerman (1993)

41

54 15 22

28

66 40 53

20

25

92 40 80 90

Welander and Pawlicki (1993) Kaushal et al. (2005)

thetic photon flux (PPF) conditions on rooting of ‘M.9’ and on the development of these plants after transplantation. Culture flasks were covered with polypropylene film in CO2 non-enriched treatment but with membrane filter in the centre in CO2-enriched treatment and 1000 µmol mol− 1 CO2 concentration was applied. CO2-enrichment increased the fresh weight of roots from 27.3 to 422.3 mg when low PPF was applied and from 244.4 to 655.3 mg under high PPF. Survival of plantlets from CO2-enriched treatment increased significantly up to 65–80% after transplantation compared to plantlets originating from non CO2-enriched treatment (30–50%). 3.3.2. Ex vitro vs. in vitro rooting Attempts have been made to root in vitro shoots under ex vitro conditions. Webster and Jones (1989) rooted ‘M.9’ directly after dipping the bases of shoots into a powder containing 0.2% IBA and 10% Captan fungicide and planted the shoots in a seed tray with sterilized horticultural sand. The tray was covered with a transparent lid and kept for 4 weeks at 22 °C. Both direct and in vitro rooting method resulted in 90–100% rooting; however, direct rooting was more effective than in vitro rooting after transfer of plants to compost: 83% of plants were established when shoots were rooted directly but only

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45% when plantlets were produced by in vitro rooting. Isutsa et al. (1998) compared the efficacy of in vitro and ex vitro rooting of microshoots in ‘G.56’, ‘G.30’ and ‘G.11’ (difficult, moderate and easyto-root). Ex vitro rooting was studied under two CO2 (450, 1350 µmol mol− 1) and three light (30, 50, 100 µmol m− 2 s− 1) levels. Highest survival occurred under high light intensity with high CO2 for ‘G.56’ (82%) and under medium light intensity with low CO2 for the other two rootstocks (77% for ‘G.30’ and 65% for ‘G.11’). Under in vitro conditions the best rooting percentage was 100% for ‘G.30’ and ‘G.11’ but not for ‘G.65’ (30%). Moreover, survival after acclimatization of rooted plantlets from these rooting treatments varied between 40 and 80%. When shoots were rooted ex vitro all rootstocks appeared to be easy-to-root, and microshoots rooted ex vitro were superior to those rooted in vitro. However, Litwińczuk (2000) found that in vivo rooting was not superior to in vitro rooting in ‘M.26’, ‘MM.106’ and ‘P 13’ rootstocks. Shoots were dipped into rooting powder with 0.3% NAA and 0.05% IBA and then placed into a peat and perlite (1: 1) mixture and fertilized by 1% Nowokont solution (N 1.4%, P 0.2% and K 1.82% with microelements). The rate of in vivo rooting was very low (17%) compared to in vitro rooting (66.4–98.5% rooting). Magyar-Tábori et al. (2001c) cultured shoots of ‘Royal Gala’ for 1 week in liquid RIM with 3 mg/l IBA and then half of the shoots were transferred to solid REM while the other half were planted into Jiffy-7® pellets. In vitro rooting was 100% and all plants survived after acclimatization. When shoots were planted after the induction phase directly into Jiffy-7® they were less vigorous but the rate of survival was nearly the same (96%). In vivo rooting of ‘M.9’ and ‘M.26’ shoots grown in a vessel with or without a ventilation stopper was studied (Kwon et al., 2004). The concentration of ethylene and CO2 was lower and the leaf area and chloroplast index were higher in vessels with a ventilation stopper. In vivo rooting of both rootstocks was highest after treatment with 300 mg/l IBA and 3% sucrose; it was 81.7 and 86.5% for ‘M.9’ in vessels with or without a ventilation stopper, respectively and 85.4% for ‘M.26 in vessels with a ventilation stopper. Survival after acclimatization was higher (up to 71.7%) after using vessels with a ventilation stopper; stomata of plantlets from vessels with a ventilation stopper closed immediately after exposing them to room humidity unlike plantlets from non-ventilated vessels whose stomata opened after 20min exposure to room humidity. In vitro elongated shoots of ‘M.7’ and ‘MM.106’ rooted successfully ex vitro in coco-peat after inducing rooting in liquid medium (Modgil et al., 2009). Direct transfer of induced shoots to peat was successful (72.67% rooting and 90% survival). 3.3.3. Adventitious rooting In vitro apple shoots are useful to study adventitious root formation. The involvement of ethylene was examined by Harbage and Stimart (1996b) in adventitious root formation of ‘Gala’ (easy-toroot) and ‘Triple Red Delicious’ (difficult-to-root). Increasing IBA (from 0.15 up to 150 µM) increased ethylene evolution and IBA up to 15 µM increased root number, as well. No significant differences were detected between cultivars in ethylene evolution and it was not associated with the root formation zone. Moreover, treatment with ethylene gas up to 1000 µl l− 1 did not eliminate the requirement of IBA for root formation. These results strongly suggested that ethylene is not involved in auxin-dependent root formation of apple microcuttings. However, Ma et al. (1998) observed that ethylene inhibitors, such as silver nitrate (AgNO3) and aminoethoxyvinylglycine (AVG) significantly improved the rooting ability of ‘Royal Gala’ by inhibiting ethylene synthesis and/or accumulation. In order to avoid the influence of stem, apex and leaves in in vitro plantlets and their interactions on adventitious root regeneration, several authors (De Klerk et al., 1993; van der Krieken et al., 1993; Seifert et al., 1994) developed a model system which uses stem discs (1 mm thick) cut from in vitro apple shoots, for studying root regeneration in apple. Although not yet tested in apple, it is highly likely that these sections,

or thin cell layers, would be a highly successful system for micropropagation, as has been proved for several other difficult-to-propagate woody plants (Nhut et al., 2003). Sedira et al. (2005) investigated the sensitivity of stem discs to auxin from untransformed and rolBtransformed in vitro shoots of ‘Jork 9’ and the expression of the ARRO-1 gene during the root induction phase of adventitious rooting in stem discs and microcuttings. The optimal time for root induction was 7 h with 24.6 µM IBA in transformed plants and 7 h with 49.2 µM IBA in untransformed ones on stem discs. Higher expression of the ARRO-1 gene was detected in the transformed plants during adventitious rooting which suggests that its expression may be rooting specific. Sedira et al. (2007) improved root regeneration on stem discs of ‘Jork 9’ by temporarily blocking DNA replication by aphidicolin (AD) which occurred during dedifferentiation prior to the first mitoses. Application of 15 µM AD resulted in more roots per discs (8.4 compared to 6.7 roots on discs without AD) after 21 days of culture and more than twice as many roots appeared during the first 7 days compared to the control. In addition, AD also synchronized rooting. Temporal changes in explant competence to respond to exogenously applied auxin were also proved by BrdU labelling (Sedira et al., 2007). The rate of replicating nuclei during 28–32 h could be significantly increased by splitting the IBA (49.2 µM) treatment (0–4 h and 28–32 h) compared to a single IBA application (0–8 h). Han et al. (2009) maximized adventitious rooting of shoot cultures of M. hupehensis Rehd. var. pinyiensis Jiang by adding 75 µM sodium nitroprusside while 2 µg/l triacontanol (Tantos et al., 2001) increased root length and numbers more than the control. 3.4. Acclimatization The transfer from in vitro to ex vitro conditions is a very important step in the structural and physiological adaptation of plants; this is the beginning of the autotrophic life of plants. Commercial utilization of micropropagated plants requires their successful acclimatization (Fig. 1g,h) and subsequent transfer to the field. Difficulties during acclimatization include rapid desiccation of plantlets and their susceptibility to bacterial and fungal diseases. Webster and Jones (1989) transferred ‘M.9’ plantlets that had rooted in vitro directly to peat Jiffy pots of peat/grit (3: 1) compost and they were grown in a glasshouse with dry fog for 2 weeks. The success of establishment was rather low (45%) when rooting was proceeded in vitro but higher after direct rooting (83%). Bolar et al. (1998) developed an efficient method for hardening in vitro rooted apple microplants. Plants were potted into Jiffy-7® pellets which were soaked in a mixture containing biological plant protectant (Trichoderma harzianum) and NPK fertilizer before planting. After planting, a plastic humidity dome was used for 2–3 weeks to ensure high humidity and plants were kept in a growth chamber with the same conditions as during micropropagation. Then plants were potted in peat/perlite/vermiculite (4:1:1) and the pots were placed back into the growth chamber and covered with plastic bags which were gradually removed from plants over the period of 1 week. Using this method several apple cultivars (‘Marshall McIntosh’, ‘Golden Delicious’, ‘Liberty’, ‘Royal Gala’, ‘Galaxy’, and ‘M.26’) were successfully hardened (70–100%). Isutsa et al. (1998) successfully acclimatized apple rootstocks which were rooted ex vitro in a fog chamber. They observed that the temperature of the fog chamber affected the survival of plantlets with survival being high at 24±2 °C and low at 28 °C. Survival reached 100% when the highest light intensity (100 µmol mol− 1) was applied although the effect of CO2 enrichment was inconsistent and dependent on genotype. To prevent fungal contamination, it was recommended that fungicides be incorporated into the acclimatization protocol. Modgil et al. (2009) examined the survival of ‘M.7’ and ‘MM.106’ potted into media containing soil as well as soilless media in a glasshouse. They observed that coco-peat was the best medium as it ensured 90% survival compared to other media with 0–50.25% survival (0% in vermicompost/sand 1:1, 20.13% in saw dust, 21% in coco-peat/vermicompost

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1:1, 25% in vermicompost, 25.17% in soil/sand/perlite 1:1:1, 39.6% in coco-peat/saw dust 1:1, 40.3% in coco-peat/perlite 1:1 and 50.27% in soil/ sand/compost 1:1:1). Soil-containing media caused higher fungal infection than soilless media. Moreover, a significant effect of season on acclimatization was also detected. Maximum establishment (90–95%) occurred when hardening took place between October and March compared to 40–50% survival observed in the other months possibly due to high (N28 °C) temperature and rain. Survival following field transplantation reached 95% during March–April. It has been reported that the application of arbuscular mycorrhizal fungi (AMF) could shorten the acclimatization period and could improve post-transforming performance of plants. AMF helps the development of a stronger root system, increases the rooting intensity and root surface, improves the nutrient uptake of plants and can protect them from root pathogens (reviewed by Rai, 2001 and Kapoor et al., 2008). Successful application of mycorrhization has been reported in apple, as well. Endomycorrhizal infection with Glomus fasciculatum, G. mosseae and G. intraradices during a very early weaning stage of acclimatization improved the growth and homogeneity of ‘M.26’ and ‘Golden’ plants but the growth of ‘M.9’ was not affected although their homogeneity was also increased (Branzanti et al., 1992). Inoculation of ‘MM.106’ with G. mosseae at the transplant stage (Schubert and Lubraco, 2000) or that of M. prunifolia with Glomus etunicatum or with Scutellospora pellucida (Pereira Cavallazzi et al., 2007) increased the nutrient content and growth of plants. 4. Current applications Nowadays, the micropropagation of several rootstocks and scions of apple is a widely used technique. While the original aim for the development of reliable micropropagation methods of different cultivars included rapid, commercial scale production of healthy, disease-free plants, in vitro apple cultures, as tools, have been utilized for other purposes as highlighted next. In vitro shoot cultures or plantlets (rooted shoots) have also served as useful tools for basic and applied research taking advantage of in vitro systems with a standard and well-controlled culture environment and the uniformity in the physiological state of test plants. Furthermore, experiments conducted in vitro are quicker, cheaper and safer in contrast to field experiments. 4.1. Long-term cultures, automation and culture stability Mass micropropagation of different apple cultivars, mainly rootstocks, has been widespread in order to have healthy, uniform propagation materials in large quantities within a short time (Zimmerman, 1991). While some doubts have been reported regarding the use of micropropagated materials because of their juvenile-like characters, such as delay and reduced efficiency in cropping, increased sucker production due to rejuvenization was caused by in vitro propagation (Jones and Hadlow, 1989; George, 1996b), a more recent report (Czynczyk et al., 2008) justifes the use of micropropagated apple for commercial orchards. Jesch and Plietzsch (2000a,b) studied the field performance (morphological, phenological and physiological characteristics) of Prunus plants originating from different propagation methods, such as plants from in vitro propagation, cuttings from in vitro propagation, from conventionally propagated plants and from grafts. They concluded that the growing and flowering periods were influenced rather by soil and climatic conditions than by propagation method. Field performance of ‘Jonagored’, ‘Elstar’ and ‘Gala Must’ apples grafted onto ‘P 14’ was evaluated over an 8-year period, when the rootstock had been propagated in three ways: in stoolbed, in stoolbed from in vitro propagated plants or by micropropagation (Czynczyk et al., 2008). Cumulative yield, fruit size and weight on in vitro propagated rootstocks proved to be similar to those on trees grown on conventionally propagated rootstocks. Moreover, no differences were detected in growth vigour between the plants from different propagation methods.

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A possible promising way of reducing the costs of micropropagation includes automation of organogenesis in bioreactors which contain liquid media. Woody plants are sensitive to liquid environments with hyperhydricity being one of the main problems in tissue culture, especially using bioreactors. Bioreactor techniques for apple are at the research scale yet and there are some problems associated with these techniques, different from the problems in conventional micropropagation techniques, such as hyperhyricity caused by liquid medium, dissolved oxygen or mixing. Marga et al. (1997) found that hydrolised agar (0.7%) from Difco was effective against the occurrence of hyperhydricity when it was added to liquid proliferation medium and the oligosaccharide fraction (molecular weight of about 1900 D) of hydrolised agar was determined to be the active component in overcoming hyperhydricity of ‘M.26’ apple shoots. Chakrabarty et al. (2003) selected a suitable method for multiplication of nodal segments of ‘M.9 EMLA’. A comparison was made between two types of bioreactor cultures: temporary immersion (ebb and flood, with and without air flow inside the chamber of bioreactor) and continuous immersion cultures. Shoot growth and multiplication was most effective in continuous immersion culture; however, the rate of hyperhydricity was 2–3-fold higher in this bioreactor compared to ebb and flood systems. In the temporary immersion system hyperhyricity could be further decreased by compressed air supply inside the bioreactor over the culture period using air-lift-balloon type bioreactor. Zhu et al. (2003, 2005) developed a two-step method using the RITA® temporary immersion system (Teisson and Alvard, 1995) and optimized conditions (no medium exchange; tissue immersion 16-times daily) for shoot multiplication (explant size of 0.5–1.0 cm; 4.4 µM BA and 0.5 µM IBA) and subsequent shoot elongation (1.1 µM BA and 0.25 µM IBA) of ‘M.26’. Advances in the research of clonal propagation through bioreactors are described and summarized by Paek et al. (2005) and Welander et al. (2007). The raw material for breeding of new cultivars requires a wide genetic base, which depends on collections of genetic resources (Peil et al., 2009). Traditionally, conservation of genetic resources in apples is based on field planting, which requires too much space and costs. Apple tissue culture combined with cold storage or cryopreservation has been a useful method for long-term storage of germplasm with minimal space and maintenance costs. Effects of PGRs (BA, IBA, and ABA), nitrate-N content (25, 50, and 100% of standard MS medium), carbon source of the medium (sucrose or/and mannitol), and effects of container type (test tube, jar, and tissue culture bag) on cold storage of ‘Grushovka Vernenskaya’ and TM-6 selection of M. sieversii were studied by Kovalchuk et al. (2009). A significant interaction was proved between medium, container and genotype. Cold storage (4 °C) was successful either for 21 months in tissue culture bags with 25% MS nitrate-N or with 0.5 mg/l ABA, or for 33 months in test tubes or jars on MS medium (100% nitrate-N content) with 3% sucrose, 0.5 mg/ l BA and 0.1 mg/l IBA. Randomly amplified polymorphic DNA (RAPD) analysis showed no changes in DNA after 39-month-long storage. A similar finding was reported earlier (Liu et al., 2004) regarding genetic stability in surviving shoots of ‘Gala’ after cryopreservation by the vitrification method using RAPD and amplified fragment length polymorphism (AFLP) analysis. Other studies by Modgil et al. (2005) and Gupta et al. (2009) showed the efficient use of RAPD to confirm the lack of somaclonal variation in short-term cultures of MM.106 and EMLA.111 rootstocks, respectively. While no change could be observed in DNA fragment and number detected by AFLP after cryopreservation by encapsulation-dehydration method in ‘M.26’, ‘Gala’ and ‘Hokkaido No. 9’, cryopreservation reportedly induced a decrease in DNA methylation level which could be determined by methylation-sensitive amplified polymorphism (MSAP) (Hao et al., 2001). Kushnarenko et al. (in press-a) studied the ultrastructure of shoot tips from micropropagated ‘Voskhod’ and ‘Grushovka Vernenskaya’ apple shoots before and after cold acclimatization and cryopreservation by the PVS2 vitrification method (Luo

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and Reed, 1997; Matsumoto and Sakai, 2000) and concluded that cryopreservation, cold acclimatization and rewarming steps affected the cell size and caused some structural and functional intracellular changes. Kushnarenko et al. (in press-b) described that the germination and viability of M. sieversii seeds after cryopreservation in liquid nitrogen were higher than those of control seeds (stored at room temperature) and confirmed that cryopreservation in liquid nitrogen has the potential for long-term storage of apple germplasm. True-to-type clonal identity is one of the most important requirements of mass clonal propagation. Micropropagation of plants from shoot tip or axillary bud cultures has been reported to maintain clonal identity (Pierik, 1991; Wang and Charles, 1991; McMeans et al., 1998) but there is the possibility of somaclonal variation (Jain, 2001). Genomic changes depend both on genotype and culture conditions, such as long duration of cultures, in vitro process, composition of media, etc. Examination of DNA methylation profiles of field- and in vitro-grown ‘Gala’ indicated that culture period itself was a likely mutagenetic factor due to detected variation in methylation and methylation changes that occurred during tissue culture even without previous adventitious shoot regeneration (Li et al., 2002). Using RAPD markers, Modgil et al. (2005) studied the genetic stability in micropropagated ‘MM.106’ when in vitro shoot cultures were previously maintained for 4 years. They demonstrated that genetic differences could occur during micropropagation of apple therefore screening of plants derived from micropropagation is necessary.

4.2. Synthetic seeds Synthetic seed (= synseed) technology, based on the use of different micropropagules like somatic embryos, axillary shoot buds, apical shoot tips, embryogenic masses and protocorms, provides methods for production of seed analogues. It can be an alternative to traditional micropropagation for production of cloned plantlets in the future although it still has limitations for wider commercial uses. Artificially encapsulated micropropagules are able to convert into plant under in vitro and ex vitro conditions and can be used for sowing as a seed after storage. Earlier, somatic embryos, as bipolar structures, were considered for synthetic seed production, however in fruit species, such as apple, encapsulated unipolar explants could also be useful. When unipolar structures are used during synthetic seed production, they should be induced to regenerate roots before encapsulation (Ara et al., 2000). In apple several successful experiments were reported on synthetic seed production of ‘M.26’. Piccioni (1997) examined the rooting and shoot elongation from encapsulated micropropagated single nodes and concluded that the addition of PGRs (0.5 µM IBA, 1.4 µM GA3, 2.2 µM BA, and 2.5 µM adenine hemisulfate) to the artificial endosperm and culturing auxin-treated (24.6 µM IBA for 24 h) single nodes for 3–9 days in the dark before encapsulation resulted in 66% plantlet production (conversion) after sowing. Comparing synthetic seeds from apical and axillary buds, 3.4-times higher conversion rate was achieved from apical buds (Capuano et al., 1988) (Table 8). Sicurani et al. (2001) studied the encapsulation

Table 8 Summary of work on synthetic seed production in apple rootstock ‘M.26’. Propagules used for encapsulation

Conversion References rate (%)

Single nodes Apical buds Axillary buds Adventitious shoot clusters cut by hand Apical and axillary cuttings from proliferated shoots cut by hand Adventitious shoot clusters cut by machine Apical buds

66 85 25 13 25.3 10.7 70

Piccioni (1997) Capuano et al. (1988) Sicurani et al. (2001) Brischia et al. (2002)

Micheli et al. (2002)

capacity of adventitious differentiating propagules. When proliferated shoot clusters were cut into 3–5 mm fragments by hand or machine and encapsulated, high regrowth (67.2–67.8%) could be achieved and some capsules had elongated more than one shoot. Brischia et al. (2002) evaluated the plantlet producing capacity of encapsulated, machine ground explants (DP) and compared with synseeds that contained hand-cut, unipolar micropropagated explants (MC). More than twice as high conversion was reached by using synthetic seeds with MC, however the shoot number was higher using DP synseeds (2.3 compared to 1.0 shoot per MC synseed). Micheli et al. (2002) was able to increase the conversion rate of synseeds that contained apical buds of ‘M.26’ from 40 to 70% when a thin external coating layer was applied over the alginate. These results are promising and suggest that synseed technology offers great potential both in micropropagation and in germplasm conservation of apple, although further research is needed to increase the rate of conversion and to develop steps towards automatization. Moreover, the occurrence of somaclonal variation is also an important aspect to be considered mainly if adventitious propagules are used. 4.3. Disease control, biotic and abiotic stress Virus elimination is the other application area which is of great importance from a production point of view, since a virus-free status is one of the prerequisites for the production of healthy plants. Meristem culture (Huth, 1978; O'Herlihy et al., 2003) was also used to eliminate viruses although growing in vitro shoot tips are more suitable for this purpose. In vitro culture can be combined with thermo- or chemotherapy. O'Herlihy et al. (2003) successfully eliminated Apple chlorotic leaf spot virus (ACLSV) from in vitro cultures of ‘Jonagold’ by chemotherapy using Ribavirin (1-β-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide), a nucleoside analogue with antiviral activity. They also showed that cytokinin could affect the virus titre: 0.44–1.6 µM BA increased ACLSV titre and the efficiency of Ribavirin could be increased by increasing the concentration of BA in the medium. ACLSV was eliminated when 100–150 µM Ribavirin was applied in the presence of 0.18–0.009 µM BA. Heat therapy (39 ± 0.5 °C) of in vitro cultures can be an alternative way for eliminating viruses (Paprstein et al., 2008). Thermotherapy was applied on in vitro culture of ‘Sampion’ and ‘Idared’ for 6 days and subsequently apical meristems were removed. The success of elimination depended on the apple cultivar. ‘Idared’ was freed from all tested viruses however, ‘Sampion’ was infected by ACLSV, ASPV (Apple stem pitting virus) and ASGV (Apple stem grooving virus) even after thermotherapy of in vitro cultures. A similar technique was applied for testing resistance against powdery mildew (Podosphaera leucotricha) in shoot cultures of apple cultivars (‘Jonathan’, ‘McIntosh’, and ‘Prairiefire’) with different susceptibility to pathogens by Korban and Trier (in: Korban and Chen, 1992); in vitro results correlated with the field reaction of the apple genotypes. Eleven apple proliferation (AP)-resistant cultivars could be micropropagated (Ciccoti et al., 2008): Malus sieboldii; 4551 (Laxton's Superb×M. sieboldii); 4556 (Laxton's Superb × M. sieboldii); D2118 (Laxton's Superb × M. sieboldii open pollinated); D2212 (Laxton's Superb×M. sieboldii open pollinated); H0801 [4556 (Laxton's Superb× M. sieboldii) 447×M9]; H0901 [4556 (Laxton's Superb x M. sieboldii)× M9]; H0909 [4556 (Laxton's Superb×M. sieboldii)×M9]; 4608 (M. purpurea cv. Eleyi×M. sieboldii); Gi477/4 (4608 open pollinated); C1907 (4608 open pollinated). M. domestica cv. ‘Golden delicious’ was included in that study as control. In doing so, AP, a serious disease in Central and Southern Europe caused by Candidatus Phytoplasma mali, could be eliminated. Based on the quick testing method on in vitro shoot cultures of pear (Duron et al., 1987), Hevesi et al. (2000) developed an in vitro testing method (cutting of leaves with infected scissors) in shoot cultures of apple rootstocks. This method could be used for the characterization of the process of disease development and for the evaluation of virulence of different Erwinia amylovora strains originating from different hosts,

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such as M. domestica, Pyrus domestica, Cydonia oblongata, Crataegus monogyna, Cotoneaster sp. and Pyracantha sp. They also detected differences in the susceptibility between the two rootstocks examined (‘M.26’ and ‘MM.106’) at 4 weeks after infection. Later, they performed a comparative evaluation of other apple cultivars regarding their fire blight susceptibility using this in vitro screening method and the results correlated well with field reactions of cultivars examined (data not published). Apple somaclonal variants of ‘Greensleeves’ originating from in vitro regeneration were tested for resistance to fire blight both in vitro (inoculation of excised in vitro leaves, or microcuttings) and in vivo (inoculation of greenhouse-grown and field-grown plants) experiments (Chevreau et al., 1998). In these experiments, greenhouse inoculation was found to be more reliable than in vitro tests because of the extensive variability in the results from in vitro examinations. Different evaluation of applicability of in vitro methods for rating fire blight susceptibility by different authors can be explained by several factors, among which differences in the virulence of Erwinia strains, different CPU (cell per unit) of bacterial suspension used for inoculation (108 by Duron et al., 1987 and Hevesi et al., 2000 and 104–107 by Chevreau et al., 1998), different growing conditions for in vitro shoots or variant numbers of treated shoots. Shoot tip cultures of ‘Jonathan’ and ‘Prairiefire’ were inoculated with a suspension culture of Gymnosporangium juniperi-virginianae to test cedar–apple rust resistance (Joung et al., 1987). Symptoms (pycnial lesions) were observed on the leaves of the susceptible cultivar (‘Jonathan’) 10–14 days after inoculation but no symptoms were noticed on the leaves of the resistant one (‘Prairiefire’). Micropropagated ‘Fuji’ apple plants were subjected to waterdeficit treatments by adding PEG-6000 and changes in non-structural carbohydrate composition and the activity of related metabolic enzymes in the leaves originating from the treated shoots were examined (Li and Li, 2005). Accumulation of photosynthates, such as sorbitol, sucrose and glucose and simultaneously a reduction in the starch concentration were detected in the leaves. Also the activities of metabolic enzymes, such as ADP-glucose-pyrophosphorylase, amylase, sorbitol dehydrogenase and sucrose phosphate synthase were affected in response to water stress. In vitro shoot cultures of apple were exposed to 100 or 200 mM NaCl for 10 days (Liu et al., 2008) in order to evaluate the role of PAs in long-term salt stress. Salt treatment had a negative effect on the increase in shoot length and led to a significant increase in electrolyte leakage indicating cell injury. Free Put level was significantly reduced by both types of salt stresses and 100 mM NaCl also reduced the level of spermine (Spm) significantly. However, no changes were detected in the level of Spd. Transcriptional profiling of nine key enzymes responsible for PA biosynthesis was examined. Four of these genes (MdADC, MdSAMDC1, MdSPDS1, and MdSPMS) were induced mainly at higher salt concentration (100 mM NaCl), although transcript levels of MdACL5 decreased with increasing salt concentration (from 100 to 200 mM NaCl) indicating that the genes may play different roles in the salt stress response. Some of them, such as MdADC and MdSPMS, might take part in stress perception, although others, such as MdACL5, were related to growth. The genes not affected by salt treatment in this study may play a role in early response to salt stress corresponding with earlier reports in apple (Hao et al., 2005). 4.4. Micrografting Micrografting is defined as the transplantation of small shoot apices onto rootstocks, which could be conducted both in vitro and in vivo (George, 1993). It can be used for virus elimination, rejuvenation and improvement or for study of physiological connections between rootstocks and scions, such as (in)compatibility, root to shoot communication, or transport; methods and utilization of in vitro micrografting are reviewed by Dobránszki and Jámbor-Benczúr (2006) in different herbs and woody plants. Graft incompatibility and root to shoot communication

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were examined in detail in apple by Zeller et al. (1991) and Soumelidou et al. (1994). In apple, a reliable micrografting method between in vitro propagated scion and rootstock was reported (Dobránszki et al., 2000d) where grafting partners were stuck together by agar–agar (1%) until fusion took place. Huang and Millikan (1980) applied micrografting for virus elimination in apple and found that the size of the scion was inversely related to the probability of obtaining virus–free plants but the rate of graft success increased with the size of the scion. The required size of shoot tips used as scions was an apical dome with 2 leaf primordia in apple. Micrografting was successfully used for establishment of in vitro culture from scions (‘Remo’, ‘Rewena’, and ‘Reanda’) whose culture establishment was unsuccessful earlier because of high tissue browning (Dobránszki et al., 2005).

4.5. Physiological, biochemical and molecular studies Examining three strains of ‘McIntosh’ with different growth habits (standard, compact, and extremely compact) under field conditions, Lane and Looney (1982) reported that BA tolerance to its supraoptimal concentration in meristem-tip culture can be used as a tool for selecting cultivars with different growth habit. Sarwar et al. (1998) studied the effects of other cytokinins (BA, 2-iP (2-isopentenyladenine), TDZ, and KIN) on in vitro shoot proliferation and growth using cultivars with different growth habits (‘McIntosh’, as standard, ‘Macspur, as intermediate and ‘Wijcik’, as dwarf). They demonstrated that all examined cytokinins can be used to differentiate genotypes according to their growth habit. Végváry et al. (2000) studied the bioavailability of BA using different BA concentrations in the culture medium (0.25, 0.5 and 1.0 mg/l) in the in vitro culture of ‘JTE-F’ by high performance liquid chromatography. They found that all available BA was taken up from the medium and accumulated by plantlets. The BA content of shoots increased until the 14th day but on the 30th day a significant decrease was observed. This decrease may be due to different reasons; either plantlets became unable to take up more BA or the decrease was caused by an increase in plant weight during the 4-week-long culture of in vitro shoots or BA was conjugated or metabolized. The real cause of decrease in the BA content of plantlets at the end of the subculture was not studied in this work, although its knowledge could give some information regarding BA metabolism of apple during in vitro culture. A cDNA library of proliferating in vitro apple plantlets (‘McIntosh Wijcik’) was screened by differential hybridization in order to obtain insight into the molecular mode of cytokinin action during axillary bud stimulation (Watillon et al., 1991). Two cDNAs (pSD3 and pSD4) were isolated and characterized corresponding to transcripts up- or down-regulated by exogenous cytokinin (BA) added to the culture medium. pSD3 was more abundant in plantlets grown on media with BA, and the protein encoded accumulated in leaves and stems; pSD4 was more abundant in leaves of plantlets grown in the absence of cytokinin. Biochemical and/or molecular events responsible for the mechanisms by which plants respond to different environmental effects can also be analyzed in in vitro shoot cultures. Molassiotis et al. (2006) examined the mechanism of boron-induced damage in in vitro shoot culture of ‘EM.9’ and found enhanced lipoxygenase activity, lipid peroxidation and hydrogen peroxide (H2O2) accumulation which indicated oxidative damage induced by exposure to excess boron. Furthermore, an increase in the activities of SOD, POX and depression in catalase activity of leaves and stems, a decrease in proline content and an increase of nucleolytic activities in leaves were detected. Excess boron caused oxidative damage and affected RNase and DNase activities.

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4.6. Genetic transformation For overcoming conventional, sexual hybridization systems, the use of genetic transformation is essential in the genetic improvement of apple. Genetic engineering plays an important role in the efficient transfer of horticulturally useful features, such as resistance to different bacteria, insects, fungi and herbicides, rooting ability, or dwarfism of rootstocks, environmental stress resistance or modified metabolism, from various Malus species or other genera as recently reviewed by Aldwinckle and Malnoy (2009). Successful application of genetic transformation is based on the development and existence of tissue culture techniques, such as efficient shoot regeneration systems and micropropagation techniques. The lack of a reliable regeneration method can hinder the development of a transformation system. Several factors can influence the success of in vitro shoot regeneration (Gahan and George, 2008) among which cytokinins have proven to be one of the most important factors (Magyar-Tábori et al., 2010). The availability of effective micropropagation methods for a wide range of different apple scions and rootstocks is a necessary precondition for the propagation of transgenic plants. The specific media and protocols empoyed in the genetic transformation of different cultivars and rootstocks has been covered by Aldwinckle and Malnoy (2009) and Hanke and Flachowsky (2010) and thus lies beyond the scope of this review. 5. Conclusions and future prospects This review of advances in the micropropagation of apple indicates that micropropagation is feasible for rapid propagation of superior cultivars, faster introduction of new cultivars with desirable traits and for rapid multiplication of disease-free, healthy propagation material. Currently many reproducible protocols have been developed for micropropagation of different apple rootstocks and scions. Clonal fidelity should be controlled during commercial propagation despite shoot multiplication mainly being based on pre-existing meristems, since somaclonal variation could be manifested or methylation changes could occur during tissue culture even without previous adventitious shoot regeneration (Li et al., 2002; Modgil et al., 2005). The studies carried out during the last few decades on different stages involved in apple micropropagation has led to considerable improvement of protocols and methods. Shifting of medium status from gelled to liquid medium during shoot multiplication and rooting would be a strategic step for automation and cost efficiency in micropropagation using bioreactors. Results reviewed on propagation of apple in bioreactors (Marga et al., 1997; Chakrabarty et al., 2003; Paek et al., 2005; Zhu et al., 2005; Welander et al., 2007) indicate that it can be the one of the most promising ways for large-scale propagation of apple in the future. Several authors took advantage of in vitro systems with a standard and well-controlled culture environment for using them as tools for studying different physiological, biochemical and molecular processes, speeding up breeding works by short-term selection of different traits under in vitro conditions. Acknowledgements The authors wish to thank Kasumi Shima for creating Fig. 1. We also wish to extend our sincerest thanks to the following individuals who provided valuable advice and updated information in the final stage of the revision process: Dr. Ahmad M. Abdul-Kader (Biotechnology Department, General Commission for Scientific Agricultural Research (GCSAR), Damascus, Syria), Dr. Sean Bully (The New Zealand Institute for Plant & Food Research Ltd., Auckland, New Zealand), Drs. Magda-Viola Hanke and Henryk Flachowsky (Julius Kühn-Institute, Institute for Breeding Research on Horticultural and Fruit Crops, Dresden, Germany) and Dr. Amit Dhingra (Department of Horticul-

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