Anthurium in vitro: A review

G Model

ARTICLE IN PRESS

HORTI-5644; No. of Pages 33

Scientia Horticulturae xxx (2015) xxx–xxx

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Review

Anthurium in vitro: A review Jaime A. Teixeira da Silva a,∗ , Judit Dobránszki b,∗∗ , Budi Winarto c,∗∗ , Songjun Zeng d,∗∗ a

P.O. Box 7, Miki-Cho Post Office, Ikenobe 3011-2, Kagawa-ken 761-0799, Japan Research Institute of Nyíregyháza, University of Debrecen, P.O. Box 12, Nyíregyháza H-4400, Hungary c Tissue Culture Department, Indonesian Ornamental Crops Research Institute, Jl. Raya Ciherang, Pacet-Cianjur 43253, West Java, Indonesia d Key Laboratory of South China Agricultural Plant Molecular Analysis and Gene Improvement, South China Botanical Garden, Chinese Academy of Sciences, 510650, China b

a r t i c l e

i n f o

Article history: Received 26 November 2013 Received in revised form 22 September 2014 Accepted 24 November 2014 Available online xxx Keywords: Anthurium Organogenesis Somatic embryos Winarto–Teixeira medium

a b s t r a c t Anthurium (Anthurium spp.) is an ornamental that is widely appreciated around the world, primarily for its showy and colorful spadix. A successful tissue culture protocol for anthurium would allow for the mass clonal propagation of this plant to serve the floriculture pot-plant and cut-flower markets. Success has been achieved in anthurium tissue culture using several explant types, and the morphological and cytogenetic stability of such regenerants has also been tested. This review provides a detailed analysis of the conditions required for the successful culture of anthuriums in vitro. Besides micropropagation, this review also highlights selected current applications of in vitro anthurium biotechnology such as anther culture, polyploidy production, genetic transformation, and their importance in breeding work, synthetic seed technology and cryopreservation. © 2014 Elsevier B.V. All rights reserved.

Contents 1. 2.

3. 4.

Why are in vitro methods important in Anthurium breeding and propagation? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In vitro culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. The basal medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Explant choice and its sterilization for the in vitro environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. The explant and its interaction with the in vitro milieu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Anther culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acclimatization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Current applications of tissue culture and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Cytogenetic stability and modification in chromosome number of in vitro regenerants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. In vitro selection of pest resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Protoplasts and somatic hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Genetic transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Cryopreservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6. Synthetic seed technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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∗ Corresponding author. Tel.: +81 878988909. ∗∗ Co-Corresponding authors. E-mail addresses: [email protected] (J.A. Teixeira da Silva), [email protected] (J. Dobránszki), budi [email protected] (B. Winarto), [email protected] (S. Zeng). http://dx.doi.org/10.1016/j.scienta.2014.11.024 0304-4238/© 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: Teixeira da Silva, J.A., et al., Anthurium in vitro: A review. Sci. Hortic. (2015), http://dx.doi.org/10.1016/j.scienta.2014.11.024

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1. Why are in vitro methods important in Anthurium breeding and propagation? Anthurium is a popular genus of the Araceae (order Spathiflorae) as a cut-flower and pot-plant ornamental due to its attractive long-lasting inflorescences (Hamidah et al., 1995, 1997a,b; Zeng et al., 2004) and long vase life (Elibox and Umaharan, 2010). The sales volume of Anthurium is ranked second in the world, following orchids (Rikken, 2010; Hua, 2014). Anthurium is one of the main products in Dutch flower auctions with a turnover of D 50 million (Rikken, 2010). On the Chinese mainland, the sales volume of Anthurium was 20 million pots in 2013, 6.5 million of which were sold during the Chinese New Year (Hua, 2014). The flower consists of a protruding spadix containing numerous florets, subtended by a brightly colored modified leaf, the spathe (Higaki et al., 1984, 1995; Croat, 1988). Spathe color is correlated with vacuolar pH (Avila-Rostant et al., 2010) and color throughout development is a complex response to developmental and environmental signals (Collette et al., 2004). The development of different organs within the flower follows a strict set and order of events (Bai et al., 2004). Anthuriums are bisexual and protogynous, and since the spadix first produces a female phase followed by a male phase a month after, this reduces the frequency of self-pollination of flowers and induces cross-pollination at a high frequency (Callotte, 2004). Zygotic embryogenesis has been studied in detail (Matsumoto et al., 1998). Seed-derived progeny are often heterogeneous, which can result in non-uniform color, quality, yield and the time to first flowering (Bejoy et al., 2008; Jahan et al., 2009). Anthurium andraeanum (Hort.) is a collective name that refers to modern anthurium cultivars that are complex interspecific hybrids between A. andraeanum Linden ex André and other species within the section Calomystrium that were initially imported to Hawai’i from Colombia where an intensive breeding program was implemented (Nakasone and Kamemoto, 1962; Kamemoto and Kuehnle, 1996). The genus Anthurium, which consists of approximately 1500 species (900 published) endemic to the neotropical zones of northern Mexico and south through Central America to southern Brazil, and on the Caribbean Islands (Croat, 1988; Mayo et al., 1998; Frodin and Govaerts, 2002; Boyce and Croat, 2012), is conventionally propagated by seed but seed storage is difficult (Zeng, 2000; Dufour and Guérin, 2003, 2006). All Anthurium spp. are perennial, epiphytic or (infrequently) terrestrial herbs (Franz, 2007). It takes approximately three years from pollination to seed maturity and for plants to develop sufficiently to be selected as female parents in a breeding program (Higaki et al., 1995). Seed-propagated plants tend not to be uniform (Geier, 1990; Dufour and Guérin, 2006) and the length of seed development and the long juvenile phase make the breeding of uniform improved cultivars a laborious and time-consuming task (Geier, 1986). Using ovule and lamina culture, Hodgin (2006) could induce callus and shoots from 19 Anthurium species (A. amnicola, A. antioquiense, A. formosum, A. bakeri, A. bicollectivum, A. folsomianum, A. garagaranum, A. gracile, A. jefense, A. kamemotoanum, A. lindenianum, A. nymphaeifolium, A. pallidiflorum, A. ravenii, A. roseospadix, A. sanctifidense, A. scandens, A. trinerve, A. watermaliense), in a bid to safeguard important Anthurium germplasm, indicating the importance of this aspect of biotechnology. The propagation aspects of tissue culture methods were previously covered by Gantait and Mandal (2010) and Atak and C¸elik (2012). However, these reviews failed to report details of important in vitro conditions essential for successful growth and development and did not discuss the developed tissue culture methods of importance to breeding, such as anther culture or polyploidy production. The present review, which aims to summarize the results achieved to date in Anthurium biotechnology, has two clear objectives. Firstly, this information allows for a better understanding of the cost-effective production of healthy and uniform propagation

material that can feed ornamental markets. Secondly, the precision and detail would allow for the production of hybrids and breeding lines within a short period of time based on in vitro anther culture. This review also covers current and future aspects of applied tissue culture techniques, such as somatic hybridization, in vitro selection, genetic transformation, cryopreservation, and synthetic seeds. 2. In vitro culture Anthurium was first propagated in vitro by Pierik et al. in 1974. The early studies by Pierik and colleagues (1974–1979) (Pierik, 1975, 1976; Pierik and Steegmans, 1975, 1976; Pierik et al., 1974, 1979) and Geier (1982, 1986, 1990) in fact established most basal interpretations regarding the important factors for regeneration in vitro, including the importance of the level of ammonium, the mode of sterilization, leaf explant size and the importance of carbon source. Most protocols that followed were simply derivations of these basal studies or the application to new cultivars or to new species. Leffring and Soede (1979) established that the regenerability of various genotypes of A. andraeanum differed. Anthuriums have been propagated in vitro via direct and indirect (i.e., through callus induction) organogenesis (primarily shoot induction) having been successfully established using different explants: seedlings, shoots, stems, leaves, petioles, spathes, and spadices (Table 1), although regeneration protocols remain very dependent on in vitro factors (Teixeira da Silva et al., 2005) and genotype (Nhut et al., 2006). According to Geier (1982, 1986), even though regeneration from the spathe is better than from leaf tissue, spathe tissue is not always available, explaining why more protocols exist for leaf tissue than for reproductive tissue (Table 1). In 1986, Geier claimed that multiple shoot formation was only possible after several sub-cultures, but no further details or quantification of those claims were made. Only in the Geier (1986) protocol was it clearly stated that the leaf lamina should be plated abaxial side down on the medium. None of the other protocols outlined in Table 1 indicate explant orientation following sterilization. Somatic embryogenesis is less commonly reported (Xin et al., 2006; Beyramizade et al., 2008; Liu et al., 2010). However, Xu et al. (2010) reported some physiological and biochemical characteristics of different developmental stages of somatic embryogenesis of A. andraeanum ‘Amigo’ and found that the embryogenic callus formation stage was important for the regulation of somatic embryogenesis. A recent study by Pinheiro et al. (2014) on A. andraeanum ‘Eidibel’ showed how the choice of explant, in this case nodal segments, being a superior explant, could influence the outcome of somatic embryogenesis. Plant tissue culture of Anthurium spp. is now well established although earlier studies were often hampered by high levels of bacterial contamination (Brunner et al., 1995). As for other ornamentals, the success of propagation in vitro is influenced by the culture medium, which serves as the source of inorganic nutrients and carbohydrate(s), organic compounds such as vitamins, and plant growth regulators (PGRs) allowing the plant to grow, photosynthesize and develop in the artificial in vitro environment (Teixeira da Silva et al., 2014). Most plant species, even at the level of cultivar, have a characteristic set of conditions and medium additives that would stimulate an optimum growth response in tissue culture (George et al., 2008). This section of the review has the objective of summarizing the work that has been conducted with Anthurium tissue culture and related in vitro studies. The factors that most influence the in vitro propagation of Anthurium spp. include basal medium, genotype, explant type, PGRs and illumination.

Please cite this article in press as: Teixeira da Silva, J.A., et al., Anthurium in vitro: A review. Sci. Hortic. (2015), http://dx.doi.org/10.1016/j.scienta.2014.11.024

Basal medium

PGR (type and concentration, mg/l or molar)a

Other medium additivesa

Other culture conditionsa

Effect on tissue culture, development, etc. (regeneration type)b

Productivity, somaclonal variation and abnormalities

Acclimatization (% survival)

References

38 genotypes (not specified)

Leaf, embryo

BCM: modified MS with ½ MS macro

1.0 mg/l PBA from embryos and 1.0 mg/l PBA + 0.1 mg/l NAA from adult plant leaves; 0.08 mg/l 2,4-D + 1.0 mg/l BA (leaves; 1979)

3% sucrose if explants originated from embryos but 4% glucose if exlants originated from adult plants; 0.7% Difco Bacto agar

Callus formation, and shoot regeneration in 30% of the genotypes of adult plants (2–3 years old)

Leaf to complete plant = 12 months: callus induction (3 months), callus multiplication (2 months), shoot induction (4 months), chlorophyllation and leaf development (1 month), root formation (2 months)

Not performed

Pierik et al. (1974, 1979) and Pierik (1975, 1976)

28 genotypes (not specified)

Leaves

½ MS

3 mg/l 2iP

3% sucrose; 0.6% agar

Dark and 25 ◦ C for callus induction and subculture at rotation of 120 rpm (1975); placing under continuous fluorescent light (Philips TL 40 W/57) and on solid medium for shoot development NR

Callus, shoots (I)

10.2 shoots/explant

Not performed

Marian Seefurth

Axillary buds (nodal cuttings) Leaves, petioles, spathe, spadix, roots

37.5% MS (solid and liquid)

0.2 mg/l BA

PP NR; 1100 lx; 25–28 ◦ C

Shoots

3.1 shoots/explant

Not performed

½ MS (only macronutrients halved); 4 other media tested

0.1 mg/l NAA + 5.0 mg/l BA (CIM, SIM)

15% CW; 2% sucrose; 0.8% agar–agar 3% sucrose; 0.8% Difco-Bacto agar

Leffring et al. (1976) and Leffring and Soede (1979) Kunisaki (1980)

Callus and shoots (I)

72% of petioles formed callus No clear protocol for individual shoots or rooting

Acclimatized performed but not quantified

Finnie and van Staden (1986)

Nitsch

PGR-free

2% sucrose; 0.9% agar (VW)

16-h PP; 27 ␮E m−2 s−1 ; 20 or 25 ± 2 ◦ C; continuous light or darkness also tested 16-h PP; 2000 lx; 25 ◦ C; 80 rpm shaking for Kunisaki medium

Seed germination

93.3% of seeds germinated and 1.5% formed callus

8% (leaf-derived) 10% (spadixderived) 6% (axillary bud-derived)

Nirmala (1989)

Geier (1986) (leaf) Kunisaki (1980) (spadix) VW (axillary bud) Nitsch

0.1 mg/l 2,4-D + 1 mg/l BA (Geier)

2% sucrose + 15% CW (Kunisaki); 2% sucrose + 0.9% agar (VW, Geier)

Shoots (I)

10% (leaf-derived); 15% (spadix-derived); 12% (axillary bud-derived)

0.05–1.0 mg/l 2,4-D (CIM); 0.2–1.0 mg/l BA (SIM); 0–800 mg/l NH4 NO3 (RIM) 4.4 ␮M BA + 0.36 ␮M 2,4-D

INA

INA

Callus, shoot and root

3–5 shoot per callus

Not performed

Lightbourn and Deviprasad (1990)

Sucrose and Gelrite of Pierik (1976) medium substituted for glucose and Bacto-agar, respectively

Darkness for 2–3 months; 23 ◦ C

Callus or embryogenic callus

63.2% callus from leaves, 49.3% from petioles

Not performed

Kuehnle and Sugii (1991) (used for Kuehnle et al., 2001)

16-h PP; 54 ␮mol m−2 s−1 ; 25 ◦ C

Shoots (I)

Not specified

IIHR 77, IIHR 83

Tropical Pink, Premium Red, White and Tulip Kaumana, Kozohara, Marian Seefurth, Mauna Kea, Nitta, Ozaki, Paradise Pink

Leaves (1 cm2 ) of greenhouse plants; spadix 0.8–1.0 cm segments; nodal sections (0.5–1.0 cm3 )

Leaf explants

Leaf (1–2 cm with one major vein), petiole

Modified (1976) or Finnie and van Staden (1986) media

Leaf-derived callus clusters (30–40 mg)

Kunisaki (1980) medium

2.2 or 22 ␮M BA

ARTICLE IN PRESS

Explant used (type, size, origin)

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Cultivar(s)

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Please cite this article in press as: Teixeira da Silva, J.A., et al., Anthurium in vitro: A review. Sci. Hortic. (2015), http://dx.doi.org/10.1016/j.scienta.2014.11.024

Table 1 Summary of Anthurium andraeanum Hort in vitro studies (listed chronologically).

3

Long-term callus (12–13 months) Shoot, stem with axillary bud

UH780, UH965 (intraspecific hybrids), UH1003, UH1060 (tulip-type hybrids)

Petiole (1 cm long), whole lamina (1.0–1.7 cm long), and bi-sectioned lamina

PGR (type and concentration, mg/l or molar)a

Other medium additivesa

Other culture conditionsa

Effect on tissue culture, development, etc. (regeneration type)b

Productivity, somaclonal variation and abnormalities

Acclimatization (% survival)

References

MS, ½ MS

MS + 0.5 mg/l BA + 0.5–1.0 mg/l Kin (SIM); MS + 1–2 mg/l BA + 0.5–1.0 mg/l Kin (shoot proliferation); ½ MS + 1.0–1.5 IBA + 0.5 mg/l NAA (RIM) 1–4 mg/l 2,4-D + 0.33–1.0 mg/l Kin (somatic embryo induction)

0.7% agar

pH 5.8; 10–12 h PP; 2000 lx; 25–30 ◦ C

Shoots

Proliferation rate was 3.03 every 60-d subculture

Plantlet acclimatized in vermiculite, sand, soil (1:1:1) with 90% survival rate

Mao et al. (1991)

4% sucrose +2% glucose 0.18% Gelrite 100 mg/l myo-inositol

Magenta® GA-7 vessels; darkness; 23 ◦ C

Somatic embryogenesis

Maximum of 38.8% of explants formed somatic embryos. Only whole lamina (or to a less extent petioles) but not sectioned lamina formed somatic embryos.

16-h PP; 54 ␮mol m−2 s−1 ; 25 ◦ C

Plant regeneration

Not quantified

3% glucose was most suitable for callus induction among other concentration glucose and sucrose, light was beneficial for callus induction Blight pathogen: (1) survived in or on callus for over 4 months without producing symptoms in callus or turbidity in the medium; (2) survived for more than 1 year on or within stage II shoots without producing symptoms and was successively transferred three times as latently infected shoots were multiplied; (3) did not grow or survive for more than 2 weeks in MS medium lacking plant material; (4) the addition of coconut water enhanced bacterial growth and produced turbidity in culture media. 65% of explants contaminated

½ MS (only macronutrients halved)

MS

0.2 mg/l BA

MS + 1.0 mg/l BA + 0.08 mg/l 2,4-D (CIM, SIM); ½ MS + 0.1 mg/l NAA (RIM) No PGRs

1–6% sucrose or 1–3% glucose

pH 5.8; 12-h PP; 1000 lx; 25 ± 1 ◦ C

Callus, shoots and roots

Different levels of sucrose, glucose, CW and other additives 0.18% Gelrite

Dark culture for 2 weeks then inoculated (100 callus clumps + 2 ␮l of a 10 cfu/␮l solution of four bacterial strains)c ; 24 ◦ C

Organogenesis not the focus, only bacterial contamination

Not specified

Leaf of in vitro plantlets

MS or ½ MS

UH1060

Callus (organ from which derived and conditions under which induced unspecified)

MS; original callus proliferation on H1

Not specified

Young leaves (1 cm2 ) of greenhouse plants Young leaves

MS

NR

NR

Darkness; 27 ◦ C

Organogenesis not the focus, only bacterial contamination

MS and ½ MS

1.0 mg/l BA + 0.1 mg/l Kin + 0.5 mg/l NAA (CIM, SIM)

NR

NR

Callus, shoot and root

Not specified

79% of explants formed shoots, 4.9 shoots/explant

Kuehnle et al. (1992) (used for Matsumoto et al., 1996, cv. Anuenue and Toyama Peach) 100% acclimatization possible with no observed abnormalities 86% in perlite

Cen et al. (1993)

Not performed

Norman and Alvarez (1994)

Not performed

Brunner et al. (1995)

85%

Gao and Li (1995)

ARTICLE IN PRESS

UH965, UH1060, UH1003 Not specified

Basal medium

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Cultivar(s)

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Table 1 (Continued)

Basal medium

PGR (type and concentration, mg/l or molar)a

Other medium additivesa

Other culture conditionsa

Effect on tissue culture, development, etc. (regeneration type)b

Productivity, somaclonal variation and abnormalities

Acclimatization (% survival)

References

Marian Seefurth, Anuenue, Ozaki Not specified

Axillary buds (nodal cuttings)

MS, ½ MS, 1⁄3 MS

0.2 mg/l BA

15% CW; 2% sucrose; 0.8% agar

INA

Multiple shoots

5 shoots/explant

Acclimatized performed but not quantified

Higaki et al. (1995)

Leaf lamina, petioles, internodes, roots Single shoots without leaf blades

½ MS (all salts except for NH4 NO3 )

1 mg/l BA + 0.2 mg/l 2,4-D (CIM, SIM)

NH4 NO3 (400 mg/l)

16-h PP; 25 ◦ C

No somaclonal variation reported

96–100%

Orlikowska et al. (1995)

½ MS (only macronutrients halved)

2 ␮M BA 10 ␮M Prochloraz (SIM)

16-h PP; 40 ␮mol m−2 s−1 ; 23 ◦ C

NR

Not performed

Werbrouck and Deberg (1996)

Anuenue and ‘Toyama Peach’

Lamina

Modified MS

1.0 mM 2,4-D + 0.15 mM Kin 0.5 mM 2,4-D + 0.15 mM Kin

111 mM sucrose; 555 ␮M myo-inositol; 7 g/l BDH agar 2% sucrose + 1% glucose

Callus on roots and petioles (I); shoot propagation from leaves (D) Enhanced shoot and root number 60 new shoots/explant after 15 weeks (D) Somatic embryos

5 globular protrusions, 4 multiple somatic embryos, 7 multiple embryos with secondary embryos

Matsumoto et al. (1996)

Alii, Anuenue, Rudolph, Mauna Kea; interspecific hybrids UH1003, UH1060

Roots from in vitro plants

Modified MS

2.2 ␮M BA (CIM, SIM)

16-h PP; 4 ␮mol m−2 s−1 ; 25 ◦ C

76% of UH1060 roots formed green-yellow callus (56% for Alii) 72% of UH1060 roots formed multiple shoots (32% for Alii) (I)

Normal growth and flowering reported after 16 months except for one plant (change in flower color). Four cultivars (UH1003, UH1060, Rudolph, Mauna Kea) could not produce shoots under the transformation conditions used

Not specified

Leaf-derived callus (singly or in aggregates), homogenized with a blender

MS liquid or raft culture (shaken at 120 rpm) vs. MS solid

2.2–4.4 ␮M BA + 0.9 ␮M 2,4-D (CIM); SIM = CIM without 2,4-D

Thiamine–HCl increased to 0.4 mg/l; 25 mg/l NaFeEDTA; 100 mg/l myo-inositol; 2% sucrose; 0.18% Gelrite; 15% (v/v) CW for transformed roots 15% CW; 2% sucrose; 0.7% Difco Bacto agar

Histological study carried out, but acclimatization not performed Not performed

Adventitious shoots from callus (I)

185 shoots/vessel when inoculum was >1000 ␮m in size loosely clustered on a raft; 175 shoots/vessel when inoculum was >1000 ␮m in size firmly clustered on solid medium

Not performed

Teng (1997)

Midori, Ozaki, Mauna Kea and six Anthurium species (A. pittieri Engl., A. ravenii Croat and Baker, A. antioquiense Engl. and A. aripoense N. E. Br.). Not specified

Seeds 3–4 month old plantlets used for in vitro nematode assays

H1

No PGRs

0.3% Gelrite

Magenta® GA-7 vessels; leaf explants in the dark at 25 ◦ C for 2–3 months; yellow, firm callus with shoot primordia transferred to light at 25 ◦ C; 16-h PP; 20 ␮mol m−2 s−1 Magenta® GA-7 vessels; constant light (before nematode infection); 14.34 ␮E m−2 s−1 (after nematode infection); 23 ◦ C

Organogenesis not the focus, only testing tolerance and resistance to burrowing nematode

Alfalfa root callus with approx. 400 nematodes in each callus cluster used to infect one vessel with 4 plants/vessel. Root and leaf damage was observed, but this was cultivar-dependent: highest tolerance and resistance in A. pittieri and A. ravenii

Not performed

Wang et al. (1997, 1998)

Leaves

¼ MS

0.5 mg/l BA + 2.0 mg/l IAA

INA

Continuous darkness

Callus

INA

INA

Sreelatha et al. (1998)

Rother 2

Darkness; 23 ◦ C

Chen et al. (1997)

ARTICLE IN PRESS

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Table 1 (Continued)

5

PGR (type and concentration, mg/l or molar)a

Other medium additivesa

Other culture conditionsa

Effect on tissue culture, development, etc. (regeneration type)b

Productivity, somaclonal variation and abnormalities

Acclimatization (% survival)

References

Sonate

Young leaves from 1-year-old plants

Modified MS

0.08 mg/l 2,4-D + 1.0 mg/l BA

1 g/l AC; 0.950 g/l KNO3 + 0.825 g/l NH4 NO3

pH 5.7; 16-h PP; 37 ␮mol m−2 s−1 ; 26 ◦ C

Callus

87.5% of explants formed callus

90% after 2 months in sugarcane: rice husks (1:1)

Trujillo et al. (1999, 2000)

pH 5.8; 10–12 h PP; 1200–1500 lx; 25 ± 2 ◦ C

Shoots (I) Roots Callus induction; shoot differentiation and root formation

62.5% of callus formed shoots 2.36 roots/shoot 83.3% of explants formed callus; 4.94 shoots/explant; 100% shoots with roots

92.3% survival in topsoil

Pan et al. (2000)

0.5 mg/l vitamin B5; 200 mg/l PVP; 15% CW

INA

Shoots

4.66 shoots/explant

Mahanta and Paswan (2001a,b)

15% CW; 100 mg/l each of banana pulp, wheat malt and millet malt 2–3% glucose; 0.6% agar

INA

Callus (I); somatic embryogenesis; shoot regeneration

INA

Plantlet acclimatized in soilrite-perlite (10:1) with 60% survival rate INA

12-h PP; 1000 lx; 26 ◦ C

Callus, shoots, roots

88.2% callus formation; 80% shoot regeneration

Plantlet acclimatized in vermiculite, coconut chaff, soil with 85% survival rate Not performed

Zhang et al. (2001)

3 mg/l BA MS + 0.5 mg/l BA + 0.8 mg/l 2,4-D (CIM); N6 + 2.5 mg/l BA + 0.1 mg/l 2,4-D, or 2.5 mg/l ZT + 0.1 mg/l 2,4-D (SIM); ½ MS + 0.5 mg/l BA or 0.5 mg/l NAA, or 0.5 mg/l 2,4-D (RIM) 0.0 mg/l BA + 0.1 mg/l IAA (SIM); 0.1 mg/l IAA (RIM)

50% nitrates 3% sucrose; 0.7% agar

Not specified

Young leaf

MS, ½ MS, N6

Agnihorti

Axillary buds

MS

Mauritius Orange, Liver Red

Leaf petiole

Nitsch

INA

Not specified

Young stems

Modified MS

1.0 mg/l BA (CIM); 0.8 mg/l BA + 0.05 mg/l NAA (SIM); 0.1 mg/l IBA or NAA (RIM)

Casino, Tropical, SHP, Samangi, Maringue Lady Jane, White, Magic Red, Pierot Valentino

Leaf and petiole explants

MS

1 mg/l BA and 0.2 mg/l IAA (CIM); 0.2–0.5 mg/l IAA and 1.0–2.0 mg/l BA (SIM)

INA

Darkness; 25 ◦ C

Callus, shoots

6.75% of explants produced callus; 2.7–2.9 shoots/explant

Young leaf

MS, ½ MS

3% glucose; 0.6% agar

pH 5.6; 14-h PP; 1500 lx; 26 ± 1 ◦ C

Callus induction; shoot differentiation; root formation

The frequencies of callus formation from petiole section, leaf slices without or with midrib were 25%, 27% and 30%, respectively

Plantlet acclimatized in: perlite:peat (1:1) with 96% survival rate

Lv et al. (2002)

Singapore hybrid

Leaf petiole from in vitro plantlets

MS

½ MS + 4 mg/l BA + 1 mg/l 2,4-D (CIM); MS + 8 mg/l BA (SIM); ½ MS + 2 mg/l BA + 0.2 mg/l NAA (RIM) 2 mg/l IBA + 4 or 6 mg/l Kin (CIM and SIM); 0.25 or 1.5 mg/l IBA + 1.5 mg/l Kin (RIM)

INA

INA

Callus, shoots

90% of explants formed callus; regeneration frequency: maximum of 20.5 shoots/explant; 3–5 roots/shoot

Dhananjaya and Sulladmath (2003, 2006)

Atlanta

Shoots

MS

10.0 mg/l BA or 1.0 mg/l TDZ (CIM)

Unclear

pH 5.8; 16-h PP; 40 ␮mol m−2 s−1 ; 25 ◦ C

Callus (D)

74.9% of shoots formed callus

Plantlet acclimatized in coffee cherry husk: FYM: soil: sand (2:1:1:1) with 80% survival rate 99.3% (in 1:1 perlite: vermiculite)

Prakash et al. (2001, 2002)

Weerasekara and Yapabandara (2002)

Han and Goo (2003)

ARTICLE IN PRESS

Basal medium

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Table 1 (Continued)

Lima White, Tropical White, Tropical Red

Explant used (type, size, origin) Shoot-derived callus (7 × 10 mm) Leaf (lamina + petiole) of mature plants; lamina = 1 cm2 , petiole = 1 cm

Basal medium

½ MS

½ MS

Leaf, petiole from in vitro plantlets

Pink, Oilclothflower, Rosetta, Atlanta, Fantasia, Afrikanerin

Stem, leaf, petiole

Pierik macroelements and MS microelements, ½ MS

Midori, Kalapana

Axillary buds (2 cm long, 1 cm in diameter, 0.5 cm thick) Leaf lamina at young brown and green stages (1 cm2 )

Tinora Red, Senator

Osaki, Nitta, Anouchka

Young, tender leaf (1–2 cm2 )

10.0 mg/l BA or 10.0 mg/l BA + 1.0 mg/l 2,4-D (SIM) 0.88 ␮M BA + 0.9 ␮M 2,4-D + 0.46 ␮M Kin (CIM)

0.88 ␮M BA + 0.54 ␮M NAA + 0.46 ␮M Kin (SIM) 0.44 ␮M NAA (RIM) 2 mg/l BA + 0.2 mg/l 2,4-D (CIM)

Other medium additivesa

3% sucrose; 0.6% agar

Other culture conditionsa

pH 5.5; 16-h PP; 25 ␮mol m−2 s−1 ; temperature NR

Acclimatization (% survival)

References

Tropical White produced more callus (67.2%), shoots (21.2 shoots/culture) and roots (8.8 roots/shoot) than the other two cultivars Flowering after 12–15 months. Morphology similar to mother plants

97% in vermicompost: sand (1:3)

Joseph et al. (2003)

Petiole showed significantly better results than leaf in callus induction, and bud differentiation. Continuous light and 10-h PP obviously improved callus formation and bud differentiation than the treatment with no light. N6, KC and ½ MS suitable for petiole and Pierik, N6 and ½ MS for leaf The frequencies of callus formation were significantly different among varieties. The tendency of frequencies of callus formation and shoot differentiation were stem > petiole > leaf 10 or 15 shoots/explant (Midori/Kalapana)

Not performed

Lan et al. (2003a)

100% in peat moss or Dragon’s blood slag

Lan et al. (2003b)

100% on peat moss

Lee-Espinosa et al. (2003)

Not performed

Martin et al. (2003)

Not performed

Puchooa and Sookun (2003) and Puchooa (2005)

Effect on tissue culture, development, etc. (regeneration type)b

Productivity, somaclonal variation and abnormalities

Shoots (I)

12.9 shoots/callus clump

Callus (I)

Multiple shoots

Roots Callus induction; shoot differentiation

NR

0, 10, or 24-h PP; 2000 lx; 28 ± 2 ◦ C

Pierik + 2.0 mg/l BA + 0.2 mg/l 2,4-D (CIM and SIM); ½ MS + 0.2 mg/l IAA (RIM)

NR

14-h PP; 2000 lx; 28 ± 2 ◦ C

Callus induction; shoot differentiation; root formation

37.5% MS (liquid at 1 rpm)

0.8 mg/l BA

100 mg/l myo-inositol; 2% sucrose; 0.2% Phytagel®

16-h PP; 222 ␮mol m−2 s−1 ; 25 ◦ C (day), 18 ◦ C (night); 55% RH

Shoot proliferation

½ MS

1.11 ␮M BA + 1.14 ␮M IAA + 0.46 ␮M Kin (SIM)

3% sucrose; 0.6% agar

16-h PP; 25 ␮mol m−2 s−1 ; 25 ◦ C

Shoot induction (D)

Proximal end of lamina of brown or green leaves gave most shoots (both cvs)

½ MS

0.44 ␮M BA

Multiple shoots

½ MS

0.54 ␮M NAA + 0.93 ␮M Kin (RIM) 1 mg/l BA + 0.1 mg/l 2,4-D (CIM); 0.5 mg/l BA (SIM); 1 mg/l IBA (RIM)

Roots

12.2/explant (Tinora Red); 5.4/explant in Senator Almost 100% rooting for both cultivars 5 Gy gamma rays increased callus proliferation; RAPD analysis revealed no variation; 100% callus induction and >10 shoots/callus piece possible for all three cultivars

MS + Nitsch vitamins

NH4 NO3 (200 mg/l for callus and 720 mg/l for shoots); 0.04% AC for rooting; 0.8% agar

pH 5.8; 16-h PP; 5.0 W m−2 ; 25 ◦ C

Callus, shoots, rooted plantlets; same protocol as the Geier (1986) protocol used for A. scherzerianum and the Puchooa and Sookun (2003) protocol for A. digitatum

ARTICLE IN PRESS

½ MS MS, ½ MS, Pierik, N6, B5, KC

Not specified

PGR (type and concentration, mg/l or molar)a

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Table 1 (Continued)

7

Explant used (type, size, origin)

Basal medium

PGR (type and concentration, mg/l or molar)a

Other medium additivesa

Other culture conditionsa

Effect on tissue culture, development, etc. (regeneration type)b

Productivity, somaclonal variation and abnormalities

Acclimatization (% survival)

References

Southern Blush, Marian Seefurth

Whole plants

NR

NR

NR

Magenta® GA-7 vessels; 20–40 E m−2 s−1 ; 12-h PP; 25 ◦ C; 35% RH

Plantlet maintenance (D)

NR

Marian Seefurth grown in greenhouse under 78% shade, 20–28 ◦ C, 80–95% RH

Hayden and Christopher (2004)

Avanti (orange flower)

Shoot tips (2 per 200 ml medium) Shoots 10 mm long

MS

0.5 mg/l BA

3% sucrose; 0.8% agar

pH 5.6; 16-h PP; 40 ␮mol m−2 s−1 ; 25 ◦ C

MS

0.5 mg/l 2iP

½ MS

2.5 g/l IBA (shoot tips 15 mm long dipped in IBA then implanted into MS)

3% sucrose; 1.0% agar 3% sucrose; 0.8% agar or 1.5 g/l Phytagel® ; 8 g/l AC

Shoot tip culture; sub-culture every 4 weeks Shoot elongation until 15 mm Root induction

MS

4.4 ␮M BA + 0.36 ␮M 2,4-D for 60 days then onto 8.9 ␮M BA for 4× 45-d sub-cultures (SIM)

3% sucrose; 100 mg/l myo-inositol; 0.28% agar

Sonate, Tropical, Merengue

Shoot tips

NR

PGR-free (CIM); 1 or 2 mg/l BA + 0.08 mg/l 2,4-D (SIM)

Rubrun

Seed from spadices and micro-cuttings

MS

4.4 ␮M BA + 0.05 ␮M NAA (SIM)

3% glucose + 2% sucrose; 100 mg/l myo-inositol; 0.2% Gelrite 6% sucrose; 1.2 mM thiamine; 0.6 mM myo-inositol; 0.2% Gelrite

Callus from above explants or 8-week-old plantlets Leaf, stem, petiole

MS

8.9 ␮M BA + 2.7 ␮M NAA (CIM)

MS, ½ MS

MS + 1.0 mg/l BA + 0.5 mg/l 2,4-D (CIM); MS + 1.0 mg/l BA + 0.1 mg/l IBA (SIM); ½MS + 0.5 mg/l NAA (RIM) 1 mg/l BA + 0.1 mg/l 2,4-D (CIM); 0.01 mg/l TDZ (SIM)

Unspecified

Jolanba

In vitro plantlets (including shoots, leaves and petioles)

mMS

pH 5.8; 16-h PP; 80 ␮mol m−2 s−1 ; 25 ± 1 ◦ C. Then temporary immersion bioreactors (one immersion for 3 min every 3 h) pH 6; 16-h PP; 2000–2500 lx; 27 ◦ C pH 5.8; continuous light at 50 ␮mol m−2 s−1 ; 25 ◦ C

87% of shoots formed roots; 3.91 roots/shoot when agar was 1.2%; 93.33% induction with Phytagel® at 3.0 roots/shoot Highest multiplication in 8.9 ␮M BA + 10 mg/l Pectinomorph (new PGR of pectin oligosaccharides)

100% in peat moss: vermiculite

Not performed

Lara et al. (2004)

Callus induction; shoot induction (I)

Sonate and Tropical: 83% of explants formed callus and a maximum of 20 shoots/explant

Not performed

Montes et al. (2004)

74% seed germination; multiple shoots (D); 3.6 shoots/explant

No somaclonal variation observed

80%

Vargas et al. (2004)

Shoot induction (D)

43.8 plantlets/cm2 of callus (I)

3% sucrose; 0.75% agar

pH 5.8; 14-h PP; 25 ± 2 ◦ C

Callus induction; shoot differentiation; root formation

The frequencies of callus formation from stem cuttings were higher than those from leaf or petiole

96% survival rate in coco coir (or peat): perlite (1:2)

Yuan et al. (2004)

3% sucrose; 0.6% agar

12-h PP; 1500–2000 lx; 25 ± 2 ◦ C

100% of shoots and 43.8% petioles generated callus

2.38 shoots/callus cluster

Not performed

Zhao et al. (2004)

ARTICLE IN PRESS

Shoot tips

Keatmetha and Suksa-Ard (2004) J.A. Teixeira da Silva et al. / Scientia Horticulturae xxx (2015) xxx–xxx

Tropical

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Table 1 (Continued)

Basal medium

PGR (type and concentration, mg/l or molar)a

Other medium additivesa

Other culture conditionsa

Effect on tissue culture, development, etc. (regeneration type)b

Productivity, somaclonal variation and abnormalities

Acclimatization (% survival)

References

Unspecified

Young inflorescences

MS, ½ MS

3% sucrose

pH 5.6; 12-h PP; 1000 lx; 25 ± 1 ◦ C

Callus induction; shoot differentiation and proliferation; root formation

NR

Not performed

Zhu et al. (2004)

Cananéia

Seedless fruits

MS

MS + 1.0 mg/l BA + 0.2 mg/l 2,4-D (CIM); MS + 0.5 mg/l BA + 0.1 mg/l NAA (SIM); MS + 1.0 mg/l BA + 1.0 mg/l Kin + 0.1 mg/l NAA (shoot proliferation medium); ½ MS + 0.5 mg/l NAA (RIM) 2.22 ␮M BA or 18.09 ␮M 2,4-D (CIM)

NR

16-h PP; 24 ◦ C; light intensity NR

63.4% of fruits generated callus

NR

Not performed

Alves dos Santos et al. (2005)

Most and longest roots and tallest shoots with 5.71 ␮M IAA and 4% sucrose for both cultivars Not quantified

Not performed

del Rivero-Bautista et al. (2005)

Not performed

Nitayadatpat and Te-chato (2005)

21 shoots after 2 months; Gelrite superior to agar–agar (20 vs. 7 shoots after 2 months) ∼65% explants with callus; shoot multiplication rate = 5.17 ± 2.30

100% survival in peat: perlite (4:6, v/v)

Ruffoni and Savona (2005)

0.89 ␮M BA (SIM)

Sonate, Lambada

Shoots from nodal segments

MS (liquid), half-stregnth macronutrients

IAA (0–5.71 ␮M)

3% or 4% sucrose

pH 5.8; PP NR; 48–62.5 ␮mol m−2 s−1 ; 26 ± 2 ◦ C

Unspecified

Leaves

MS

0.1 mg/l BA + 0.1 mg/l 2,4-D

3% sucrose

pH 5.7; 14-h PP; 3000 lx; 27 ◦ C

½ MS (liquid) overlaying MS solid MS (solid)

PGR-free

1 mg/l AS; 0.75% Gelrite

2 mg/l BA for initial culture and 3 mg/l for subculture

3% sucrose

Unspecified

Meristems from commercial in vitro mother plants placed in immersion system using RITA® vessels

3 mg/l BA

Plantlets

MS liquid

Only a single callus cluster formed shoots (unquantified) Shoot and root induction (D)

Callus induction (D)

Shoot induction (I)

Dark at 15 ◦ C for 3 months and subcultured every 15 days for initial culture; then culture room with 24 ± 1 ◦ C, 16-h PP; 30 ␮mol m−2 s−1 ; subcultured every 40 days 16-h PP; 30 ␮mol m−2 s−1 ; 24 ◦ C; subculture every 40 days Immersion in RITA® (TIS) for 3 min every 3 h

Shoot establishment

Shoot development (D)

∼63% callused explants with shoots

Bioreactor growth

3.7 shoots/explant (explant = plantlet?)

ARTICLE IN PRESS

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Table 1 (Continued)

9

Explant used (type, size, origin)

Basal medium

PGR (type and concentration, mg/l or molar)a

Other medium additivesa

Other culture conditionsa

Effect on tissue culture, development, etc. (regeneration type)b

Productivity, somaclonal variation and abnormalities

Acclimatization (% survival)

References

Elizabeth

Shoot tips

½ MS (liquid) poured evenly over Rockwool® block in Milliseal® ventilated vessel or Neoflon® PFA film Culture Pack®

PGR-free Only aeration

Aerated micropropagation 3% glucose

pH 5.3; 16-h PP; 45 ␮mol m−2 s−1 ; 25 ◦ C

Shoot development, rooting enhancement, plantlet growth

6.8 leaves/plant

Teixeira da Silva et al. (2005)

Arizona, Pink Champion, Valentino, Ardor, Cancan

Young leaf

Modified MS (205 or 410 g/l NH4 NO3 )

1.0 mg/l IBA + 0.1 mg/l 2,4-D (SIM); 0.1–0.5 mg/l IBA (RIM)

3% sucrose or 3% glucose; 0.7% agar

pH 5.8; darkness or 14-h PP; 1000–2000 lx; 25 ◦ C

Callus induction; shoot differentiation; root formation

Pink Champion, Tropical, Sweet Dream, Toscane, Baleno, Pohris, Michigan, Fantasy Love Eidibel

Leaf, petiole

Nitsch, MS

Many exogenous hormones combinations for CIM and SIM

Solid or liquid culture

24-h PP; 2000 lx, or dark; 28 ◦ C

Callus induction; shoot differentiation

Not performed

Jiang et al. (2006)

Nodal segments

½ MS (liquid)

4.44 mM BA + 2.89 mM GA3

NR

16-h PP; 50 ␮mol m−2 s−1 ; 25 ◦ C

Shoot induction (D)

The frequencies of callus formation were higher (76.7%) on modified MS (205 or 410 g/l NH4 NO3 ) than on MS (10%). mMS with 3% glucose was higher (80) than with 3% sucrose (46.6). In darkness was higher (76.7% for Arizona, 87.6% for Pink Champion) than on light (12.2% for Arizona, 18.8% for Pink Champion) the induction rate of callus and the browning rate of explants between varieties were significantly differen. The sorts of explants, exogenous hormones and the medium significantly affected the callus induction, proliferation and bud differentiation 4.7 leaves/plantlet; 6.5 roots/plantlet in liquid medium

100% acclimatization possible by direct transfer of gas-permeable vessel from in vitro to greenhouse or transfer to Metromix® for 60 days Plantlet acclimatized in vermiculite, perlite, peat, with 95% survival rate

Lima et al. (2006)

Tropical, Choco, Pistache, Carnaval, Neon, Sonate, Midori, Safari, Arizona, Cancan

Leaf segments (1 cm2 ) 0.5 cm2 callus pieces for callus proliferation

½ MS

1.0 mg/l BA + 0.08 mg/l 2,4-D

0.8% agar (all media); 3% glucose

pH 6.0; 10-h PP; 45 ␮mol m−2 s−1 ; 25 ◦ C; 70–75% RH

Callus induction (all 10 cvs)

Acclimatization with AMF (Gigaspora albida, Glomus etunicatum, Acaulospora longula) resulted in 13.6 leaves/plant 100% (first 3 cvs)

1.0 mg/l BA

2% glucose

Nhut et al. (2006) (similar protocol reported for ‘Tropical’ in Nhut et al., 2004)

ARTICLE IN PRESS

10.1 shoots/callus piece in Tropical (4.3 for Choco, 3.5 for Pistache)

Xia et al. (2005) J.A. Teixeira da Silva et al. / Scientia Horticulturae xxx (2015) xxx–xxx

2% glucose; 206 mg/l NH4 NO3

Callus proliferation (all 10 cvs) Shoot regeneration (SIM) (first 3 cvs)

65.1% in Pistache after 100 days (strong genotype dependence)

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Table 1 (Continued)

Explant used (type, size, origin)

Basal medium

PGR (type and concentration, mg/l or molar)a

¼ MS Leaves, nodes, internodes of 2-week-old in vitro plantlets

MS

0.5 mg/l BA + 0.5 mg/l TDZ

Sonate

Leaf

MS

XP, ARZ, ATL, KLT, AYZ

Leaf or stem with petiole from in vitro plantlets

MS, ½ MS

Unspecified

Leaf, shoot, petiole, root

MS, ½ MS, White’s, B5, N6

2–3 mg/l 2,4-D + 0.5 mg/l Kin (CIM); 0.5 mg/l BA (somatic embryos) ½ MS + 1.0 mg/l BA + 0.1 mg/l 2,4-D + 0.1 mg/l Kin (CIM); ½ MS + 0.5 mg/l 2,4-D + 0.5 mg/l NAA (RIM) 1.0 mg/l BA + 0.2 mg/l NAA (CIM)

Unspecified

Seed

3 ml/l Albert’s solution (fertilizer)

0.8 mg/l NAA + 2.0 mg/l 2,4-D

MS

1.5 mg/l BA + 0.5 mg/l 2,4-D (shoot proliferation); 1.0 mg/l BA + 0.5 mg/l IBA (RIM) MS + 1.0 mg/l BA + 0.1 mg/l 2,4-D + 0.2 mg/l NAA (CIM); MS + 1.0 mg/l BA (SIM); ½ MS (RIM) 2 mg/l 2,4-D + 0.5 mg/l Kin

Pink Champion, Robino, Sweet Heart Red, Toscane, Alpine Sultan

Leaf, stem, petiole, young spadices

MS, ½ MS

Callus

MS

3% glucose; 1 g/l AC 3% glucose; 0.75% agar

Other culture conditionsa

pH 5.7; darkness

Effect on tissue culture, development, etc. (regeneration type)b

Productivity, somaclonal variation and abnormalities

Root induction (RIM) (first 3 cvs) Nodular and embryogenic callus

Roots not quantified

3% sucrose

16-h PP; 20 ␮mol m−2 s−1 ; 25 ◦ C

Callus induction; somatic embryogenesis

3% sucrose or 3% glucose; 0.8% agar

pH 5.8; 14–16 h PP; 25 ◦ C

Callus induction; shoot differentiation; root formation

NR

pH 5.8; 1200–1500 lx; 22–25 ◦ C

Callus induction; shoot differentiation; root formation

3% sucrose

pH 5.4; light, temperature, PP, RH conditions NR

Seed germination

Acclimatization (% survival)

References

MS (86.6% of explants formed callus from leaves) was superior to Nitsch (40%) and WPM (66.7); 100% callus on nodes in MS; genotype-dependence; wounding of explants increased callus production by 33–100% 90.3% of leaves formed callus, 62.9% leaves formed somatic embryos

Not performed

Te-chato et al. (2006)

Not performed

Xin et al. (2006)

The frequencies of callus formation from shoot cuttings with petiole were higher than those from leaves with petiole. ½ MS, sucrose was better than MS or glucose The trendency of callus formation frequencies was ½ MS > MS > N6 > B5 > White’s; young leaf > middle-age leaf > old leaf, leaf with margin > leaf without margin 90% germination

100%

Yao et al. (2006)

Not performed

Cai et al. (2007)

90% of plants grown on Albert’s solution vs. 65% of plants grown on MS when acclimatized in sand: coir dust (1:2)

Fernando and Subasinghe (2007)

Plantlet acclimatized in perlite with 80% survival rate Not performed

Jia et al. (2007)

Plantlet development

48.4 shoots/seed?; 15.1 roots/plant with 7 ml/l Albert’s solution

3% sucrose or 3% glucose

12-h PP; 2000 lx; 25 ± 1 ◦ C

Callus induction; shoot differentiation; root formation

Callus formation did not occure from young spadices

3% sucrose; 0.75% agar

pH 5.7; 14-h PP; 1300 lx; 26 ± 2 ◦ C

Somatic embryos; shoot regeneration (I)

Maximum 67% callus formation; 100% of callus formed shoots with max. of 8.7 shoots/callus cluster; somaclonal variation shown by esterase assay.

Sontikul and Te-chato (2007)

ARTICLE IN PRESS

Plew Thien Phuket, Sonat, Valantino

Other medium additivesa

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Table 1 (Continued)

PGR (type and concentration, mg/l or molar)a

Other medium additivesa

Other culture conditionsa

Effect on tissue culture, development, etc. (regeneration type)b

Productivity, somaclonal variation and abnormalities

Acclimatization (% survival)

References

Flamingo

Recently expanded leaves from 3 to 4 year old greenhouse plants

½ MS (only macronutrients halved)

0.08 mg/l 2,4-D + 1.0 mg/l BA + 1.0 mg/l 2iP (CIM)

0.75% agar

16-h PP; 25 ◦ C

Callus

Maximum 0.41 callus clumps/leaf explant

100%

Viégas et al. (2007)

Shoots (I)

Maximum 8.65 shoots/callus cluster after 70 days Callus induction after 1 month, shoot differentiation after two months

Plantlet acclimatized in vermiculite, reed residue (1:1) with 80% survival rate Not performed

Xu et al. (2007)

0.5 mg/l BA (SIM) Unspecified

Spathe

Modified Nitsch

Pink Champion, Tropical

Leaf

Modified Nitsch medium.

Agnihothri

Leaf lamina segments (1–1.5 cm2 ) from leaves of mature plants 5–10 days after unfolding

½ MS

Tera (Terra?)

Leaves 1–3 after unfolding (1 cm2 )

½ MS (full micronutrients)

MS

Lambada

Leaf segments (1 cm2 ) from in vitro plantlets propagated using the Pierik (1976) protocol

½ MS (full micronutrients)

0.4 mg/l 2,4-D + 1.0 mg/l BA (CIM); 0.25 mg/l BA + 0.1 mg/l Kin (SIM); 0.25 mg/l BA + 0.2 mg/l IBA (RIM) 0.5 mg/l 2,4-D + 0.5 mg/l BA + 0.5 mg/l NAA (CIM); 0.5 mg/l 2,4-D + 0.5 mg/l Kin + 0.2 mg/l NAA (SIM); 0.2 mg/l BA + 1.0 mg/l IBA (SIM) 1.0 mg/l BA + 0.5 mg/l 2,4-D (CIM)

0.3 mg/l BA (SIM) 0.3 mg/l BA + 0.5 mg/l NAA (shoot maintenance, RIM) 0.08 mg/l 2,4-D + 1.0 mg/l BA (CIM)

2% sucrose, 15% CW (CIM, SIM); 3% sucrose and 2 g/l AC (RIM)

16-h PP; 1500–2000 lx; 25–28 ◦ C

Callus induction; shoot differentiation and root formation

NH4 NO3 720 mg/l, 5% CW, 3% sucrose (CIM, SIM); 1 g/l AC + 0.15% sucrose (RIM)

28 ± 2 ◦ C

Callus induction; shoot differentiation and root formation

NR

3% sucrose; 0.6% agar

pH 5.5; darkness

Callus

53% of explants responsive, best from pale green leaves

16-h PP; light intensity NR

Adventitious shoots (I)

9.7 shoots/callus clump 98% of shoots formed roots

Darkness; 25 ◦ C

Callus

75.2% of explants induced callus

16-h PP; 35 ␮mol m−2 s−1 ; 25 ◦ C

Somatic embryos (I)

50% of explants formed somatic embryos

Shoot regeneration (I) Callus

23.3 shoots/explant 48.7% of explants formed callus

3% sucrose; 0.25% Gelrite

3 mg/l 2,4-D + 0.33 mg/l Kin PGR-free (SIM) 2.32 ␮M Kin + 6.79 ␮M 2,4-D (CIM)

3% sucrose; 0.25% Gelrite

pH 5.8; 27 ◦ C

Zhang et al. (2007)

89%; 14,000 plantlets produced/leaf explant

Bejoy et al. (2008)

100% on perlite at 25 ◦ C, 75% RH, 70% shading; total of 750 plants

Beyramizade et al. (2008) (overlapping data set with Beyramizade and Azadi, 2008)

Not performed

del Rivero-Bautista et al. (2008)

ARTICLE IN PRESS

Basal medium

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Table 1 (Continued)

Explant used (type, size, origin)

Basal medium

PGR (type and concentration, mg/l or molar)a

Other medium additivesa

4.44 ␮M BA (somatic embryo induction) 1.78 ␮M BA (somatic embryo germination)

CanCan

Arizona, Mississippi, Colorado, Fiestra, Vitara, Tender Lover, Anouk, Sacha Arizona, Sumi

Red Star

Mississippi, Abason, Alabama Unspecified

Leaf, petiole, young spadices, aerial root from in vitro plantlets Young leaf

Leaf (1 cm2 ) from greenhouse plants

MS

Effect on tissue culture, development, etc. (regeneration type)b

Somatic embryos (I) Induction in darkness; germination in light; PP NR; 100–125 ␮mol m−2 s−1 ; 25 ◦ C Multiple apical shoots 0.7% agar; PP NR; 3000 lx; 25 ◦ C; (D) 60% RH

0.1 mg/l NAA + 0.25 mg/l BA (SIM)

29.4 somatic embryos/callus after 80 days at 8.88 ␮M BA (embryos at cotyledonary stage) 64.5% of somatic embryos germinated 60 shoots/shoot tip in 103 days; no variation between in vitro and mother plants shown by ISSR analysis (10 primers) although considerable morphological variation was observed in in vitro plantlets in response to different PGR levels/combinations

0.5 mg/l BA 0.5 mg/l IAA (RIM) 0.1 mg/l Kin + 1.0 mg/l BA (CIM); 0.5 mg/l IBA (RIM)

60 mg/l AS 2 g/l AC 0.6% agar

MS or modified MS (½ NH4 NO3 + ½ CaCl2 )

Modified MS + 0.5 mg/l BA + 0.1 NAA (CIM)

0.7% agar

pH 5.8; 26 ◦ C (day), 21 ◦ C (night)

Callus induction

The frequencies of callus formation were significantly different among varieties; cv. Mississippi is highest (66.7)

½ MS

0.6 mg/l 2,4-D + 1 mg/l BA (CIM)

3% sucrose; 0.6% agar

Darkness; 27 ◦ C

Callus induction

Explants formed callus after 1 month in the dark

0.1 mg/l 2,4-D + 1 mg/l BA (SIM)

250 mg/l NH4 NO3 ; 2% sucrose; 0.6% agar 0.04% AC 2% sucrose; 0.1 g/l myo-inositol; 0.16% Gelrite; 0.005% ClO2 3% sucrose; 0.7% agar

16-h PP; 27 ◦ C

Shoot regeneration (I)

33.7 or 26.96 shoots/explant (Arizona vs. Sumi)

NR 16-h PP; 40 ␮mol m−2 s−1 ; 26 ◦ C; 60% RH

Roots Shoot and plantlet multiplication (D)

NR NR

pH 5.8–6.0; 12-h PP; 1000–1500 lx; 25–28 ◦ C pH 5.8; 16-h PP; 2000 lx; 26 ◦ C; subculture every 5 weeks

Callus differentiation

More suitable medium for bud induction was ½ MS than MS or ¼ MS; 7.44 shoots/callus 85% of leaf segments formed callus (82% for spadix segments)

½ MS

1 mg/l IBA (RIM) No PGRs (SIM)

pH 5.8; 16-h PP; 1500–2000 lx; 26 ◦ C

Multiple shoots Roots Callus induction; shoot differentiation and root formation

Productivity, somaclonal variation and abnormalities

1-cm tall shoots with 2 leaves and no roots Callus

½ MS (only macronutrients halved) ¼ MS, ½ MS, MS

2.0 mg/l BA, 0.1 mg/l NAA (SIM)

Leaf, spadix segments (3–4 months old)

Nitsch

2.5 mg/l BA + 0.2 2,4-D (CIM)

MS

1.0 mg/l BA (SIM)

Multiple shoots (I)

½ MS

1.0 mg/l IBA (RIM)

Roots

Carbon source NR; 0.4% Phytagel®

Callus

Callus did not form from young spadices, however, aerial root was suitable explant for callus formation

Acclimatization (% survival)

References

85%

Gantait et al. (2008) and Gantait and Sinniah (2011)

Plantlet acclimatized in perlite, peat (v:v = 1:1) with 91% survival rate Not performed

Ge et al. (2008)

Yang et al. (2008)

Not performed

Atak and C¸elik (2009)

NR

Cardoso (2009)

Not performed

Duan et al. (2009)

85% in sand, loamy soil and coco-peat (1:1:1)

Jahan et al. (2009)

18 shoots/leaf segment; 14 shoots/spadix segment 3.8 roots/shoot

ARTICLE IN PRESS

Unspecified

Shoot tip derived from young (30-day-old) plants

Other culture conditionsa

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Table 1 (Continued)

Basal medium

PGR (type and concentration, mg/l or molar)a

Other medium additivesa

Other culture conditionsa

Effect on tissue culture, development, etc. (regeneration type)b

Productivity, somaclonal variation and abnormalities

Acclimatization (% survival)

Nicoya

Terminal cuttings from in vitro mother plants 3–4 cm long with 3–4 leaves

MS

1.0 mg/l BA (SIM)

3% sucrose

Shoot development (D) 16-h PP; 67.7 ␮mol m−2 s−1 (SIM) and 135.1 ␮mol m−2 s−1 (RIM); 26 ◦ C

4.17 axillary shoots/cutting

83% in a mist Liendo and chamber or Mogollón 77% in a (2009) humidity chamber in commercial Promix at 27.8 ␮mol m−2 s−1 and 28 ◦ C

Arizona, Colorado, Champion, Pink champion, Robino, Sweetheart Red

Young leaf

MS, ½ MS, MP (modified Pierik), B5, N6

Modified Pierik: 412.5 mg/l NH4 NO3 , 440 mg/l CaC12 ·2H2 O

14-h PP; 1000–1500 lx; 25 ± 2 ◦ C

Red Hot

Young leaf laminas of greenhouse plants at the olive-brown unopened stage and petioles Laminas 1 cm2 Petiole 1 cm long

½ MS (only macronutrients halved)

2% sucrose; 0.3% Gelrite

pH 5.5; 16-h PP; 45.9 ␮mol m−2 s−1 ; 25 ◦ C; subculture every 3 weeks

½ MS ½ MS

2.26 ␮M 2,4-D + 4.44 ␮M BA (SIM) 0.90 ␮M 2,4-D + 8.88 ␮M BA (CIM) 0.90 ␮M 2,4-D + 4.44 ␮M BA 4.44 ␮M BA PGR-free

Shoot tip

MS

0.5 or 1.0 mg/l BA

3% sucrose

pH 5.7; 16-h PP; 8000 lx; 25 ◦ C

Shoot tip

MS

0.5 mg/l NAA + 2 mg/l 2iP (SIM and shoot proliferation); 2.0 mg/l IBA (RIM)

3% sucrose; 0.8% agar

pH 5.7; 16-h PP; 45 ␮mol m−2 s−1 ; 25 ◦ C

Leaf, petiole

½ MS

½ MS

Donna, Bianca, Red Queen, Bijing Success, Keny, Kim, Orange Queen, Anouk, Venus, Cleopatre Paradi

3% sucrose; 0.7% agar

12-h PP; 25–30 ␮mol m−2 s−1 ; 25 ◦ C

Root induction (D) Callus induction; shoot differentiation and proliferation, root formation

5.49 roots/shoot The most suitable base medium of callus induction was MP for Champion and Sweetheart red, ½ MS for Arizona. The frequencies of callus formation were high Pink in champion, Sweetheart red and Robino, low in Arizona

Callus induction

33.3% of leaf lamina formed callus (64.9% and 75% with 2.26 ␮M 2,4-D and 5.71 ␮M IAA, respectively)

Shoot induction (I)

21 shoots/callus

Callus induction

89% of explants formed callus after 8 weeks

Plantlet acclimatized in perlite or mixture of perlite and peat (1:1 or 3:1) with 100% survival rate

Liu et al. (2009)

Not quantified although growth assessed in detail Endopolyploidy analyzed

Seah (2009)

Not quantified

Yu et al. (2009)

Callus proliferation PLB production PLB germination (i.e., shoot formation) Adventitious shoots (D)

85.5 PLBs/callus cluster

3.57 shoots/shoot tip

Not performed

Haddad and Bayerly (2010)

Adventitious shoots (D)

16 shoots/shoot tip; 9.7 roots/shoot

97%

Harb et al. (2010)

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Valentino

0.1 mg/l NAA (RIM) MP or ½ MS + 1.0–2.0 mg/l BA + 0.1–0.2 mg/l 2,4-D (CIM); MS + 0.5 mg/l BA + 0.5 mg/l NAA or MP + 1.0 mg/l BA (SIM); MS + 1.0 mg/l BA (shoot proliferation); ½ MS + 0.5 mg/l IAA (RIM) 4.44 ␮M BA (CIM)

References

ARTICLE IN PRESS

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Table 1 (Continued)

Basal medium

PGR (type and concentration, mg/l or molar)a

Other medium additivesa

Other culture conditionsa

Effect on tissue culture, development, etc. (regeneration type)b

Productivity, somaclonal variation and abnormalities

Acclimatization (% survival)

References

Nitta

Mid-ribs of young leaves

MS

1.0 mg/l NAA + 1.0 mg/l BA (SIM)

NR

Darkness (shoot induction); 16-h PP; light intensity NR; 25 ◦ C

Adventitious shoots (D)

3.37 shoots/leaf mid-rib

Not performed

Islam et al. (2010)

Adventitious roots (D)

4.29 roots/shoot Plantlet acclimatized in soil, peat, sand (2:1:1) with 85% survival rate Acclimatized in soil and organic humus (1:1). Not quantified. Acclimatization not performed, only in Stancato and da Silveia (2010) in which acclimatization with AMF (Glomus intraradices) performed more poorly than with NPK fertilizer (15:10:10) Not performed

Liu et al. (2010)

1.0 mg/l IBA + 1.0 mg/l BA (RIM) 0.5 mg/l 2,4-D + 0.05 mg/l BA (SIM); 0.5 mg/l BA (somatic embryogenesis); 0.2 mg/l NAA (SIM) 2 mg/l BA + 0.5 mg/l NAA (CIM)

3% sucrose; 0.64% agar

pH 5.8; 12-h PP; 2500 lx; 25 ± 2 ◦ C

Callus; somatic embryogenesis; differentiation; root formation

73.3% seedling formation from somatic embryo

Carbon source NR; 0.2% Gelrite

pH 5.8; PP NR; 50 ␮mol m−2 s−1 ; 25 ◦ C; subculture every 4 months Light intensity, PP and temperature NR

Seedling development; callus proliferation and shoots (I)

Maximum 74% seed germination. Callus formed after 5 weeks in culture and shoots after 4 months. Total soluble sugars and reducing sugar levels decreased in all aeration/light treatments with 0, 15 or 60 mM sucrose; chlorophyll content, shoot and root dry mass and leaf area improved with aeration and special lamps at 60 mM sucrose

0.25% sucrose; 0.5% agar

pH 5.8; 25 ± 1 ◦ C

Callus induction

The frequencies of callus formation were highest from leaves base blades

3% sucrose; 0.02% Gelrite

pH 5.8; darkness for ± 2 months for callus induction; 12-h PP under cool flourescent lamp with 13 ␮mol m−2 s−1 ; 23.5 ± 1.1 ◦ C; 60.6 ± 3.8% RH

Callus induction and adventitious shoots

20% callus regeneration derived from half-anther culture; 76% callus formation; 6–13 and 20 shoots/callus cluster for regeneration and multiplication; 22.5 days for root initiation time with 3.8 roots/shoot; morphological variation in plant size, leaf size, shape and length, spathe and spadix color, shape and size; cytological variation: haploid, diploid, triploid and aneuploid formation

Arizona

Young leaf

MS

Rubrun

Seed from spadices

MS

Eidibel

Plantlets exposed to aeration and lighting with distinct emission spectrum irradiation

½ MS

PGR-free, autotrophic growth by aeration

60 mM sucrose

Dakota

Young leaves blade (top, center and base) Anther, half-anther

¼ MS

0.9 mg/l BA + 0.9 mg/l 2,4-D + 0.5 mg/l Kin (CIM)

WT-1

0.01 mg/l NAA + 0.5 mg/l TDZ + 1.0 mg/l BA (CIM)

NWT-3

0.25 mg/l 2,4-D + 0.02 mg/l NAA + 1.5 mg/l TDZ + 0.75 BA (SIM)

Tropical, Casino, Laguna, Safari

Plantlet growth

64–100% with 83% in average in raw rice husk + bamboo moss and organic manure (1:1:1, v/v/v)

Maira et al. (2010)

Stancato and Tucci (2010)

Tao et al. (2010)

Winarto et al. (2010a,b, 2011b) and Winarto and Teixeira da Silva (2012)

Callus induction and multiple shoot regeneration

ARTICLE IN PRESS

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Table 1 (Continued)

Explant used (type, size, origin)

Basal medium

PGR (type and concentration, mg/l or molar)a

NWT-3

0.2 mg/l NAA + 1.0 mg/l Kin (RIM) MS + 1.0 mg/l BA + 0.1 mg/l 2,4-D (CIM); MS + 1.5 mg/l BA + 0.1 mg/l NAA (SIM); MS + 2.0 mg/l BA + 0.2 mg/l NAA (shoot proliferation); ½ MS + 0.2 mg/l NAA (RIM) 0–4 mg/l BA

MS, ½ MS

Not specified

Axillary buds

Arizona

Petiole

MS, Nitsch and Nitsch (N2) and Schenk and Hildebrand (SH) ½ MS

0.5 mg/l BA + 0.1 mg/l 2,4-D (CIM)

1.0 mg/l BA + 0.1 mg/l 2,4-D (SIM) 0.5 mg/l 2,4-D (RIM) 0.2 mg/l IAA + 1 mg/l BA (CIM); PGR-free (SIM, RIM)

Effect on tissue culture, development, etc. (regeneration type)b

Productivity, somaclonal variation and abnormalities

Acclimatization (% survival)

References

Roots 3% sucrose; 0.5% agar

pH 5.8; 12-h PP; 1500–2000 lx; 25 ± 1 ◦ C

Callus induction; shoot differentiation and proliferation; root formation

The frequencies of callus formation was 80%, shoot differentiation rate was 93%, the shoot d proliferation rate was 7.1, the rooting rate was 96.5%

Plantlet acclimatized in perlite, peat with 95% survival rate

Wu (2010)

3% sucrose; 0.7% agar

16-h PP under cool fluorescent lamps; 13 ␮mol m−2 s−1 ; 24 ± 2 ◦ C 16-h PP; 5000 lx; 25 ◦ C

Shoots

5.7 shoots/explants produced on N2 containing 2 mg/l BA

Not performed

Yuniastuti et al. (2010)

Callus induction

Petioles and callus sensitive (death) to Kan >100 mg/l; in controls, 100% of callus and 89.9% of petioles produced new callus

Not performed

Zhao et al. (2010) (based on Yao et al., 2006)

Shoot induction from 1 cm3 callus pieces Root formation Callus formation; shoot and root regeneration

2.08 shoots/callus clump

Acclimatization in perlite assessed with incomplete information Plantlets transferred ex vitro into pots in cocopeat: perlite (1:1) with 80% survival Not performed

Bakhsi-Khaniki et al. (2011)

3% sucrose; 0.7% agar; AS for callus recovery; 400 mg/l cefotaxime + 100–125 mg/l Kan for elimination of Agrobacterium and selection of transgenic tissue (no Kan in RIM)

Rooting not quantified Not indicated

3% sucrose; 0.8% agar; 0.1% AC

Darkness

1.0 mg/l BA for germination; 0.5 mg/l BA + 1.0 mg/l GA3 for shoot multiplication; 1.0 mg/l IBA for root formation

3% sucrose; 0.2% Gelrite

pH 5.8; PP NR; 50 ␮E m−2 s−1 ; 25 ◦ C

Seed germination, shoot regeneration and root formation

5.9 shoots/explant

Modified MS

2.2 ␮M BA (CIM); PGR-free (RIM)

3% sucrose; 0.28% Gelrite

Callus, shoots, roots

Not quantified

Nitsch/MS

2 mg/l BA (on Nitsch) (CIM) 0.01 mg/l TDZ (on MS) (SIM)

5 mg/l adenine; 3% sucrose

Dark incubation for callus induction; light incubation for shoot multiplication (60 ␮mol m−2 s−1 ); 26 ◦ C Darkness; 25 ◦ C

Callus induction; shoot production (I)

83.43% of cultures were disinfected Pumasillo callused more than Corallis

Not spesified

Leaf explants

MS

Temptation

Seed

MS (seed germination); Nitsch (plantlet growth)

Midori, Marian Seefurth

Laminae, petioles, internodes, nodes, and root sections

Pumasillo, Corallis

Young brown leaf lamina

90%

Chitra et al. (2011)

Fitch et al. (2011)

Kumari et al. (2011)

ARTICLE IN PRESS

Leaf, petiole

Other culture conditionsa

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Not specified

Other medium additivesa

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Table 1 (Continued)

Terra

Leaf (1 × 1 cm )

CanCan

Shoot tip derived from young (30-day-old) greenhouse plants

2

Basal medium

PGR (type and concentration, mg/l or molar)a

MS

3 mg/l BA (CIM)

MS

0.1 mg/l 2,4-D + 1.5 mg/l BA (CIM) PGR-free (SIM) 15 ␮M 2iP

3% sucrose

10 ␮M Kin

100 ␮M AS

5 ␮M IBA (RIM)

500 ␮M AC

MS MS

Other medium additivesa

Other culture conditionsa 16-h PP; 1000 lx; 25 ◦ C pH 5.7; 16-h PP; light intensity NR

0.8% Gelrite; 16-h PP; 60 ␮mol m−2 s−1 ; 25 ◦ C

Alabama, Sierra

Leaf (1 cm2 )

MS (CIM), mMS (SIM, RIM)

1.82 ␮M TDZ (CIM); 0.89 ␮M BA + 2.32 ␮M Kin + 0.98 ␮M IBA (SIM); 0.98 ␮M IBA (RIM)

3% sucrose

Not specified

Adventitious buds

½ MS

1 mg/l NAA + 3 mg/l BA (SIM); 1 mg/l NAA + 2 mg/l IBA (RIM)

15% CW

Tropical Red

Shoots 3 cm long

MS

0.5 mg/l BA

Eidibel

Nodes 1 cm long of leaf-derived in vitro plants

mMS (Pierik, 1976) solid

mMS (Pierik, 1976) liquid at 100 rpm mMS (Pierik, 1976) semi-solid

Effect on tissue culture, development, etc. (regeneration type)b

Productivity, somaclonal variation and abnormalities

Acclimatization (% survival)

References

Callus induction

Not quantified

Not performed

Farsi et al. (2012)

Shoot induction PLB induction (D)

Not quantified 98% of explants induced PLBs 120 PLBs/shoot tip within 50 days

98% after 15 days

Gantait et al. (2012)

PLB proliferation; shoot induction (D) Rooting of shoots

17 shoots/PLB within 30 days

Acclimatization not performed but growth of in vitro plantlets was greater under red + blue LEDs (1:1) Plantlets acclimatized with 84% survival Not performed

Gu et al. (2012)

No variation in acclimatized plants shown by leaf and flower morphology and RAPD analysis (12 primers) 83.3% and 77.8% of Alabama and Sierra leaf explants produced callus while 24.9 and 24.7 adventitious shoots were produced per callus piece

pH 5.8; 0.6% agar; darkness for callus induction; for shoot induction: 12-h PP; 40 ␮mol m−2 s−1 ; 25 ◦ C INA

Callus induction; shoot production (I)

Shoot regeneration and root formation

84.5% bud regenetaion; root established 39.1 days after culture

3% sucrose; 0.1 g/l myo-inositol; 15% CW

0.8% agar vs. 0.46% Phytagel®

20% NaOCl resulted in more shoots (24.65) that the autoclaved control (20.40)

10.0 ␮M NAA

2% sucrose; 0.65% agar

pH 5.8; darkness; 25 ◦ C

Shoot growth in NaOCl-sterilized glassware was superior to growth in autoclaved glassware Somatic embryos (induction)

Developed in 60 days (unquantified)

0.47 ␮M Kin

2% sucrose

Somatic embryos (maturation)

Matured in 45 days (unquantified)

2.32 ␮M Kin

Unspecified

Somatic embryos (germination)

Germinated in 45 days (unquantified)

16-h PP; 36 ␮mol m−2 s−1 ; 25 ◦ C

Maitra et al. (2012)

Peiris et al. (2012)

65%. Rooted Pinto de shoots 1.5 cm Carvalho et al. tall with 2 (2012) leaves planted ® in Plantamax at 28 ◦ C under 50 ␮mol m−2 s−1 .

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Table 1 (Continued)

PGR (type and concentration, mg/l or molar)a

Other medium additivesa

Other culture conditionsa

Effect on tissue culture, development, etc. (regeneration type)b

Productivity, somaclonal variation and abnormalities

Acclimatization (% survival)

References

Casino, Antadra

Leaf (2 cm2 ), petiole (1.5 cm) from 15 to 20-day-old greenhouse plants

MS

3 mg/l BA + 0.5 mg/l NAA (CIM)

3% sucrose; 0.8% agar

16-h PP; 50 ␮mol m−2 s−1 ; 25 ◦ C

Callus

Antadra performed better than Casino

96%

Raad et al. (2012a,b)

Shoots (I)

22.83 shoots/cm2 of callus

Rooting of shoots

11.5 roots/plantlet Not performed

Reddy and Bopaiah (2012)

Not performed

Sedaghati et al. (2012)

Not performed

Zhou et al. (2012)

Not performed

Gao et al. (2013)

Plantlets converted from somatic embryos were acclimatized.

Pinheiro et al. (2013)

Not performed

Wen (2013)

Sucrose and agar NR; 50 ml/l CW

NR

Callus and shoots

0.12 mg/l 2,4-D + 1.0 mg/l BA (CIM) 1.0 mg/l BA (SIM)

3% sucrose; 0.7% agar

Callus

Shoots (I)

Quantification unclear

½ MS

2.0 mg/l BA + 0.2 mg/l 2,4-D (SIM)

3% sucrose; 0.7% agar

Darkness for 4 weeks at 25 ◦ C; 75% RH 16-h PP; light intensity NR; 25 ◦ C; 50% RH pH 5.8; 12-h PP; 1500–2000 lx; 25 ± 2 ◦ C

High number or regenerated shoots (quantification not performed) 14.64% at 1 mg/l BA

Callus

½ MS

1.0 mg/l BA + 0.2 mg/l 2,4-D or 2.0 mg/l BA + 0.1 mg/l 2,4-D (CIM); 0.5 mg/l BA + 0.1 mg/l 2iP + 0.05 mg/l NAA (SIM)

2.5% sucrose

Darkness for 30 days then 12-h PP at 2500 lx; 26 ◦ C

Callus induction, shoot differentiation

Pierik medium was applied for embryo induction; for embryo maturation: Pierik or AA2 with 300 mg/l L-glutamine; PGRs tested: 2,4-D at 0, 4.52 or 9.05 ␮M + Kin 0, 0.47 or 2.32 ␮M MS + 2.0 mg/l BA + 1.0 mg/l NAA + 1.0 mg/l IBA (shoot proliferation); ½ MS + 0.5 mg/l NAA or 1.0 mg/l IBA (RIM)

pH 5.8; 25 ± 2 ◦ C

Maturation of somatic embryos

The tendency of frequencies of callus formation were leaf > petiole > spathe and spadices > lateral bud Suitable CIM was ½ MS + 1.0 mg/l BA + 0.2 mg/l 2,4-D for Fiesta; frequency of callus formation was 55.26%, and on ½ MS + 2.0 mg/l BA + 0.1 mg/l 2,4-D, the frequency of callus formation for Altimo was 63.9% Highest embryo yield was detected and the embryo development was mostly favored on Pierik medium with 0.47 ␮M Kin. Embryo development and maturation were histologically proved.

pH 5.8; 12-h PP; 2000 lx; 25 ± 1 ◦ C

Shoot proliferation; root formation

Not specified

Leaves and spathes

½ MS and Nitsch medium

Midori

Leaf segments (1 cm2 )

mMS

YNG5

Fiesta and Altimo

Leaf, petiole, spathe, spadices, lateral bud Petiole

1 mg/l BA + 0.01 mg/l NAA (SIM) 1 mg/l IBA + 0.2 mg/l Kin (RIM) 2 mg/l BA + 2 mg/l NAA (CIM, SIM)

Eidibel

Nodal segments

Pierik, AA2

Pink

In vitro seedling

MS, ½ MS

3% sucrose; 0.7% agar

Shoot proliferation rate was 5.35%; rooting rate was >95%.

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Table 1 (Continued)

Basal medium

PGR (type and concentration, mg/l or molar)a

Other medium additivesa

Other culture conditionsa

Effect on tissue culture, development, etc. (regeneration type)b

Productivity, somaclonal variation and abnormalities

Acclimatization (% survival)

References

Red One, Red Dark, White Beauty, Snow White

Leaf segments

½ MS (NH4 NO3 was reduced to 250 mg/l)

1.0 mg/l BA + 0.1 mg/l 2,4-D (CIM, SIM)

3% sucrose; 0.6% agar (Algagel, type 900)

pH 5.8; darkness for 60 days, then 16-h PP, 35 ␮mol m−2 s−1 for 30 days; 26 ± 1 ◦ C

Callus, shoot development

93–98% rooting of multiplied shoots and 89–96% acclimatization in Red Dark and White Beauty, respectively

Cardoso and Habermann (2014)

White Beauty

Leaf segments from juvenile (6-month-old) and adult (12month-old) donor plants

Genotype-dependence: No shoots from White Beauty; 8.13, 0.63 and 1.25 shoots/leaf segment in Red Dark Red One and White Snow, respectively; explant position had decisive role: shoot regeneration occurred only if leaf segments were placed with adaxial side onto the medium 1.0 mg/l BA induced shoot development in White Beauty. 2.8 shoots/leaf segment from juvenile leaf but only 0.3 shoots from adult leaf

Dakota, Alabama

Base leaf, leaf, petiole

Modified ¼ MS (CIM); MS (SIM)

Whole leaves, half leaves, petiole, nodal segments, root segment

Pierik

Plantlets with 4–5 leaves transferred ex vitro into pots in peat: perlite (3:1) with 100% survival at 18–25 ◦ C and 90% RH Plantlets conversed from embryos were successfully acclimatized

Li et al. (2014)

Eidibel

Callus induction rates: for Dakota, petiole > leaf base > leaf, for Alabama, leaf base > petiole > leaf. The organ regeneration and somatic embryo regeneration rates of Dakota were 91% and 54%, and of Alabama were 62% and 57% on ¼ MS with 1.0 mg/l BA Somatic embryo development was the highest on Pierik medium supplemented with 10 ␮M NAA and using nodal segments

Four combinations of PGRs: 0.1 mg/l 2,4-D + 1.0 mg/l BA; 1.0 mg/l BA; 0.1 mg/l 2,4-D + 1.0 mg/l Kin; 1.0 mg/l Kin 0–0.5 mg/l Kin + 0.6–1.0 mg/l BA + 0.2–0.9 mg/l 2,4-D (CIM); 0.1–2.0 mg/l BA (SIM)

Testing auxins of IAA, IBA, NAA, 2,4-D and Picloram at 2.5, 5.0, 7.5 and 10 ␮M

Callus, shoot development

2.5% white sugar; 0.7% agar

pH 5.8; 25 ± 2 ◦ C, darkness for callus induction

Callus, shoot development; somatic embryogenesis

2% sucrose; 0.65% agar

pH 5.8; 25 ± 2 ◦ C for 60 days

Somatic embryogenesis

Pinheiro et al. (2014)

2,4-D, 2,4-dichlorophenoxy acetic acid; 2iP, N6 -2(isopentenyl)adenine; AA2, AA2 medium (Abdullah et al., 1986); AC, activated charcoal; AMF, arbuscular mycorrhizal fungi; AS, adenine sulphate; BA, N6 -benzyladenine (also represents BAP or 6-benzylaminopurine if so reported in the literature originally, according to Teixeira da Silva, 2012); CIM, callus induction (and development) medium; ClO2 , chlorine dioxide; cv(s), cultivar(s);CW, coconut water; GA3 , gibberellic acid; H1 medium (Kuehnle and Sugii, 1991); IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; 2iP, N6 -[2 -isopentenyl]adenine; INA, information not available; Kan, kanamycin-sulphate; Kin, kinetin (6-furfurylaminopurine); KM medium, Kao and Michayluk (1975); LED, light-emitting diode; MS, Murashige and Skoog (1962) medium; mMS, modified MS medium; NAA, ␣-naphthaleneacetic acid; NaOCl, sodium hypochlorite; NH4 NO3 , ammonium nitrate; NOA, ␣-naphthoxyacetic acid; NR, not reported; NWT, new Winarto–Teixeira No. 3 medium (Winarto and Teixeira da Silva, 2012); PBA, 6-(benzylamino)-9-(2-tetrahydropyranyl)9H-purine; PGR, plant growth regulator; PLB, protocorm-like body; PP, photoperiod; RAPD, random amplified polymorphic DNA; RH, relative humidity; RIM, root induction (and development) medium; SIM, shoot induction (and development) medium; TDZ, thidiazuron (N-phenyl-N -(1,2,3-thidiazol-5-yl)urea); TIBA, 2,3,5-triiodobenzoic acid; TIS, temporary immersion system; WPM, Woody Plant medium (Lloyd and McCown, 1980); WT, Winarto–Teixeira No. 1 medium (Winarto and Teixeira da Silva, 2012). a Concentration units reported as the original units in the literature; % values indicate w/v, except where otherwise indicated. The original light intensity reported in each study has been represented since the conversion of lux to ␮mol m−2 s−1 is different for different illuminations (main ones represented): for fluorescent lamps, 1 ␮mol m−2 s−1 = 80 lx; the sun, 1 ␮mol m−2 s−1 = 55.6 lx; high voltage sodium lamp, 1 ␮mol m−2 s−1 = 71.4 lx (Thimijan and Heins, 1983). b Regeneration type is either direct (D) or indirect, i.e., via callus (I). c The four strains were: Xanthomonas campestris pv. dieffenbachiae, X. c. pv. dieffenbachiae, Pseudomonas fluorescens, Erwinia herbicola.

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Table 1 (Continued)

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2.1. The basal medium In vitro cultured tissues and organs of plants are grown and developed on artificial media. The composition of basal media containing both inorganic and organic components strongly affects the growth and development of plant parts in vitro and therefore determines the success of plant tissue culture (George and de Klerk, 2008). Murashige and Skoog (MS, 1962) is the most commonly used basal medium in the in vitro propagation of Anthurium, but the use of other media was also reported, including modified MS medium, Nitsch (1969), Geier, Kunisaki, VW, Pierik (modified Pierik), and Finnie and van Staden, H1 (Wang et al., 1997) (Tables 1 and 2). The high-nutrient MS medium was originally developed for tobacco (Nicotiana tabacum) callus but an optimized medium for Anthurium anther culture, including callus induction, was developed by Winarto and Teixeira da Silva (2012), termed Winarto–Teixeira (WT) medium. Even though the original MS medium or modified MS has served for the tissue culture of several explants (including apical shoots, leaves, petioles, spadices, young inflorescences and roots), WT is essential for anther or half-anther culture. Depending on the PGR used and cultivar, WT or new WT medium (NWT) could support callus induction from anthers. WT-1 and NWT-3 were better than at least 10 other basal media for the culture of anthers of A. andraeanum cv. ‘Carnaval’ (Winarto and Mattjik, 2009a) and local Indonesian Anthurium cultivars (Winarto and Mattjik, 2009b). In those studies, WT-1 containing 0.5 mg/l thidiazuron (TDZ) and 0.01 mg/l ␣-naphthaleneacetic acid (NAA) was best for callus induction while WT-1 supplemented with 1.5 mg/l TDZ and 0.02 mg/l NAA produced the highest number of shoots. In the anther culture of A. andraeanum cultivars ‘Casino’, ‘Laguna’ and ‘Safari’, NWT-3 (but not WT-1) supplemented with 0.25 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D), 0.02 mg/l NAA, 1.5 mg/l TDZ and 0.75 mg/l N6 -benzyladenine (BA) was also successfully applied to induce callus and regenerate shoots (Winarto, 2009, 2014). Lan et al. (2003a) reported that N6, KC and 1/2 MS were suitable media for callus induction from petioles while Pierik, N6 and 1/2 MS were suitable for callus induction from leaves. Duan et al. (2009) reported that the most suitable medium for bud induction of three A. andraeanum cultivars ‘Mississippi’, ‘Abason’ and ‘Alabama’ was 1/2 MS with 2.0 mg/l BA and 0.1 mg/l NAA, performing better than MS or 1/4 MS medium with the same PGRs. Cui et al. (2007) reported a gradient in callus formation frequency: 1/2 MS > MS > N6 > B5 > White’s medium. Xia et al. (2005) indicated that callus formation frequency was higher (76.7%) on modified MS with a low concentration of NH4 NO3 (205 or 410 mg/l) than on MS medium with 1650 mg/l NH4 NO3 . Cai (2002) reported that the frequency of callus formation was highest (89%) on modified Nitsch (200 mg/l NH4 NO3 ) containing 1.0 mg/l BA and 0.1 mg/l 2,4D, while Nitsch (720 mg/l NH4 NO3 ) containing 0.5 mg/l BA was suitable for callus differentiation and shoot formation, and Nitsch (720 mg/l NH4 NO3 ) was appropriate for rooting. 2.2. Explant choice and its sterilization for the in vitro environment One of the critical steps of tissue culture is the establishment of in vitro culture. Two main problems are associated with this stage (Stage I), the right choice of explants and the sterilization procedure. Correct choice of explant allows differentiation and growth in a defined set of culture media and conditions. Surface sterilization should ensure that explants placed into the in vitro environment are free of any microbiological contamination (Te-chato et al., 2002; George, 2008; George and Debergh, 2008). There are different possible sources of explants in Anthurium: spadices, spathes or seeds (Wang et al., 1998; Vargas et al., 2004;

Alves dos Santos et al., 2005; Jahan et al., 2009; Maira et al., 2010; Winarto et al., 2011a; Ancy et al., 2012), leaves (Eapen and Rao, 1985; Geier, 1986; Teng, 1997; Joseph et al., 2003; Lan et al., 2003b; Martin et al., 2003; Puchooa, 2005; Nhut et al., 2006; Viégas et al., 2007; Bejoy et al., 2008; Atak and C¸elik, 2009; Seah, 2009; Islam et al., 2010; Kumari et al., 2011; Reddy et al., 2011; Farsi et al., 2012), petioles and pedicels (Eapen and Rao, 1985; Raad et al., 2012a), and shoot tips (Gantait et al., 2008, 2012; Liendo and Mogollón, 2009). Different methods were applied to surface sterilize explants, but these depended on the explant type and Anthurium species. For seeds, spadices or spathes, 70% ethanol was used most often for 2 min and combined with 1–3% calcium or sodium hypochlorite [Ca(OCl)2 , NaOCl] for 10 min (Vargas et al., 2004; Alves dos Santos et al., 2005). NaOCl (1%) or Clorox (5%, v/v) were used also alone for 20–30 min (Wang et al., 1998; Maira et al., 2010). NaOCl (1–5% w/v) for 12–30 min alone (Teng, 1997; Farsi et al., 2012) or in combination with 70% ethanol for a few seconds (Puchooa, 2005; Viégas et al., 2007; Jahan et al., 2009; Atak and C¸elik, 2009; Seah, 2009; Kumari et al., 2011; Raad et al., 2012a) were applied in general after washing explants in tap water and using some detergent, like some drops of Tween-20, for surface sterilization of leaves. Mercuric chloride (HgCl2 , 0.1%) solution for 10–15 min combined with dipping into hydrogen peroxide for 30 s was also a successful surface sterilization method (Ancy et al., 2012). Application of HgCl2 (0.1%) for 8–10 min alone (Joseph et al., 2003; Martin et al., 2003; Reddy et al., 2011) or combined with ethanol (70%) for a few seconds (Eapen and Rao, 1985; Islam et al., 2010) were suitable, as well. In some cases, antibiotics, fungicides or pesticides were used for the surface sterilization of spadices and spathes (Alves dos Santos et al., 2005; Winarto et al., 2011a), leaves (Lan et al., 2003b; Puchooa, 2005; Atak and C¸elik, 2009; Kumari et al., 2011), or shoot tips (Liendo and Mogollón, 2009). 2.3. The explant and its interaction with the in vitro milieu The competence and capacity of different plant tissues and organs for regeneration differ. Therefore, the type, size, age, position and orientation of explant used in plant tissue culture and in the multiplication stage (Stage II) of micropropagation are all of great importance (George, 2008; Raad et al., 2012b; Pinheiro et al., 2013; Cardoso and Habermann, 2014; Pinheiro et al., 2014). Explants also have different demands for in vitro conditions, like PGRs, nutrient composition of the medium, temperature or light (Gahan and George, 2008; Pinheiro et al., 2013; Cardoso and Habermann, 2014). Many studies showed that the Anthurium genotype had a significant effect on callus induction (Kuehnle et al., 1992; Lan et al., 2003b; Xia et al., 2005; Jiang et al., 2006; Yao et al., 2006; Jia et al., 2007; Yang et al., 2008; Duan et al., 2009; Liu et al., 2009; Li et al., 2014). Jiang et al. (2006) also found that the level of callus induction and explant browning differed significantly among different Anthurium varieties, the latter showing a significant negative correlation. Therefore, callus formation can be promoted by controlling explant browning. The frequency of callus formation can differ significantly among different explants. Lan et al. (2003a, 2003b), Li et al. (2014) and Yuan et al. (2004) reported that the callus induction capacity of stem tissue was best while that of petioles was better than leaves. Lan et al. (2003a) reported that petioles showed significantly better callus induction and bud differentiation than leaves. Liu and Xu (1992) found a higher frequency of callus formation from petiole sections near the stem rather than near the leaf apex, and the frequency of callus formation from petiole sections, leaf slices without or with a midrib was 25, 27 and 30%, respectively when cultured on 1/2 MS containing 4 mg/l BA and 1 mg/l 2,4-D. Tao et al. (2010) also

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Basal medium

PGR (type and concentration, mg/l or molar)a

Other medium additivesa

Other culture conditionsa

Effect on tissue culture, development, etc. (regeneration type)b

Productivity, somaclonal variation and abnormalities

Acclimatization (% survival)

References

A. bakeri

Seed, leaf explants

Nitsch

0.1 mg/l BA + 0.1 mg/l 2,4-D (CIM)

INA

INA

Not assessed

Not performed

Kumar (1993)

A. bicolor

Seed

MS

2 mg/l BA + 0.5 mg/l NAA

5% CW

Not performed

Hypocotyl from seeds

MS and ½ MS

MS + 1.0 mg/l BA + 1.0 mg/l 2,4-D (CIM); MS + 1.0 mg/l BA + 0.1 mg/l NAA (SIM); ½ MS + 0.1 mg/l NAA (RIM)

Shoot multiplication rate 5–6 times; 90% adventitious buds with roots in rooting medium

2–3 cm plantlets tramsplanted in sawdust: perlite (1:1) with 100% survival

Ancy et al. (2012) Chen (2003)

A. cubense

Seed

MS

0.1–0.88 ␮M BA, 2.32 or 4.64 ␮M Kin, 2.27–9.08 ␮M 2,4-D (1999), or 4.7 ␮M pectinomorph (2000)

1.5% or 3% white sugar, 0.75% carrageenan powder 2% sucrose; 2% Gelrite

90 days required for seed to germinate Callus formation; shoot and root regeneration

Not assessed

A. clarinervium

Dark incubation for callus initiation 1599–2000 lx; 24 ◦ C pH 5.8; 10-h PP; 1500 lx; 25 ± 2 ◦ C

Plantlets with 3–4 leaves transferred to soil: organic compost (1:1) with 90% acclimatization (1999)

Montes et al. (1999, 2000)

A. cultorum

Shoot tips (0.5 cm)

½ MS

0.125–2.0 mg/l of BA, Kin, Zea, 2iP

100% callus formation; 19 or more shoots/explant within 60 days on 0.44 ␮M BA, 4.64 ␮M Kin and 2.27 ␮M 2,4-D (1999), or pectinomorph (2000) Callus formed in response to BA, Kin, Zea and 2iP, but not quantified; maximum of 3.4 shoots/explant with 0.5–1.0 mg/l Kin

Not performed

Soczek and Hempel (1989)

Seed and axillary buds

MS

Red Hookeri

Shoots

Kurnianingsih et al. (2009) Sari (2010)

A. parvispathum

A. patulum

A. hookerii INA

16-h PP; 1500–2000 lx; 27 ◦ C

Callus and shoot formation (I)

2% sucrose; agar NR

16-h PP; 4.3 W m−2 ; 24–26 ◦ C

Callus, shoots (I)

0.2 mg/l BA (SIM, shoot multiplication)

3% sucrose; 0.7% agar

Shoot formation and multiplication

8.9 shoots/explant

Not performed

MS

0.5 mg/l NAA (SIM, RIM)

Shoot and root

2.9 shoots/explant; 6.78 roots/shoot

Not performed

Seed

MS

2 mg/l BA + 0.2 mg/l NAA (shoot multiplication); 0.25 mg/l IBA (RIM)

50 mg/l casein hydrolysate NR

11-h PP; 10 ␮mol m−2 s−1 ; 21 ◦ C; 50–60% RH pH 5.6–5.8; 1000–2000 lx; 20–25 ◦ C NR

Shoot multiplication; root induction

4-fold increase in number of shoots; 3.6 roots/shoot

Leaf, spathe (1.5 cm2 ), petiole, pedicel (2 cm) from 3-year-old plants

½ MS + Lin and Staba (1961) vitamins

0.1 mg/l 2,4-D + 1 mg/l BA (CIM); 1 mg/l NAA (RIM)

Callus in 6 weeks (83.3% on leaf with mid-vein; 80.4% on leaves without mid-vein; 41.6% on pedicels; 55.9% on spathes; 34.2% on petioles); after 3 sub-cultures on callus-induction medium, shoots formed (I) after 6 months (max. 7.4 shoots/leaf explant)

Shoot production possible from callus after 2 years; no somaclonal variation reported.

Acclimatization performed successfully in media containing peat, bark and soil. 98% plantlet survival 75%

6% sucrose

pH 5.8; darkness; 25 ◦ C

Atta-Alla et al. (1998)

Eapen and Rao (1985)

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Table 2 Summary of in vitro studies of other Anthurium spp. (listed alphabetically and then, within each species, in chronological order).

PGR (type and concentration, mg/l or molar)a

Other medium additivesa

Other culture conditionsa

Effect on tissue culture, development, etc. (regeneration type)b

Productivity, somaclonal variation and abnormalities

Acclimatization (% survival)

References

A. podophyllum

Young leaves and apical bud

MS, ½ MS

MS + 2.0–4.0 mg/l BA + 0.2–1.0 mg/l NAA + rifampicin (CIM); MS + 3.0 mg/l BA + 1.0 mg/l ZT + 0.01 mg/l NAA + 0.5 mg/l GA3 (SIM); MS + 1.0 mg/l BA + 1.0 mg/l Kin (shoot proliferation); ½ MS + 0.2 mg/l NAA (RIM)

3% sucrose; 0.75% carrageenan

12-h PP; 1000–2000 lx; 25 ± 2 ◦ C

Callus formation; shoot and root regeneration

Shoot multiplication rate 5 times; 100% adventitious buds with roots in rooting medium

3–5 cm plantlets tramsplanted in peat: perlite: coco coir (3:1:1) with 96–100% survival

Liao et al. (2005)

A. powmanii Croat Not specified

Seed

MS, ½ MS, Hyponex

13.32 ␮M BA + 0.45 ␮M 2,4-D (SIM); 6.66 ␮M BA + 0.45 ␮M 2,4-D (shoot multiplication)

3% sucrose; 0.7% agar

pH 5.9

Shoots and roots

Not performed

Catrina et al. (2008)

Not specified

Seed

MS

1.0 mg/l Kin + 1.0 mg/l NAA (SIM, RIM)

3% sucrose; 0.7% agar

pH 5.8

Shoot and root

Shoot initiation occurred 6.8 days after culture, with 100% regeneration; 7.5 shoots/explant; 13.9 roots/shoot 3.8 shoots/explant; 3.0 roots/shoot

Not performed

Not specified

Node explants

MS, ½ MS, Hyponex

13.32 ␮M BA + 0.45 ␮M 2,4-D (CIM, SIM)

3% sucrose; 0.7% agar

pH 5.8

Callus, shoot, root

100% explants regenerated callus; number of shoot per explant not assessed; 13.9 roots/shoot

Not performed

Hernanto et al. (2008) SirongoRingo (2009)

Leaf, callus

mMS with ½ MS macro; liquid and solid

1.0 mg/l PBA + 0.1 mg/l 2,4-D (CIM); CIM + 0.1 mg/ml 2,4-D (callus proliferation)

3% glucose (CIM); 2% sucrose for callus proliferation 3% glucose; 100 mg/l myo-inositol; 0.7% Difco Bacto agar in solid medium NH4 NO3 (200 mg/l) for callus and at 720 mg/l for shoot formation; 2% sucrose

Darkenss; 25 ◦ C

Callus

Callus and shoot formation not quantified, but taking place after 3 months in 3 of 4 genotypes. Root formation after 8 weeks.

Not performed

Pierik and Steegmans (1975, 1976)

Light; 23 ◦ C

Shoots (I) and root formation

Continuous dark culture for callus; 14-h PP, 40–50 ␮E m−2 s−1 for shoot induction; 25 ◦ C

Callus and shoot induction (I); low level of NH4 NO3 (200 mg/l) improved the regeneration (callus and shoots) of all genotypes; high level of NH4 NO3 (720 mg/l) accelerated foot formation; regeneration strongly genotype-dependent, ranging from 0 to 100% callus formation and from 0 to 96% shoot formation; 100% of R-4, F-0 and F-92 formed callus 96% of AS-10c and R-83 formed shoots

10–30% losses due to contamination; depending on genotype, callus formation could take from 2 weeks (AS-10c) up to 6 months; young leaves significantly more leaves (7.6/explant) than old leaves (1.4/explant) for AS-1; glucose at 2% decreased shoot numbers/explant; only two genotypes formed >10 shoots/explant: AS-10c and F-45I; TIBA reduced shoot formation; 0.5 g/l NOA or 1 mg/l Kin were just slightly less effective than BA in callus and shoot formation

Success of rooting depended on initial callus-induction and shoot-induction medium

Geier (1986)

A. scherzerianum Schott 4 genotypes (not specified)

0.1 mg/l IAA

18 cultivarsc

Leaf segments (10–14 mm2 ) young and old) of seed-derived plants of from spathe-derived (Geier, 1982) micropropagated plants; zygotic embryos

Nitsch

1 mg/l BA + 0.1 mg/l 2,4-D (CIM); 0.5 mg/l BA (SIM); no PGRs (RIM)

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Table 2 (Continued)

Basal medium

PGR (type and concentration, mg/l or molar)a

Other medium additivesa

Other culture conditionsa

Effect on tissue culture, development, etc. (regeneration type)b

Productivity, somaclonal variation and abnormalities

Acclimatization (% survival)

References

Unspecified var.

Leaf segments with vein (1 cm2 ) and petioles (6 mm thick) Shoot and young stem

VW

1 mg/l 2,4-D + 1 mg/l BA

NR

Darkness

Callus (D)

30% of leaves formed callus after 3 months; petioles were unresponsive

Not performed

Ravindra (1992)

MS, ½ MS

3% sucrose; 1% agar

5–7 multiplication coefficient

98% plantlet survival on peat with 90% RH at 25–30 ◦ C

Haorentaben and Yu (1995)

Leaf

MS

pH 5.8–6.0; 10–12 h PP; 1500–2000 lx; 25 ± 3 ◦ C 16-h PP; 50 ␮mol m−2 s−1 ; 25 ◦ C

Callus formation; shoot and root regeneration

Unspecified red var.

MS + 1–2 mg/l BA + 0.1–0.5 mg/l NAA (CIM, SIM); ½ MS + 0.5 mg/l NAA (RIM) 0.45 ␮M 2,4-D + 0.44 ␮M BA

Somatic embryo induction and germination

73% of explants formed somatic embryos

Transfer to peat with 98–100 RH

Hamidah et al. (1995, 1997a,b)

Unspecified var.

Shoot

MS, ½ MS

MS + 5.0 mg/l BA + 1.0 mg/l IBA (CIM); MS + 1.0 mg/l BA + 0.1 mg/l IBA (callus proliferation); ½ MS + 0.5–1.0 mg/l IBA (RIM)

pH 5.8; 16-h PP; 1500–2000 lx; 25 ± 3 ◦ C

Callus formation; shoot and root regeneration

100% shoots had roots

Li (1997)

Unspecified var.

Shoot and stems

MS

1.0 mg/l BA + 0.5 mg/l IBA (CIM, SIM); 1.5–2.0 mg/l BA + 0.5 mg/l IBA (shoot proliferation); PGR-free ½ MS (RIM)

3% sucrose; 6.0 g/l agar

pH 6.1; 14–16 h PP; 1000–2000 lx; 26 ± 2 ◦ C

Callus and plantlets

10.8 shoots/explant for primary culture, 15.3 shoots for subculture

A. warocqueanum

Leaves with petiole

MS, mMS

mMS + 2.0 mg/l BA + 1.0 mg/l Kin (CIM, SIM); ½ MS + 1.0 mg/l IBA (RIM)

INA

12–14-h PP; 1000–2000 lx; 26 ± 4 ◦ C

Callus formation; shoot and root regeneration

4.9 shoots/explant; 3–5 roots/shoot

AS-1

Leaf

Modified Nitsch

Nitsch (200 mg/l NH4 NO3 ) + 1.0 mg/l BA + 0.1 mg/l 2,4-D (CIM); Nitsch (720 mg/l NH4 NO3 ) + 0.5 mg/l BA (SIM); Nitsch (720 mg/l NH4 NO3 ) (RIM)

6.5 g/l agar

pH 5.8; darkness for callus induction; 14-h PP; 3000 lx for shoot formation; 25 ± 1 ◦ C

Callus induction; shoot differentiation and proliferation, root formation

89% explants formed callus; 15.4 shoots/primary culture; 34.1 shoots/callus

Acclimatization performed successfully in media containing peat, vermiculite, river sand, 94% plantlet survival Acclimatization performed successfully in media containing river sand, vermiculite (2:1). 90% plantlet survival 2–3 cm plantlets tramsplanted in peat, perlite sand, ot their combination with 98% survival after 15 d transplanting Transfer to saw dust and sand (3:1) with 85% RH at 25–30 ◦ C, the regenerated plants flowering after 18 months transplanting

Unspecified var.

2% sucrose; 2.5 mM NH4 NO3 ; 0.35% Bacto agar + 0.35% BDH agar 3% sucrose (CIM, SIM); 2% sucrose (RIM)

Zhao et al. (1999)

Huang et al. (2001)

Cai (2002)

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Table 2 (Continued)

PGR (type and concentration, mg/l or molar)a

Other medium additivesa

Other culture conditionsa

Effect on tissue culture, development, etc. (regeneration type)b

Productivity, somaclonal variation and abnormalities

Acclimatization (% survival)

References

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Protoplasts from somatic embryos and leaves

No PGRs

0.5% cellulase, 0.3% macerase and 0.5% driselase for optimum protoplast isolation; 4.26 g/1 MES, 0.6 M mannitol in alginate bead

16-h PP; 40 ␮mol m−2 s−1 ; 23 ◦ C

Somatic embryos

Maximum of 1 × 105 protoplasts/g embryo; intergeneric protoplast fusion was possible with Spathiphyllum wallisii ‘Alain’ but only until colony stage

Not possible; not performed

Duquenne et al. (2007)

Unspecified var.

Young and old leaves; spathe

KM medium in 1.2% low meltingpoint agarose beads cultured in liquid KM medium; stock plants maintained on MS MS

2 mg/l BA + 0.5 mg/l 2,4-D or 2 mg/l BA + 2 mg/l NAA (CIM) 2 mg/l BA + 0.5 mg/l 2,4-D (SIM)

5% CW

Dark culture for 25 days then 1500–2000 lx; 24 ◦ C

Callus and plantlets (I)

Not performed

Reddy and Bopaiah (2012)

A. digitatum unspecified var.

Leaves (different stages of development)

2 mg/l BA + 0.5 mg/l 2,4-D (CIM); 2 mg/l BA (SIM); 2 mg/l NAA (RIM)

5% CW (CIM, SIM); 2 g/l AC (RIM)

Dark culture for 25 days then 1500–2000 lx; 24 ◦ C

Callus, shoots, rooted plantlets (I) Shoot production not quantified

Callus induction possible from young leaves and spathe, but not from old leaves; callus induction possible from MS, ½ MS and Nitsch Abnormalities claimed but not quantified

Not performed

Reddy et al. (2011)

½ MS

2,4-D, 2,4-dichlorophenoxy acetic acid; 2iP, N6 -[2 -isopentenyl]adenine; AC, activated charcoal; AMF, arbuscular mycorrhizal fungi; AS, adenine sulphate; BA, N6 -benzyladenine (also represents BAP or 6-benzylaminopurine if so reported in the literature originally, according to Teixeira da Silva, 2012); CIM, callus induction (and development) medium; ClO2 , chlorine dioxide; CW, coconut water; GA3 , gibberellic acid; H1 medium (Kuehnle and Sugii, 1991); IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; INA, information not available; Kin, kinetin (6-furfurylaminopurine); KM medium, Kao and Michayluk (1975); MS, Murashige and Skoog (1962) medium; mMS, modified MS medium; NAA, ␣-naphthaleneacetic acid; NH4 NO3 , ammonium nitrate; NOA, ␣-naphthoxyacetic acid; NR, not reported; NWT, new Winarto–Teixeira No. 3 medium (Winarto and Teixeira da Silva, 2012); PBA, 6-(benzylamino)9-(2-tetrahydropyranyl)9H-purine; PGR, plant growth regulator; PLB, protocorm-like body; PP, photoperiod; RAPD, random amplified polymorphic DNA; RH, relative humidity; RIM, root induction (and development) medium; SIM, shoot induction (and development) medium; TDZ, thidiazuron (N-phenyl-N -(1,2,3-thidiazol-5-yl)urea); TIBA, 2,3,5-triiodobenzoic acid; TIS, temporary immersion system; WPM, Woody Plant medium (Lloyd and McCown, 1980); VW, Vacin and Went medium (1949); WT, Winarto–Teixeira No. 1 medium (Winarto and Teixeira da Silva, 2012); ZT, zeatin. a Concentration units reported as the original units in the literature; % values indicate w/v, except where otherwise indicated. The original light intensity reported in each study has been represented since the conversion of lux to ␮mol m−2 s−1 is different for different illuminations (main ones represented): for fluorescent lamps, 1 ␮mol m−2 s−1 = 80 lx; the sun, 1 ␮mol m−2 s−1 = 55.6 lx; high voltage sodium lamp, 1 ␮mol m−2 s−1 = 71.4 lx (Thimijan and Heins, 1983). b Regeneration type is either direct (D) or indirect, i.e., via callus (I). c 18 cultivars were: AS-1, AS-1 (4x), AS-2, AS-10c, R-1, R-2, R-3, R-4, R-83, F-0, F-16I, F-26I, F-31I, F-45I, F-63I, F-63Ia, F-74I, F-92 in which AS-1, AS-2, R-1 and R-2 were spadix-derived clones, AS-1 (4x) was a tetraploid plant, AS-10c was a zygotic embryo-derived clone and the remainder were individual seed-derived plants.

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found that the frequency of callus formation was highest from leaf blades at the base of the stem (i.e., older leaves) than from the center or the top. Cui et al. (2007) noted a trend in callus formation: young leaves > middle-aged leaves > old leaves and leaves with a margin > leaves without a margin. Ge et al. (2008) noted that callus did not form from young spadices and that aerial roots were more suitable than leaves or petioles as explants. Zhou et al. (2012) found a ranking in callus formation ability: leaves > petioles > spathes or spadices > lateral buds. However, Jiang et al. (2006) reported that the most suitable medium for callus induction varied for different varieties, or for different explants of the same variety. For example, the most suitable medium for callus induction from the leaves of A. andraeanum ‘Sweet Dream’ or ‘Toscine’ was modified Nitsch (200 mg/l NH4 NO3 ) with 1.0 mg/l BA and 0.1 mg/l 2,4-D, or modified Nitsch (200 mg/l NH4 NO3 ) with 0.5 mg/l BA, 1.5 mg/l 2,4-D and 1.5 mg/l NAA for ‘Pink Champion’. In addition, the callus induction frequency of ‘Pink Champion’ leaves was significantly higher than from petioles. Therefore, the optimal medium for the rapid propagation of Anthurium spp. is dependent upon the variety and explant used. Most frequently, differences in the choice of PGR and medium additives used have determined the success of the organogenic outcome in Anthurium spp. in vitro studies. A wide range of PGRs, including BA, 2,4-D, TDZ, zeatin (ZT), kinetin (Kin), NAA, indole3-butyric acid (IBA), indole-3-acetic acid (IAA) and others, have been used, either alone or in combination, for the in vitro propagation of Anthurium. The most suitable concentration of PGRs or their combinations differed for different varieties, explants or stages of culture, primarily including callus induction, shoot differentiation and proliferation, root formation and other organogenic processes (Tables 1 and 2). Pan et al. (2000) and Cui et al. (2007) reported that single PGRs such as BA, Kin and 2,4-D were unable to induce callus and that a combination of auxin and cytokinin(s) was necessary for callus induction from leaf tissue. Huang et al. (2001) found that callus induction of A. warocqueanum leaf explants improved as BA concentration increased from 0.5 to 2.0 mg/l. Yuan et al. (2004) reported that callus induction of A. andraeanum leaf explants improved as BA concentration increased from 0.2 to 1.0 mg/l. However, Yang et al. (2008) reported that the most effective BA concentration was 0.5 mg/l for callus induction of three A. andraeanum varieties. Zhang et al. (2001) and Liu et al. (2009) reported that callus differentiation and shoot formation of A. andraeanum needed a low BA concentration (0.5 or 0.8 mg/l). Jiang et al. (2006) reported that the ratio of auxin (2,4-D) and cytokinin (BA) was more important than the individual influence of each for callus induction of A. andraeanum ‘Pink Champion’, the combination of 0.1–0.2 mg/l 2,4-D and 0.5 mg/l BA being most suitable. Zhao et al. (1999) reported the improved proliferation of A. scherzerianum as BA concentration increased from 1.0 to 2.5 mg/l, and when Kin or ZT concentration ranged from 0.5 to 10 mg/l. Wu (2010) reported the improved proliferation of A. andraeanum when BA concentration was 1.0–3.0 mg/l, but 2.0 mg/l was most suitable. Prochloraz, an imidazole fungicide, strongly stimulated A. andraeanum shoot production, but only in the presence of BA (Werbrouck and Deberg, 1996). Carbohydrate serves as the energy source in a plant tissue culture medium and, as an osmoticum, affects Anthurium tissue culture. Cen et al. (1993) and Xia et al. (2005) reported glucose to be better for callus induction of A. andraeanum than sucrose, but the result of Yao et al. (2006) indicated the exact opposite. Kuehnle et al. (1992) reported that a combination of 2% sucrose with 1% glucose in the medium favored embryogenesis more than 3% sucrose alone. The effect of light on Anthurium callus induction has resulted in mixed results. Lan et al. (2003a) reported that continuous light or a 10 h photoperiod improved callus formation and bud

25

differentiation, respectively, more than the treatment with no light. Lan et al. (2003b) reported that light could promote callus induction but Kuehnle et al. (1992) and Xia et al. (2005) reported that darkness was conducive to callus formation. Xia et al. (2005) reported higher frequencies of callus formation in the dark (76.7% for ‘Arizona’, 87.6% for ‘Pink Champion’) than under light at 2000 lx (12.2% for ‘Arizona’, 18.8% for ‘Pink Champion’). Jiang et al. (2006) found that callus induction in the dark was significantly higher than in the light, being significantly inhibited at 2000 lx. Chen et al. (2013) reported that light-emitting diodes (LEDs) promoted plantlet growth and improved their quality, with the combination of 50% red light and 50% blue light being the most appropriate. The pH of the basal medium affects the organogenic outcome of A. andraeanum (Martin et al., 2003). The pH range of Anthurium in vitro was 5.3–6.1, but pH 5.8 was most commonly reported (Tables 1 and 2). A pH 6.0 of half-strength MS containing 1.0 mg/l BA and 0.08 mg/l 2,4-D induced callus from leaf segments of A. andreanum cv. ‘Tropical’, ‘Choco’, ‘Pistache’, ‘Carnaval’, ‘Neon’, ‘Sonate’, ‘Midori’, ‘Safari’, ‘Arizona’ and ‘Cancan’ (Nhut et al., 2006). Callus formation was also reported at a lower pH of 5.5 for A. andreanum cv. ‘Red Hot’ (Seah, 2009). A pH 5.8 was successfully applied for callus and shoots derived from young leaves of A. andreanum cv. ‘Osaki’, ‘Nitta’ and ‘Anouchka’ (Puchooa and Sookun, 2003; Puchooa, 2005), ‘Arizona’, ‘Pink Champion’, ‘Valentino’, ‘Ardour’ and ‘Cancan’ (Xia et al., 2005), XP, ARZ, ATL, KLT and AYZ (Yao et al., 2006), and ‘Alabama’ and ‘Sierra’ (Gu et al., 2012). pH was also an important factor for the successful stimulation of callus and shoot regeneration in anther culture of ‘Tropical’, ‘Carnaval’, ‘Laguna’, ‘Casino’, ‘Safari’ and Indonesian anthurium local accessions (Winarto and Mattjik, 2009a,b; Winarto et al., 2010b, 2011b; Winarto and Teixeira da Silva, 2012). Plantlet formation derived from callus has shown varied responses, as reported in several studies. Vargas et al. (2004) noted that callus, which could form at the base of explants (8-week-old plantlets derived from seed), could form plantlets within 6 weeks, but for callus derived from leaf segments of ‘Agnihothri’, 10 weeks were needed for plantlet formation (Bejoy et al., 2008), 15 weeks for ‘Flamingo’ (Viégas et al., 2007), 24 weeks for ‘Valentino’ (Yu et al., 2009), and one year for ‘Local Pink’ (Maitra et al., 2012). Callus derived from anther culture of ‘Tropical’, Carnaval’, ‘Casino’ and ‘Laguna’ produced plantlets approximately 9 months after culture (Winarto and Mattjik, 2009a; Winarto et al., 2011a,b; Winarto and Teixeira da Silva, 2012; Winarto, 2014), but even longer periods, as much as 11 months, for local Indonesian Anthurium accessions (Winarto and Mattjik, 2009b) or even more than 1.5 years for ‘Safari’ (Winarto, 2014). 2.4. Anther culture Anther culture is a supplementary in vitro technique for breeding work in many plant species (Jain et al., 1996). Anthers cultured in vitro develop into plantlets either by direct microspore embryogenesis or by organogenesis, providing valuable breeding material, such as haploid plants, or homozygous diploid lines. From these plantlets, double haploids, new hybrids and varieties can be produced (Murovec and Bohanec, 2012). To develop and establish an anther or half-anther culture system for A. andraeanum, ‘Tropical’ serves as an appropriate plant model. Half-anthers were the most responsive and suitable explant and responsive anthers were isolated from the transition area of the spadix with 50% of the stigmas being receptive (Rachmawati, 2007; Winarto and Rachmawati, 2007). The best culture media for callus induction, formation and regeneration were either WT medium supplemented with 0.5 mg/l TDZ, 1.0 mg/l BA and 0.01 mg/l NAA or NTW medium containing 1.5 mg/l TDZ, 0.75 mg/l BA and 0.02 mg/l NAA (Winarto, 2009;

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Fig. 1. Shoot development from anther culture of Anthurium andraeanum cv. ‘Carnaval’. Induced callus (A), initial shoot formation (B) and regenerated shoots (C) form in about 4, 5 and 6.5 months after culture, respectively.

Winarto et al., 2009; Winarto, 2010; Winarto et al., 2010a,b,c,d, 2011a,b). Histological studies showed that anther wall cells produced callus following culture (Winarto et al., 2010a). When WT medium was modified (1) by increasing sucrose concentration from 30 to 60 g/l (Winarto et al., 2009), (2) by applying 0.5 mg/l 2,4-D and 2.0 mg/l TDZ for callus induction or using NWT supplemented with 1.0 mg/l 2,4-D and 0.5 mg/l TDZ for shoot regeneration (Winarto et al., 2010b), (3) by adding 250 mg/l glutamine (Winarto, 2011a), or (4) by supplementing 700 mg/l ammonium nitrate in half-strength WT medium for callus induction or 205 mg/l amonium nitrate in half-strength NWT medium for callus regeneration (Winarto, 2013), the induction of callus formation can be increased by 55–70% and regeneration of shoots is successful (3.2–5.3 shoots/callus cluster). Further improvement in callus induction was achieved when half-anthers were placed abaxial side down on medium (Winarto and Teixeira da Silva, 2012). Modifying the PGR content of WT medium (0.5 mg/l 2,4-D, 1.0 mg/l TDZ, 0.5 mg/l BA and 0.02 mg/l NAA) further increased the shoot number (as many as 6 shoots/callus cluster) (Winarto, 2010). Shoots rooted well on PGR-free WT medium (Winarto, 2009, 2010) and NWT medium with 2.0 g/l Gelrite (Rachmawati, 2007; Winarto and Teixeira da Silva, 2012) or with 0.2 mg/l NAA and 1.0 mg/l Kin (Winarto et al., 2011a,b). Based on the results of ‘Tropical’ anther culture, anther cultures for A. andraeanum ‘Carnaval’ (Winarto and Mattjik, 2009a; Fig. 1), ‘Casino’, ‘Laguna’ and ‘Safari’ (Winarto, 2009, 2014), and a local Indonesian accession of anthurium (Winarto and Mattjik, 2009b), were successfully developed. Under fluorescent lamps (TL-Philips, The Netherlands) at a photosynthetic photon flux density of 13.5 ␮mol m−2 s−1 in a 12-h photoperiod, 23.5 ± 1.1 ◦ C, and 60.6 ± 3.8% relative humidity, it takes about 4 months to establish callus (Fig. 1A), about 5 months from initial culture to form initial shoots (Fig. 1B) and about 6.5 months from initial culture to form 1.5 cm-long shoots (Fig. 1C). 3. Acclimatization Acclimatization is an equally important stage of the tissue culture process as it guarantees the survival of quality plantlets in a state that would influence the quality of leaves and flowers. Despite this, most studies on Anthurium tissue culture (Tables 1 and 2) did not conduct an acclimatization stage of the experiment. Haploid Anthurium plantlets tend to die easily during acclimatization with as much as 90% mortality (Winarto et al., 2009). Thus, the acclimatization step is one of the most important aspects of the tissue culture protocol. Geier (1986) claimed that a peat + sand mixture (ratio undefined) resulted in no losses of plants. Finnie and van Staden (1986) claimed to use peat compost with low light intensity (40 ␮mol m−2 s−1 ) but the success of acclimatization was not quantified. Vargas et al. (2004) used a 1:1 mixture of soil and

organic humus under high humidity and low light intensity (the latter two undefined). Chen et al. (1997) found UH1060 plants to acclimatize well to a two-step process: first transfer to community pots containing tree-fern fiber, then into a 3:1 mix of composted peat: #3 Perlite. Kuehnle et al. (1992) had earlier used tree-fern fiber to acclimatize in vitro propagated plants. Gantait et al. (2012) acclimatized plants that had been derived from shoot-tip-induced protocorm-like bodies (PLBs) (a possible misnomer) by first washing individual plantlets in water, then by dipping the basal section in a 0.2% solution of Bavistin® DF, a fungicide, for 2 min. Roots were trimmed and plantlets were transferred into earthen pots containing a mixture of autoclaved sand and soil (2:1, v/v) and covered with plastic lids and watered intermittently. Partially acclimatized plantlets with 3–4 leaves were transferred to larger pots containing a mixture of charcoal and coconut fiber (1:1, v/v) for 4 weeks in a well-ventilated greenhouse under a 12-h photoperiod and a maximum photosynthetic photon flux density of 200 ␮mol m−2 s−1 at 28 ◦ C and 70% relative humidity (RH) for 30 d. Yu et al. (2009), after washing the roots of well-rooted plantlets with tap water, placed plantlets in community pots containing 1:1 of peatmoss and perlite. Plantlets were then planted separately into a soilless substrate (60% Canadian peat, 20% vermiculite, 20% perlite, v/v) supplemented with 4 kg/l of dolomite. Potted plants were directly grown in a shaded greenhouse under 200 ␮mol m−2 s−1 , at 20–28 ◦ C, and 70–100% RH. Harb et al. (2010) used a simple peatmoss: sand (1:2) mixture to acclimatize plants after leaving culture jars open for 3 d and leaving rooted plantlets in vermiculite in the laboratory for 7 d. In vitro-derived plantlet roots were washed in tap water, dipped in 0.1% bavistin solution and transferred to a plastic tray with a polythene cover, containing sterile cocopeat (Kumari et al., 2011). Hardened plantlets that had developed 4–5 leaves and 2–3 well-developed roots were then transplanted to a commercial potting medium (cocopeat:rice husk:sand:farmyard manure; 1:1:1:1). Peat:perlite:sand (1:1:1, v/v) under 4000 lx, at 27 ◦ C, and 70% RH was effective for two A. andraeanum cultivars (Raad et al., 2012a). Viégas et al. (2007) tested 10 different substrates and found that a 1:1 ratio of organic soil and vermiculite resulted in the tallest plants while the highest number of leaves per plant formed in a 1:1:1 ratio of organic soil, vermiculite and sphagnum moss while sawdust, turf, carbonized rice husks, xaxim, and pine cones resulted in plants with poorer performance. In contrast, Yu and Paek (1995) found fine bark to result in better shoot and root formation than a peat:perlite mix (1:1). Atta-Alla et al. (1998) found that 98% of A. parvispathum plantlets derived from in vitro culture could acclimatize in a 1:1:1 mixture of turf, tree bark and soil. Puchooa and Sookun (2003) showed that transfer to vermiculite was sufficient for plantlet survival, provided that light intensity was low. Bejoy et al. (2008) treated rooted shoots with 3 or more leaves with a 3% solution of commercial fungicide (Dithane M 45) for 5 min then

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transferred to earthen community pots containing coarse river sand and charcoal (3:1). Nhut et al. (2006) acclimatized A. andraeanum ‘Tropical’, ‘Choco’ and ‘Pistache’ plantlets (>2 well-developed roots and 2.5–3.0 cm in height) in a shaded nursery with 50–70% of daylight on a tree fern fiber substrate at 22 ◦ C and 80–85% RH. Plantlets were sprayed with water twice a day, fertilizer was added three times a week and 5 g/l of a fungicide solution mixture including Zodiac 80 WP and Manozep 80 WP was applied twice a week. Plantlets derived from A. andreanum ‘Tropical’ half-anther culture showed mixed ploidy, namely haploids, diploids and triploids (Winarto et al., 2011a), could be successfully acclimatized with as high as 100% survival. Treatments started by removing plantlets carefully from culture bottles, washing plantlet roots gently under tap water, immersing in a biocide solution containing 0.5% benomyl (w/v) and 0.2% streptomycin sulphate (w/v) for 1 min, planting in a plastic pot containing burned-rice husk, raw rice husk and organic manure (2:2:1, v/v/v), covering the pot with transparent plastic for 7 d and placing rooted plantlets in a glasshouse under low light intensity (37–74 ␮mol m−2 s−1 ). The method was successfully applied in anthuriums with 82.5% (Winarto et al., 2011b), 100% (Winarto and Teixeira da Silva, 2012) and 77.6% (Winarto, 2013) survival. Using a radical treatment, Teixeira da Silva et al. (2005) showed that aerated micropropagation would allow for the direct acclimatization of in vitro grown plantlets to an ex vitro environment while retained in the in vitro culture vessel, a specialized gas-permeable vessel or when the in vitro substrate, Rockwool® , was transferred directly to a new substrate in pots in the greenhouse with a well developed root system. The anaerobic conditions in vitro caused by root growth in agar can stunt growth and thus decrease survival of ex vitro plantlets in the greenhouse (Keatmetha and Suksa-Ard, 2004). Soilless, hydroponic culture is another effective way to grow anthuriums and reduce the risk of fungal and bacterial contaminants (Dufour and Guérin, 2005), although this study did not examine the use of hydroponics for in vitro-derived plants. In the only study that used arbuscular mycorrhizal fungi (Lima et al., 2006), in vitro-derived plantlets showed better growth and shoot/root indices than all other treatments when a mixture of three fungi were used (Table 1). 4. Current applications of tissue culture and future perspectives An observation of the ultrastructural features in the Kuehnle et al. (1992), Chen et al. (1997) and Gantait et al. (2012) papers indicates that somatic embryos and PLBs (a term used only by the last two papers) are one and the same organ. However, PLBs are generally reserved exclusively for orchids and thus the use of the term PLB might not be appropriate for Anthurium. 4.1. Cytogenetic stability and modification in chromosome number of in vitro regenerants Anthurium half-anther culture results in morphological and cytological variations of plantlets. Morphological variations clearly observed in regenerants derived from anther culture included alterations in plant size, peduncle length, spathe position compared to leaves, the type and number of buds, spathe and spadix color, and spadix length. There were also cytological variations in both in vitro and ex vitro regenerants of anther culture with 23–29% haploids, 5–10% aneuploids, 56–69% diploids, and 3–4% triploids (Winarto et al., 2011a); 33.5% haploid, 62.7% diploid and 5.7% triploid (Winarto et al., 2010d) for an in vitro study; cytological analysis of 180 acclimatized-plantlets ex vitro revealed that 34 were haploid

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(n = 14–18), 15 aneuploid (n = 20–26), 126 diploid (n = 28–34) and 5 triploid (n = 45–57) (Winarto et al., 2011b). Cytological variation was successfully detected using a modified Darnaedi method with mild heating of root tips in 1 N HCl: 45% glacial acetic acid (3:1, v/v) for 10 min and dipping the root tips in 2% aceto-orcein for 15 min (Winarto, 2011b). Counting the number of chloroplasts in a pair of stomatal guard cells is the most convenient and reliable indirect method to determine the ploidy level in regenerants derived from anther culture (r = 0.945; p < 0.01) (Winarto et al., 2010d). These studies confirm the findings in other studies in which the development of anther cultures, especially via callus formation, resulted in morphological and cytological alterations. Thus, for clonal propagation, organogenesis through an indirect route (through callus) is not advised. However, establishment of an anther culture method for producing homozygous lines in Anthurium is an important way to produce novel quality hybrids and seeds (Maluszynski et al., 2003). To address this need in Anthurium tissue culture, and to provide an alternative in vitro regeneration pathway, suitable anther culture systems were developed (Winarto and Mattjik, 2009a, 2009b; Winarto et al., 2010a,b, 2011a,b; Winarto and Teixeira da Silva, 2012). Few studies to date have employed flow cytometry and other molecular techniques to understand in more detail the genome and phylogeny of Anthurium spp., although some advances were made fairly recently (Bliss and Suzuki, 2012), while a recent study highlights the genetic mechanisms for the inheritance of color in the spathe (Gopaulchan et al., 2014). An understanding of genome size can be useful for in vitro studies and breeding since genome size is correlated with seed mass, cell and stomatal size, stomatal density, and length of the cell cycle (e.g., Beaulieu et al., 2008), all aspects that are of interest and importance to the culture of Anthurium in vitro. Seah (2009) conducted intensive studies on the occurrence of endopolyploidy in ‘Red Hot’ using flow cytometry. Only 2C and 4C DNA were detected in leaf lamina, petiole and root tissues of greenhouse-grown plants, 2C DNA accounting for over 90% of the samples. In the spathes, the 2C content reached 95% while 8C DNA was detected in spadix tissue (5.3–11.7%). Leaf petiole, lamina and root tissues of in vitro plants contained at least 85% 2C DNA in the nuclei. Interestingly, only 2C and 4C DNA was detected in any callus tissue, and no endopolyploidy was detected. This is in sharp contrast with hybrid Cymbidium (Teixeira da Silva and Tanaka, 2006) or other ornamentals (Teixeira da Silva et al., 2014), where high levels of endopolyploidy can be found in certain tissues. Geier (1986) noted that theoretically, callus-derived regenerants should show somaclonal variation, but claimed that from hundreds of regenerants observed, no variation had ever been found, although those claims were not supported by any data. Kuehnle and Sugii (1991) claimed that callus of three cultivars (UH965, UH1060 and UH1003) could remain embryogenic and regenerate plantlets even after 12–13 months without somaclonal variation. Gantait et al. (2012) used random amplified polymorphic DNA (RAPD) – a way to differentiate anthurium cultivars and to establish the genetic relationship between them (Ranamukhaarachchi et al., 2001; Pan et al., 2011) – to test for and confirm the genetic stability of regenerants and, in previous studies on the same cultivar (Gantait et al., 2008; Gantait and Sinniah, 2011), also confirmed cytogenetic stability through the use of inter-simple sequence repeat (ISSR) markers. Puchooa (2005) used seven 10-mer RAPD primers, but detected no banding variation between mutation-induced plantlets and control plantlets. However, no detailed assessment of the mutations (e.g., their epigenetic nature) was provided. Polyploid production is a breeding tool for producing new ornamental varieties of greater economic value (Eeckhaut et al., 2006). Polyploids may have different leaf shapes, larger leaves and

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flowers or even different flower colors. Polyploid A. andraeanum could be produced by treatment with colchicine (Zhang et al., 2007; Tian and Ma, 2008; Chen et al., 2011; Tian et al., 2013). Zhang et al. (2007) found that the induction of tetraploids in vitro depended on the treatment method, the concentration of colchicine and the duration of treatment. Induction was higher when low concentrations of colchicine (0.2–1.0 mg/l) were used. At the same concentration of colchicine, the induction rate of tetraploids was higher in liquid culture with longer treatment time than on solid culture or with a shorter treatment period (3–14 min). The highest induction rate of tetraploids (45.5%) was obtained when callus was cultured in modified Nitsch liquid medium supplemented with 0.2 g/l colchicine for 14 d on a rotator. The induction rate was not affected by genotype. A. andraeanum tetraploids could be identified by larger and thicker leaves and spathes, with deeper color and larger pollen, anthers and plants, while the stomatal cells on the abaxial epidermis were longer than those of diploid plants, but the stomatal density was lower than in diploids. Tian and Ma (2008) reported polyploid A. andraeanum ‘Arizona’ produced from a callus clump and young seedlings by treatment with colchicine using three methods: (1) soaking in 0.1, 0.2 or 0.3% colchicine for 3, 5 or 7 h; (2) co-culture on MS medium supplemented with 0.2 mg/l 2,4-D, 1.0 mg/l BA and 0.05, 0.1 or 0.2% colchicine; (3) threading method: placing a cotton wool thread soaked in 0.05, 0.1, 0.2 or 0.3% colchicine solution after sterilization with 70% alcohol into the junction between the root and stem for 7 or 9 d. The optimum condition for the induction of polyploidy was method 3, i.e., the use of adventitious roots that had been soaked in 0.3% colchicine for 7 h, resulting in highest polyploid induction of 63.3%. The characteristic tetraploids were similar to those obtained by Zhang et al. (2007). Chen et al. (2011) reported the highest level of tetraploids (7.6%) of A. andraeanum ‘Arizona’ obtained after a 5 h treatment with 0.3% colchicine. However, Tian et al. (2013) reported a higher induction rate of tetraploidy (17.5%) when A. andraeanum ‘Alabama’ callus was cultured in 0.2 g/l colchicine in MS liquid medium for 15 d under constant rotation using the method of Tian and Ma (2008).

4.4. Genetic transformation Genetic transformation can be a tool for introducing new agronomic traits into Anthurium species, hybrids and cultivars. Studies conducted on the introduction of new traits by genetic transformation into Anthurium are still limited and based mainly on Agrobacterium-mediated transformation methods (Fitch et al., 2005, 2006; Khaithong et al., 2006, 2007; Zhou et al., 2008; Zhao et al., 2010; Fitch et al., 2011). In most studies, the embryogenic callus of A. andreanum, A. limdeniarum and A. kamemotoanum cultivars was used as the target tissue for the genetic transformation (Kuehnle and Chen, 1994; Chen and Kuehnle, 1996; Chen et al., 1997; Kuehnle et al., 2001; Fitch et al., 2005, 2011, 2006; Khaithong et al., 2006, 2007; Zhao et al., 2010; Hosein et al., 2012). Some agronomically important genes were introduced which may potentially result in pest and disease resistance but practical results for Anthurium are still limited. Cystatin, NPRI, attacin, T4 lysozyme, cowpea trypsin inhibitor genes were introduced by Fitch et al. (2011) to A. andraeanum ‘Midori’ and ‘Marian Seefurth’ hybrids for resistance to bacterial blight and nematodes, and even though transgene insertions were confirmed by PCR and Southern hybridization, no pest-resistance assays were performed. After insect resistance genes (API-A, API-B; arrowhead proteinase inhibitor) were introduced to A. andraeanum ‘Pink Champion’, nine kanamycin-resistant plantlets (29%) were obtained, seven of which were positive for transgenes as assayed by PCR, but no insect-resistance assays were performed (Zhang et al., 2009). Two transgenic lines of ‘Paradise Pink’ selected by Kuehnle et al. (2004a,b) showed the production of the cecropin-like Shival lytic peptide and tolerance to Anthurium blight caused by Xanthomonas campestris pv. dieffenbachiae (in fact, it is Xanthomonas axonopolis pv. diffenbachiae). Future development of transformation protocols and application of genetic transformation would allow for the manipulation of flower color, induction of in vitro flowering and extension of vase life. 4.5. Cryopreservation

4.2. In vitro selection of pest resistance Aragaki et al. (1984) claimed that burrowing nematode (Radopholus similes (Cobb) Thorne) causes an estimated 50% loss in flowering yield of Anthurium spp., although heat treatment (hot water at 50 ◦ C for 12 min or hot air at 50 ◦ C, 60% RH for 35 min) may eliminate this nematode and bacterial blight from cut flowers (Tsang et al., 2010). An in vitro assay was used by Wang et al. (1997, 1998) to select Anthurium species or A. andraeanum cultivars that were resistant or tolerant to burrowing nematode.

4.3. Protoplasts and somatic hybridization Only two studies on protoplast isolation and regeneration exist for Anthurium. Kuehnle (1997) obtained 3.8 × 105 protoplasts/g fresh weight of leaves from A. andraeanum. Preliminary intergeneric hybridization between Spathiphyllum wallisii ‘Alain’ and A. scherzerianum ‘238’ was possible by protoplast fusion, but development until the colony stage was possible – only when protoplasts were held within alginate beads – but no plantlets were obtained (Duquenne et al., 2007). Protoplast fusion is an important in vitro breeding technique to produce somatic hybrids and “may be a starting point for the complete regeneration of somatic intergeneric hybrids in the Araceae family, and the establishment of a model system for asexual asymmetric fusion in monocot ornamental crops.” (p. 172, Duquenne et al., 2007).

Cryopreservation is a safe and cost-efficient technique for long term germplasm conservation. Although cryopreservation is routinely employed for conservation of many economically important plants, there are only a limited number of experiments carried out in Anthurium (Wang et al., 2010; Hanan and Sherif, 2013). Wang et al. (2010) reported a vitrification cryopreservation protocol for A. andraeanum embryonic suspension cells as follows: an embryonic suspension cell mass (2 mm in diameter) subcultured for 3–5 d was pre-cultured in 1/2 MS liquid medium containing 0.5 M sorbitol for 2 d, then treated for 24 h at 4 ◦ C, and pre-treated with 25% (protectant vitrification solution 2) PVS2 (Sakai et al., 1990) for 15 min at room temperature, dehydrated with 100% PVS2 for 10 min at 0 ◦ C. Finally, the cell mass was rapidly immersed into liquid nitrogen for cryopreservation. Cryopreserved cells were thawed in a 40 ◦ C water bath for 3 min, then washed with 1/2 MS liquid medium containing 1.2 M sucrose three times (each time for 10 min). Hanan and Sherif (2013) also reported conservation experiments with A. andraeanum plantlets that consisted of three treatments for chemical conservation and four treatments (excluding the control), for cryopreservation. In the former method, micronodes (3–4 cm long shoots with 4–5 well developed leaves) were cultured on MS medium (modified PVS2) containing different concentrations of sorbitol (0.5, 1.0 and 1.5 g/l) for 8 months. In the latter method, micronodes were treated with different protection solutions for 10 min in two treatments; while cultured on MS medium containing different protection solutions for 7 d in the other two treatments. The explants were immersed in liquid

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nitrogen for 12 d after the independent treatments then rapidly thawed in a water bath at 40 ◦ C. 4.6. Synthetic seed technology Synthetic seed (synseed) technology allows for the conservation and storage of rare, endangered and desirable genotypes while providing easy handling and transportation (Sharma et al., 2013). However, there are only a few experiments carried out with Anthurium (Deng et al., 1992; Ni et al., 1994; Yang et al., 1995; Nhut et al., 2004). Deng et al. (1992) used compact callus fragments induced from leaf explants of A. andraeanum. Fragments were precultured on MS medium containing 0.1 mg/l NAA to induce adventitious roots after 15 d. Plantlets were encapsulated with 3% sodium alginate and 1% calcium chloride (CaCl2 ·2H2 O), then cultured on MS medium for one month then re-encapsulated. Synseeds were sown directly into non-sterile soil, and 63% of plantlets survived (vs. 0% for non-encapsulated material and 26% for material encapsulated once). Ni et al. (1994) reported that 56% of synseeds formed plantlets if they were derived from adventitious shoots pretreated with 0.1 mg/l NAA before encapsulation. Shoots that could be easily dissected and encapsulated into synseeds formed plantlets on 1/2 MS medium. If the synseeds from adventitious shoots pretreated with a combination of a high concentration of auxin (1.0 mg/l NAA) and a low concentration of cytokinin (0.1 mg/l BA) before encapsulation, the shoots were not easily dissected and encapsulated, and only 11.3% of synseeds formed plantlets. Yang et al. (1995) pretreated A. andraeanum adventitious shoots obtained from leaf-induced callus with a low concentration of auxin before encapsulation into artificial seeds. The survival rate was very low (2.8–3.1%) when synseeds were sown directly into nonsterile soil. However, when synseeds were encapsulated with an outer coating of polymers, or if activated charcoal was added to the artificial endosperm, the survival rate increased to 26.7% and 42.5%, respectively, but when combing both treatments, the survival rate in nonsterile soil reached 71.4%. Nhut et al. (2004) encapsulated the leaf-derived embryogenic callus of ‘Tropical’ that had been derived in the presence of 1.0 mg/l BA and 0.08 mg/l 2,4D on 1/2 MS medium. Following the Nhut et al. (2005) synseed protocol for Cymbidium, as much as 81.6% of synseeds germinated. Acknowledgements The authors thank Femke Hoogervorst, researcher at Anthura Research B.V., The Netherlands, for critical comments on the review and for providing a copy of some older literature. The authors also thank the four anonymous reviewers for constructive suggestions on improving the manuscript. References Abdullah, R., Cocking, E.C., Thompson, J.A., 1986. Efficient plant regeneration from rice protoplasts through somatic embryogenesis. Nat. Biotechnol. 4, 1087–1090. Alves dos Santos, M.R., Timbó, A.L.de O., Pinto, O., de Carvalho, A.C.P., Morais, J.P.S., 2005. Callus induction and plant regeneration from Anthurium andraeanum Lindl. fruits. Plant Cell Cult. Micropropag. (Lavras) 1 (2), 77–79. Ancy, D., Bopaiah, A.K., Reddy, J.M., 2012. In vitro seed culture studies in Anthurium bicolor (Agnihothri). Int. J. Integr. Sci. Innov. Technol. 1 (4), 16–20. Aragaki, M., Apt, W.J., Kunimoto, R.K., Ko, W.H., Uchida, J.Y., 1984. Nature and control of anthurium decline. Plant Dis. Rep. 8, 509–511. Atak, C¸., C¸elik, Ö., 2009. Micropropagation of Anthurium andraeanum from leaf explants. Pak. J. Bot. 41 (3), 1155–1161. Atak, C¸., C¸elik, Ö., 2012. Micropropagation of Anthurium spp. In: Nabin Kumar Dhal, N.K., Sahu, S.C. (Eds.), Plant Science. InTech, Croatia, pp. 241–254. Atta-Alla, H., McAlister, B., van Staden, J., 1998. In vitro culture and establishment of Anthurium parvispathum. S. Afr. J. Bot. 64, 296–298. Avila-Rostant, O., Lennon, A.M., Umaharan, P., 2010. Spathe color variation in Anthurium andraeanum Hort. and its relationship to vacuolar pH. HortScience 45 (12), 1–5.

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