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The role of activated charcoal in plant tissue


culture

Article in Biotechnology advances · November 2008


DOI: 10.1016/j.biotechadv.2008.08.003

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Biotechnology Advances 26 (2008) 618–631

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

Research review paper

The role of activated charcoal in plant tissue culture


T. Dennis Thomas ⁎
Research Department of Botany, St. Thomas College, Pala, Arunapuram (P.O), PIN-686574, Kottayam (Dt), Kerala, India

a r t i c l e i n f o a b s t r a c t

Article history: Activated charcoal has a very fine network of pores with large inner surface area on which many substances
Received 4 February 2008 can be adsorbed. Activated charcoal is often used in tissue culture to improve cell growth and development. It
Received in revised form 1 August 2008 plays a critical role in micropropagation, orchid seed germination, somatic embryogenesis, anther culture,
Accepted 1 August 2008
synthetic seed production, protoplast culture, rooting, stem elongation, bulb formation etc. The promotary
Available online 22 August 2008
effects of AC on morphogenesis may be mainly due to its irreversible adsorption of inhibitory compounds in
Keywords:
the culture medium and substancially decreasing the toxic metabolites, phenolic exudation and brown
Activated charcoal exudate accumulation. In addition to this activated charcoal is involved in a number of stimulatory and
Androgenesis inhibitory activities including the release of substances naturally present in AC which promote growth,
In vitro culture alteration and darkening of culture media, and adsorption of vitamins, metal ions and plant growth
Micropropagation regulators, including abscisic acid and gaseous ethylene. The effect of AC on growth regulator uptake is still
Protoplast culture unclear but some workers believe that AC may gradually release certain adsorbed products, such as nutrients
Somatic embryos and growth regulators which become available to plants. This review focuses on the various roles of activated
Synthetic seeds
charcoal in plant tissue culture and the recent developments in this area.
© 2008 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 618
2. Micropropagation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 619
3. Somatic embryogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 622
4. Protoplast culture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 624
5. Anther and microspore culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 624
6. Synthetic seed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 625
7. Rooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 625
8. Orchid tissue culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 626
9. Lower plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 627
10. Concluding remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 628
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 628
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 628

Abbreviations: ABA, Abscisic acid; AC, Activated charcoal; BA, 6-Benzyl aminopur-
ine; BP, Banana pulp; CH, Casein hydrolysate; CW, Coconut water; 2,4-D, 2, 4- 1. Introduction
Dichlorophenoxyacetic acid; GD, Gresshoff and Doy medium; IAA, Indole 3-acetic acid;
IBA, Indole-3-butyric acid; Kn, Kinetin; MES, 2-[N-Morpholino] ethenesulfonic acid; Activated charcoal (AC) is composed of carbon arranged in a quasi-
MS, Murashige and Skoog medium; NAA, 1-Naphthalene acetic acid; NN, Nitsch
graphitic form in small particle size. It is a porous and tasteless material
medium; NOA, 2-Napthoxyacetic acid; PEG, Polyethylene glycol; PG, Phloroglucinol;
PGR, Plant growth regulator; PLB, Protocorm like bodies; PVP, Polyvinyl pyrrolidone; SE, and is distinguished from elementary carbon by removal of all
Somatic embryo; SH, Schenk and Hildebrandt medium; TDZ, thidiazuron-N-phenyl N1 noncarbon impurities and the oxidation of carbon surface (Budavari,
1,2,3-thidiazol-5-yl urea; TE, Tracheary element; WPM, Woody plant medium; WS, 1996; Mattson and Mark, 1971). AC has a very fine network of pores, an
Wolter and Skoog medium. extraordinarily large surface area and volume that gives it a unique
⁎ Corresponding author. Present address: Biozentrum Klein Flottbek, Entwicklungs-
biologie und Biotechnologie, Universität Hamburg, Ohnhorststrasse 18, 22609, Ham-
adsorption capacity (Baker et al., 1992).
burg, Germany. AC is often used in plant tissue culture to improve cell growth
E-mail address: den_thuruthiyil@yahoo.com. and development (Pan and van Staden, 1998). The addition of AC to

0734-9750/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2008.08.003
T.D. Thomas / Biotechnology Advances 26 (2008) 618–631 619

both liquid and semi-solid media is a recognized practice and its Solutes which are in contact with AC in a solution will be adsorbed
influence in growth and development may be attributed mainly to until an equilibrium between adsorbed and desorbed molecule is
the adsorption of inhibitory substances in the culture medium established (adsorption isotherm). Several factors like density, purity of
(Fridborg et al., 1978; Horner et al., 1977; Theander and Nelson, charcoal and pH affect the adsorptive capacity of charcoal. In addition to
1988; Weatherhead et al., 1978, 1979), drastic decrease in the this the inorganic salts like KCl, KI and NaCl affected the adsorption
phenolic oxidation or brown exudate accumulation (Carlberg et al., isotherms of phenol (from a dilute aqueous solution) by activated
1983; Liu, 1993; Teixeria et al., 1994), alteration of medium pH to an charcoal (Halhouli et al.,1995). X-ray diffraction studies on AC confirmed
optimum level for morphogenesis (Owen et al., 1991) and establish- that AC is amorphous in nature and has a micro-crystalline structure
ment of a darkened environment in medium and hence simulate soil which evidently affect its adsorptive capacity (Qadeer et al., 1994).
conditions (Dumas and Monteuuis, 1995). Even though the effect of The difficulty in using AC in medium is that in addition to adsorbing
AC on plant growth regulator (PGR) uptake is still unclear, some unwanted substances, it may adsorb needed hormones (Fridborg et al.,
workers believe that AC may gradually release certain adsorbed 1978; Ebert and Taylor 1990; Ebert et al., 1993; Nissen and Sutter, 1990),
products, such as nutrients and PGRs in addition to the release of vitamins (Weatherhead et al., 1978; Weatherhead et al., 1979; Pan and
substances naturally present in AC which promote plant growth van Staden,1998), or metal ions such as Cu+2 and Zn+2 (Van Winkle et al.,
(Johansson and Eriksson, 1977; Johansson et al., 1990). The growth 2003). Recently large number of reports was published regarding the
inhibitory chemicals, like 5-hydroxymethyl- furfural produced various application of AC in plant tissue culture (Table 1). Majority of the
during autoclaving from sucrose by dehydration, will be removed reports confirmed the positive role of AC in medium promoting growth
by AC (Pan and van Staden, 1998). and development of plant tissues. However, AC induced negative results
The most crucial impact of adding AC to the culture media is a were also reported in some systems. Thus, this review focuses the recent
drastic dip in concentration of PGRs and other organic supplements. developments in plant tissue culture using AC. Various aspects of the
This is due to the adsorption of these chemicals by AC. This makes the effects of AC on in vitro micropropagation, orchid tissue culture, somatic
researchers using AC unware of the actual quantity available to the embryogenesis, synthetic seed production, protoplast culture, anther
plant tissues. Obtaining target levels of adequate free exogenous and microspore culture, rooting of micropropagated shoots and
hormone may be difficult. For example, by using radioactive tracers, it influence in lower plants have been discussed.
was established that in liquid medium 99.5% of the added 100 μM 2, 4-
dichlorophenoxyacetic acid (2, 4-D) was adsorbed by 0.025 g/l AC 2. Micropropagation
within 5 days of medium preparation (Ebert and Taylor, 1990).
However, increased levels of 2, 4-D could be obtained by using semi- During micropropagation the exudation of phenol is very common
solid medium, reducing AC levels and adding higher concentrations of and it often influences the result. In Aristolochia indica leaching of
2, 4-D (Ebert and Taylor, 1990). Similar results were obtained with 6- polyphenols affected the shoot growth in vitro. However, addition of AC
Benzyl aminopurine (BA; Ebert et al., 1993). When 11.3 mg/l BA was in the medium as well as increased subculturing frequency reduced this
added to gelled medium, less than 2% of the BA was available to plant problem considerably (Soniya and Sujitha, 2006). Manjula et al. (1997)
tissue after 3 days. Further, the amount of BA added to the medium noted that 0.05 g/l AC was sufficient to reduce polyphenol exudation
influenced the availability of 2, 4-D. Thus AC in the medium can from A. indica. The polyphenols associated with Sorghum (Sorghum
considerably alter the ratio of medium components and subsequently bicolour) tissue culture resulted in blackening of inoculated tissues
influence plant regeneration (Druart and Wulf, 1993). within a day and this can result in tissue necrosis and, in severe cases,
The influence of AC on culture establishment and plant regenera- explant death also. The phenolic compounds are toxic to Agrobacterium
tion has been reported in several plant species including lower plants. cells used for genetic transformation. Several culture manipulations
The use of AC in plant tissue culture includes an enhanced the have been developed to alleviate the effects of oxidised phenolic
establishment of protoplast culture (Kunitake et al., 1995), prevention compounds like the use of antioxidants and adsorbing agents including
of development of abnormal plantlets (Ziv and Gadasi 1986) AC. Nguyen et al. (2007) tried to eradicate this problem in sorghum by
enhancing somatic embryogenesis, shoot formation, plant recovery using AC in the medium. AC (1–5 g/l) had a positive effect on reducing
and rooting (Buter et al., 1993; Dumas and Monteuuis 1995; Fuchs the black pigments released into the media by immature embryos. The
1991; Mathews et al., 1993; Patel and Thorpe 1986; Sinha and Mallick blackness of medium and explant reduced noticeably after using AC and
1991). Furthermore, the decline in regeneration ability of long-term the explant survival percentage increased after one month of culture.
calli is a common phenomenon in various systems and AC can restore The survival of the immature embryos was clearly identifiable by the
the abilities for somatic embryogenesis and consequently plant white colour of the scutellum or the callus forming scutellum. Survival
regeneration from long-term calli of different species subcultured on was only 29% without AC, while it was 80% in presence of AC. In spite of
various media combination (Zaghmout and Torello, 1988). this, the addition of AC prevented the embryos from producing callus in
Charcoal has the characteristic property of high absorptive power some cases (Nguyen et al., 2007).
for colloidal solids, gases and vapors. It is manufactured by the In Dipterocarpus alatus and D. intricatus embryo culture, the
distillation of wood and other carbonaceous materials. The nature and explant browning could be overcome by growing embryos initially
characteristic features of charcoal varies for different purposes. For on a filter paper bridge in liquid medium with AC to absorb the
instance charcoal for absorption of gases are harder and dense than oxidised phenolic compounds. The embryos were soaked in sterile
those employed for liquid purification. The surface of AC contains distilled water for 1–3 h before culture to allow leaching out of
specific areas ranging from 600 to 2000 m2 gl− 1 pore distribution phenolic compounds. The initial incubation was in darkness because
varies from 10 µm to 500 µm (Yam et al., 1990). When added to the light increases the enzyme activity associated with the oxidation of
medium, AC has certain preference for polar rather than apolar phenols leading to browning. Root growth in embryos grown on a
organics. Similarly greater absorptive capacity towards aromatic filter paper bridge in liquid medium with AC was significantly better
products like phenolics and their oxidates, auxins including indole- than all other treatments. Moreover, the number of surviving
3-acetic acid (IAA), naphthaleneacetic acid (NAA), indoel-3-butyric seedlings is greatest with this treatment (Linington, 1991). During
acid (IBA), cytokinins including BA kinetin (Kn), zeatin etc. and other the organogenesis from hypocotyl thin cell layers of Lupinus mutabilis
hormones than olefinic unsaturation products. Moreover highly polar and Lupinus albus, the presence of a piece of sterilised AC paper at the
and readily water soluble products including sugars like glucose, base of Petri dish before the addition of medium resulted in less
sorbitol, mannitol and inositol etc will not be removed from the phenolic compounds compared to the anti-oxidant diethylthio
medium or solution (Yam et al., 1990). carbamate. When L. mutabilis was cultured in a medium containing
620 T.D. Thomas / Biotechnology Advances 26 (2008) 618–631

Table 1
Some recent reports on application of activated charcoal in plant tissue culture

Common Name Botanical name Response Effect References


Cashew Anacardium occidentale Shoot multiplication P&N Boggetti et al. (1999)
Rape Brassica oleracea Microspore embryogenesis P De Silva Dias (1999)
American chestnut Castanea dentata Somatic embryogenesis N Xing et al. (1999)
Eucalyptus Eucalyptus sp. Shoot elongation P Barrueto Cid et al. (1999)
Pine Pinus heldreichii Shoot elongation P Stojicic et al. (1999)
Sycamore maple Acer pseudoplatanus Rooting P Wilhelm (1999)
Avocado Persea Americana Rooting P Barcelo-Munoz et al. (1999)
Canary Island date palm Phoenix canariensis Embryogenic callus N Huong et al. (1999)
Mangostene Garcinia mangostana Root induction P Techato and Lim (1999)
Rubber Hevea brasiliensis Somatic embryogenesis P Blanc et al. (1999)
Lavender Lavandula vera Rooting P Andrade et al. (1999)
Pearl millet Pennisetum glaucum Somatic embryogenesis P Lambe et al. (1999)
Sugarcane Saccharum hybrid Protoplast regeneration P Aftab and Iqbal (1999)
Grapevine Vitis vinifera Seed culture P Park et al. (1999)
Acacia Acacia sinuate Callus regeneration P Vengadesan et al. (2000)
Sugar beet Beta vulgaris Gynogenesis P Gurel et al. (2000)
Citrus Citrus sp. Somatic hybrids rooting P Guo et al. (2000)
Enset Ensete ventricosum Rooting P Negash et al. (2000)
Geodorum Geodorum densiflorum Micropropagation P Sheelavantmath et al. (2000)
Cotton Gossypium hirsutum Somatic embryogenesis P Zhang et al. (2000)
Gymnema Gymnema sylvestre Micropropagation N Komalavalli and Rao (2000)
Lupinus Lupinus sp. Micropropagation P Mulin and Bellio-Spataru (2000)
Phalaenopsis Phalaenopsis hybrid Bioreactor PLBs P Young et al. (2000)
Globe artichoke Cynara scolymus Rooting P Le Saos and Hourmant (2001)
Lilly Lilium longiflorum Shoot induction P Nhut et al. (2001)
Cassava Manihot esculenta Somatic embryogenesis P Taylor et al. (2001)
Minor Millet Paspalum scrobiculatum Somatic embryogenesis P Vikrant and Rashid (2001b)
Taxus Taxus mairei Prevent browning P Chang et al. (2001)
Triticale Triticale Callus regeneration P Vikrant and Rashid (2001a)
Mung bean Vigna radiate Cotyledon regeneration N Tivarekar and Eapen (2001)
Grapevine Vitis sp. Somatic Embryogenesis P Motoike et al. (2001)
Calliandra Calliandra tweedii Somatic embryogenesis P Kumar et al. (2002)
Oil Palm Elaeis guinensis Callus maintenance P Eeuwens et al. (2002)
Strawberry Fragaria x ananassa Root induction P Jemmali et al. (2002)
Bottle palm Hyophorbe lagenicaulis ZE germination P Sarasan et al. (2002)
Hybrid larch Larix hybrid Somatic embryogenesis P Aderkas et al. (2002)
Banana Musa spp. Secondry SE P Khalil et al. (2002)
Ginseng Panax ginseng Somatic embryogenesis P Kevers et al. (2002)
Minor Millet Paspalum scrobiculatum Growth of shoot, SE P Vikrant and Rasheed (2002)
Saw palmetto Serenoa repens Somatic emrbyogenesis P Gallo-Meagher and Green (2002)
Potato Solanum phureja Androgenesis P Boluarte-Medina and Veilleux (2002)
Camphor tree Cinnamomum camphora Rooting P Nirmal Babu et al. (2003)
Saffron Crocus sativus Differentiation P&N Zeng et al. (2003)
Coconut Cocos nucifera Callus regeneration and SE P Fernando et al. (2003)
Dendrobium Dendrobium nobile Seed germination P Men et al. (2003)
Lilly Lilium spp. Bulblet induction P Bacchetta et al. (2003)
Banana Musa acuminata Rooting P Jalil et al. (2003)
Nerine Nerine sarniensis Bulblet P Vishnevetsky et al. (2003)
Date palm Phoenix dactylifera Somatic embryogenesis P Fki et al. (2003)
Horse chestnut Aesculus hippocastanum Rooting P Zdravkovic-Korac et al. (2004)
Hot pepper Capsicum annuum Rooting P Anu et al. (2004)
Yam Dioscorea alata Shoot induction P Borges et al. (2004)
Enset Ensete ventricosum Phenol adsorption P Birmeta and Welander (2004)
Hepatica Hepatica nobilis Androgenesis P Nomizu et al. (2004)
Ocotea Ocotea catharinensis Somatic embryogenesis P Catarina et al. (2004)
Ophrys Ophrys sp Seed germination P Kitsaki et al. (2004)
Pine Pinus pinea Shoot elongation P Sul and Korban (2004)
Japanese pear Pyrus pyrifolia Androgenic embryos N Kadota and Niimi (2004)
Rosa Rosa hybrida Somatic embryogenesis P Kim et al. (2004)
Salix Salix caprea Micropropagation P Liesebach and Naujoks (2004)
Indian mustard Brassica juncea Microspore culture N Pream et al. (2005)
European chestnut Castanea dentata Somaticembryogenesis P Andrade and Merkle (2005)
Hinoki cypress Chamaecyparis obtusa Somatic embryogenesis P Taniguchi et al. (2005)
Cymbedium Cymbedium faberi Rooting and transplantation P Chen et al. (2005)
Cotton Gossypium sp. Protoplast callus regeneration P Sun et al. (2005)
Hagenia Hagenia abyssinica Rooting P Feyissa et al. (2005)
Zedoary Curcuma zedoaria Rooting P Loc et al. (2005)
Rice Oryza sativa Synthetic seed P Arun Kumar et al. (2005)
Date palm Phoenix dactylifera Somatic embryogenesis P Zouine et al. (2005)
Norway spruce Picea abies Somatic embryogenesis Pullman et al. (2005)
Cork oak Quercus suber Antherculture P Pintos et al. (2005)
Raspberry Rubus idaeus Prevent Browning P Wang et al. (2005)
Coast redwood Sequoia sempervirens Shoot elongation P Sul and Korban (2005)
Pineapple Ananas comosus Rooting P Firoozabady et al. (2006)
Anemone Anemone coronaria Androgenesis P Laura et al. (2006)
T.D. Thomas / Biotechnology Advances 26 (2008) 618–631 621

Table 1 (continued)
Common Name Botanical name Response Effect References
Hot pepper Capsicum annuum Microspore embryogenesis P Supena et al. (2006)
Lady’s slipper orchid Cypripedium flavum Rooting P Yan et al. (2006)
Macadamia Macadamia tetraphylla Rooting P Mulwa and Bhalla (2006)
Pine Pinus radiate TE differentaiation P Moller et al. (2006)
Thapsia Thapsia garganica Rooting P Makunga et al. (2006)
Taxus Taxus wallichiana Shoot elongation P Datta et al. (2006)
Plumbago Plumbago zeylanica Prevent callus browning N Wei et al. (2006)
Peach palm Bactris gasipaes Somatic embryogenesis P Steinmacher et al. (2007a,b)
Coconut Cocos nucifera Callus induction P Perera et al. (2007)
Dendrobium Dendrobium sp In vitro flowering P Sim et al. (2007)
Yam Dioscorea nipponica Microtuber induction P Chen et al. (2007)
Disa Disa spp Seed germination P Thompson et al. (2007)
Exacum Exacum sp. Elongation and rooting P Unda et al. (2007)
Cotton Gossypium sp. Somatic hybrids P Sun et al. (2007)
Mango tree Mangifera indica Somatic embryogenesis P Wu et al. (2007)
Mulberry Morus alba Rooting P Agarwal and Kanwar (2007)
Brazilian grape tree Myrciaria aureana Somatic embryogenesis N Motoike et al. (2007)
Phalaenopsis Phalaenopsis sp. Prevent necrosis P Shrestha et al. (2007)
Pine Pinus sylvestris Somatic embryogenesis P Lelu-Walter et al., (2008)
Burmuda grass Cynodon dactylon Callus regeneration P Zhang et al. (2007)
Litchi Litchi chinensis Somatic embryogenesis P Raharjo and Litz (2007)
Banana Musa acuminata Rooting P Xiao et al. (2007)
Mesquite tree Prosopis laevigata Rooting N Buendia-Gonzalez et al. (2007)
Sorghum Sorghum bicolor Callus induction P&N Nguyen et al. (2007)
Grapevine Vitis spp. Somatic embryogenesis P Gambino et al. (2007)
Ginger Zingiber officinale Prevent browning P Guo et al. (2007)
Rape Brassica napus Embryo germination P Shi et al. (2008)
Soybean Glycine max Embryo differentiation P Klink et al. (2008)
Grapevine Vitis vinifera. Embryo germination P Fan et al. (2008)
Salvia Salvia africana-lutea Rooting P Makunga and Van Staden (2008)

TE—Tracheary element.
P—Positive effect.
N—Negative effect.

IAA and BA in presence of sterilised AC paper, abundant shoots were acclimatization (Mohamed-Yasseen, 1993, 1995). Emergence of bulb-
formed which was not observed in diethylthio carbamate containing lets was observed in Nerine sarniensis from bulblet halves on MS
medium (Mulin and Bellio-Spataru, 2000). medium supplemented with IBA and 0.025 g/l AC (Vishnevetsky et al.,
Explant browning was a serious problem in shoot tip culture for 2003). In Muscari armeniacum bulb scale explants had two ways of
cryopreservation experiments in raspberry (Rubus idaeus). To reduce development when cultured on MS basal medium or MS + AC (1 g/l). In
this problem modified MS medium (Murashige and Skoog, 1962) was the former case explant produced yellowish calli, which gave rise to
fortified with AC, ascorbic acid, citric acid or polyvinylpyrrolidone (PVP). shoots and roots after 12–14 weeks whereas in the latter case (with
Of these various additives employed, 0.25 g/l AC supplemented medium AC) produced bulblets at the basal plate of the original bulb scale (Peck
gave rise to least browning (10%) and 90% of the explants survived on and Cumming, 1986). Similarly, AC promoted bulblet production was
this medium (Wang et al., 2005). Phenol exudation from the explant is a reported in Narcissus tazetta (Steinitz and Yahel, 1982).
major constraint in enset (Ensete ventricosum) tissue culture. Birmeta In cotton (Gossypium hirsutum) AC in medium enhanced the shoot
and Welander (2004) overcame this problem by adding 0.05–0.1 g/l AC and root induction as well as shoot length from split embryo axes as
together with dark incubation of the cultures without retarding the compared to MS basal medium. The synergistic effect of AC with
growth of explants. A monospecific rosaceae multipurpose tree Hagenia increase in incubation temperature to 30 °C increased the shoot and
abyssinica was micropropagated through terminal and lateral shoots on root formation (Hazra et al., 2002). In Vitis vinifera aneuploid plant
woody plant medium (WPM, Lloyd and Mc Cown, 1980) supplemented production by immature seed culture, AC had been employed.
with BA, indole-3-butyric acid (IBA) and 0.1 g/l AC. AC added to medium Immature seeds were cultured on Nitsch medium (NN medium,
to avoid media browning (Feyissa et al., 2005). Nitsch and Nitsch, 1969) supplemented with glutamine, serine,
In several plants the induction of bulblets is highly affected by AC cysteine, casein hydrolysate (CH) and 0.02 g/l AC and subcultured
in medium. AC not only increases the response but also the bulblet after one month on the same medium without AC for further growth
size. In Lilium sp. bulblet produced on MS medium were separated and (Emershad and Ramming, 1984; Emershad et al., 1989; Park et al.,
subcultured on MS medium supplemented with or without 0.04 g/l AC 1999). For in vitro propagation of Salix caprea, WPM supplemented
and NAA (1-naphthalene acetic acid). A significantly high percentage with 0.01 g/l AC without any PGRs was found to be the best (Liesebach
of large bulblets (b6 mm in size) were obtained when AC incorporated and Naujoks, 2004).
in media. The positive effect of AC was also evident in the medium, For black wattle (Acacia mearnsii) micropropagation nodal cuttings
which contains AC alone with out any PGRs (Bacchetta et al., 2003). were cultured on modified MS medium supplemented with 2 g/l AC
Nhut et al. (2001) developed shoots from transverse thin cell layers of gave rise to a significantly higher percent of elongated shoots (75%),
young stems of L. longiflorum on 1/2 MS medium with or without lower rate of callus induction (27%) and chlorotic shoots (31%) than
0.001 g/l AC. Maximum 4.2 shoots per section was observed on 1 g/l the media which did not contain AC. Furthermore, 12.5% of shoots
AC containing medium and the absence of AC resulted in no shoot exhibited rooting on this medium, while no rooting was observed on
regeneration. media without AC (Quoirin et al., 2001).
Bulblet was induced in garlic (Allium sativum) on MS medium with In Acacia sinuate maximum callus regeneration (85% with an
5 g/l AC under long-day photoperiod. Maximum 6.8 bulblets were average 12 shoots per culture) was observed on MS medium
produced on this medium and were transferred to soil without supplemented with coconut water (CW), 0.05 g/l AC and different
622 T.D. Thomas / Biotechnology Advances 26 (2008) 618–631

concentrations and combinations of BA and IAA (Vengadesan et al., bean (Vigna radiate) on MS medium supplemented with different
2000). In Triticale sp. the leaf base derived nodular calli were PGRs. However, the addition of 0.5 g/l AC to the medium completely
transferred to either basal medium or basal medium fortified with inhibited the shoot initiation (Tivarekar and Eapen, 2001). The callus
0.5 g/l AC for differentiation (Vikrant and Rashid, 2001a). Similarly in browning was a common phenomenon during regeneration experi-
burmuda grass (Cynodon dactylon) AC was employed to reduce ments in Plumbago zeylanica arising from the production of pigmented
browning of the medium while inducing organogenic callus and of secondary products. The addition of AC in the medium did not have
the three cultivars tried, two responded well to AC where as one any effect on reducing the browning of callus (Wei et al., 2006).
cultivar did not respond (Zhang et al., 2007). TDZ induced multiple Similarly, AC in the medium did not have any effect on regeneration of
shoots or somatic embryos (SEs) were reported in minor millet callus in Typha latifolia (Nandakumar et al., 2005). In saffron (Crocus
(Paspalum scrobiculatum) on N6 medium (Chu et al., 1975) and sativus) AC prevented petal and style explants from browning, but
complete plantlets could be regenerated on basal medium or on inhibited differentiation. Petal explants grew fast on this medium and
basal medium fortified with 0.05 g/l AC (Vikrant and Rasheed, 2002). gradually turned purple (Zeng et al., 2003).
Plantlets were obtained from a garden palm Howea forsteriana when
zygotic embryos stored at 4 °C were cultured on MS basal medium with 3. Somatic embryogenesis
0.03 g/l AC. By using in vitro techniques the time period required to
obtain plantlets could be shortened (Moura and Carneiro, 1992). During AC helps in SE induction, maturation or germination in various
the study of date palm (Phoenix dactylifera) calli derived from shoot tip systems. AC plays a crucial role in SE maturation in a deciduous woody
explants to water stress; AC was used from the culture initiation to liana Schisandra chinensis. SEs at globular and heart shaped stages
proliferation in three different stages (Al-Khayri and Bahrany, 2004). were subcultured and developed within two weeks on AC-impreg-
High rates (98 to 100%) of nodal cutting regeneration were nated filter paper discs containing WV medium (Westvaco medium;
observed in yam (Dioscorea alata) on modified D-571 medium (Borges Duchefa, Haarlem, The Netherlands) without PGRs (Smiskova et al.,
et al., 2001) with mannitol, BA and 2 g/l AC (Borges et al., 2004). 2005). In American chestnut (Castanea dentata) 5 g/l AC is routenly
Microtubers were induced in Dioscorea nipponica on MS medium with used for SE germination. The seedlings on AC containing medium
BA, NAA and 0.01 g/l AC. The growth was better than any other remained white while those without AC darkened in six weeks, also
treatments and average fresh weight of the tuber reached 3.46 g (Chen the seedlings showed a greater tendency to branch than those
et al., 2007). In sugar beet (Beta vulgaris) for inducing gynogenic medium without AC (Andrade and Merkle, 2005).
haploids, a combination of AC (0.05 g/l) and BA on MS medium gave During the transgenic soybean (Glycine max) production by
rise to a maximum (13%) response (Gurel et al., 2000). particle bombardment using SEs, AC (0.03 g/l) is necessary in MS
AC induced shoot maturation and elongation was observed in medium for initial development of embryos only and further
cotton (G. hirsutum) when cultured on MS medium fortified with 3 g/l development of embryos in to cotyledonary stage was obtained on
AC (Hemphill et al., 1998). Direct shoot induction from leaf explants of the same medium with out AC (Li et al., 2004). In Senecio hybrid 3 days
Exacum sp. had been reported by using MS medium supplemented of AC (0.05 g/l) shock in MS medium had been given to the leaf
with BA and NAA and the shoots were subcultured on modified MS explants which was treated with MS medium supplemented with BA
medium supplemented with PVP, 2iP, NAA, GA3 and 0.06 g/l AC to and 2, 4-D for 7–14 d prior to AC treatment for direct somatic
simultaneously promote both elongation and rooting (Unda et al., embryogenesis (Malueg et al., 1994). During the agrobacterium
2007). In Eucalyptus sp. the shoots regenerated from calli were mediated transformation in rosa (Rosa hybrida) embryogenic calli
subcultured to shoot elongation medium consists of MS medium were transferred to MS basal medium supplemented with 0.05 g/l AC,
containing 1 g/l AC (Barrueto Cid et al., 1999). 60 mg/l kanamycin and 125 mg/l clavamox for embryo development
In Oxalis triangularis callus regeneration was significantly influ- in dark. Cotyledonary embryos developed were continuously har-
enced by the addition of AC in the medium regardless of the culture vested from the embryogenic callus growing on the maturation
system and explant origin. Even though the regeneration was delayed medium and subcultured on the same medium to produce shoots
in AC containing medium, it produced more buds and plantlets. Callus (Kim et al., 2004). In another report on R. hybrida somatic
growth was significantly reduced by the addition of AC and the embryogenesis, the maturation and conversion medium for somatic
positive effect was obvious in solid culture medium where a 4.7 fold embryogenesis were supplemented with 0.025 g/l AC (Kamo et al.,
more regenerants were obtained as compared to 1.4 fold in liquid 2004). In oil palm (Elaeis guinensis) embryogenic suspension was
media (Teng and Ngai, 1999). In vitro regeneration in Venus fly trap transferred monthly to modified MS medium supplemented with
(Dionaea muscipula) had been achieved by culturing flower stalk on adenine sulphate, BA, 2, 4-D and 1 g/l AC. Embryogenic development
modified MS medium plus BA, NAA and 2 g/l AC. When the cultures was achieved following the transfer of cell clusters onto a liquid basal
were initiated in AC-free medium, explants turned dark brown and medium supplemented with 0.5 g/l CH (Bertossi et al., 1999). In
died within a month. AC was added to induce adventitious bud another report, the maintenance of embryogenic oil palm
initiation, but omitted in further cultures (Teng, 1999). (E. guinensis) callus was done on semi-solid Reynolds and Murashige
The effect of AC on in vitro nitrogen uptake had been evaluated in (1979) medium supplemented with 2, 4-D, N6-isopentyladenine and
Lagerstroemia indica. Explants grown in medium with AC were capable 3 g/l AC (Eeuwens et al., 2002). Teixeira et al. (1995) established
of taking up both NO3− and NH4+, although NH4+ uptake was low and E. guinensis callus on modified Y3 (Eeuwens 1976, 1978) or MS medium
the pH of the medium was maintained between 5.5 and 6.0. In containing 2, 4-D or picloram and 0.03 g/l AC.
treatments which do not have AC, NH4+ uptake was preferential and During the somatic embryogenesis in date palm (P. dactylifera) AC
the pH dropped to 3.1 (Eymar et al., 2000). had been employed in every stage. For callogenesis and embryogen-
Despite of these promotive roles, the negative effect of AC in esis 0.15 g/l was employed where as for embryo maturation and
micropropagation was also reported in some systems. In Gymnema germination 0.25 g/l were used along with MS medium (Zouine et al.,
sylvestre micropropagation, the addition of AC to overcome phenolic 2005) AC induced somatic embryogenesis was also reported in
exudation in the medium reduced the number of shoots possibly due P. dactylifera by Fki et al. (2003). In this report the presence of 0.3 g/l
to the adsorption of essential factors required for tissue growth AC in liquid medium resulted in the differentiation of large number of
(Komalavalli and Rao, 2000). In cashew (Anacardium occidentale) AC SEs.
suppressed bud sprouting from shoot nodes but shoot elongation was To reduce browning of tissues while inducing in vitro germination
better and vigorous than those without AC (Boggetti et al., 1999). High and induction of direct somatic embryogenesis in an endangered Bottle
frequency shoot regeneration from cotyledon was achieved in mung Palm (Hyophorbe lagenicaulis), Sarasan et al. (2002) cultured zygotic
T.D. Thomas / Biotechnology Advances 26 (2008) 618–631 623

embryos on MS medium supplemented with 2 g/l AC. Browning of phenolic accumulation was observed and the embryos swelled to
tissues followed by slow growth was prevented by this method. In peach approximately four times their original size in 2 weeks. After 5 weeks,
palm (Bactris gasipaes) the development of SEs and rooting was observed all explants began to develop translucent clusters of SEs. Probably AC
on MS medium supplemented with 2, 4-D, 2-iP, glutamine, CH and AC may absorb PGRs present in the medium and hence high concentration
(1.5 g/l). Plantlets were maintained in MS medium with AC until they of PGRs was used for induction (Gallo-Meagher and Green, 2002). In
were 6 cm tall, and then acclimatized (Steinmacher et al., 2007a,b). Litchi (Litchi chinensis) recovery of plants from SEs improved with 1/2
In rubber (Hevea brasiliensis) friable callus was obtained from MS medium supplemented with GA3 and AC (Raharjo and Litz, 2007).
immature seeds were induced to obtain embryogenesis and during In pearl millet (Pennisetum glaucum) maturation of embryogenic
the final stages of embryogenesis SEs measuring about 3 mm were calli occurred on MS basal medium supplemented with 0.05 g/l AC
isolatd and transferred to development medium which consists of followed by germination of SEs on medium supplemented with GA3.
modified Carron and Enjalric (1985) medium without PGRs, but with This protocol could help to restore the regeneration ability in long-
0.5 g/l AC. Once the embryo developed taproot, these embryos were term cultures (Lambe et al., 1999). In vitro somatic embryogenesis
transferred to fresh development medium without AC (Blanc et al., was induced from established internodal and petiolar segments of
1999). Somatic embryogeneis and plant regeneration of a forest tree Calliandra tweedii on MS medium supplemented with 2iP. Within
Ocotea catharinensis carried by Moura-Costa et al. (1993). SE induction 6–7 weeks, globular, heart, torpedo-shaped and dicot embryos
occurred on MS basal medium supplemented with 0.03 g/l AC in dark differentiated directly. Individual embryos were separated from the
where as globular embryos were transferred to a medium with same maternal tissues and converted into plantlets on 1/2 MS medium
formulation but with addition of 2, 4-D in dark. After two months fortified with 0.03 g/l AC (Kumar et al., 2002). In Panax ginseng SEs
globular and cotyledonary stage embryos were transferred to MS were regenerated when the calli were transferred from 1/2 MS
medium with 0.03 g/l AC and 362 μM 2, 4-D. This high concentration of 2, medium supplemented with or without auxins to regeneration
4-D in the medium was given in order to compensate for the adsorption medium which consists of 1/2 MS amended with 0.1 g/l AC for two
properties of the AC used in media (Moura-Costa et al., 1993). In Cocos months (Kevers et al., 2002). During the somatc embryogenesis from
nucifera, callus regeneration and somatic embryogenesis was achieved unfertilized ovary derived callus of coconut (C. nucifera), the
from plumule tissues on Y3 liquid medium (Eeuwens, 1978) supple- concentration of AC played a key role in callus induction and
mented with different PGRs and 0.025 g/l AC (Fernando et al., 2003). optimum response was observed on medium 72 (Karunaratne and
Catarina et al. (2004) noted that the best medium to induce repetitive Periyapperuma, 1989) fortified with 0.01 g/l AC and 2, 4-D (Perera
embryogenesis in cotyledonary somatic embryos of O. catharinensis was et al., 2007). In P. scrobiculatum SEs were germinated and developed
1/2 WPM supplemented with glutamine, 1.5 g/l AC. The mature SEs into plantlets on MS or N6 (Chu et al., 1975) medium supplemented
gradually air dehydrated showed repetitive embryogenesis after sub- with 2, 4-D. However a short duration of one-week incubation of SEs
culture on 1/2 B5 medium supplemented with 1.5 g/l AC, GA3 and NAA. in AC (0.05 g/l) supplemented basal medium, supported high rate of
AC plays very critical role in cotton (G. hirsutum) embryogenesis. embryo germination than transfer to AC-free basal medium (Vikrant
Although AC inhibited the induction of embryogenic callus, it greatly and Rashid, 2001b). In cassava (Manihot esculenta) the regenerated
promoted the formation of mature embryos. Cotton SE proliferation and SEs at cotyledonary stage were further germinated on MS medium
germination occurred on modified MS medium supplemented with 2, 4- supplemented 0.5 g/l AC. The embryos were transferred to a fresh
D and zeatin in combination with or without 2 g/l AC. On MS medium medium containing BA after 7 days (Taylor et al., 2001).
supplemented with 2, 4-D and zeatin, no cotyledonary stage SEs was In mango (Mangifera indica) embryogenic cultures were induced
observed. On medium supplemented with zeatin, only 14.3 and 13.4 from cotyledon cuts or from nucelli on modified B5 medium (Gamborg
embryos were observed per leaf and stem explants respectively. et al., 1968) supplemented with CW, Kn, 2, 4-D, and 2 g/l AC. The
However, with 2 g/l AC, an average of 28.0 and 28.1 matured embryos cultures were transferred to a fresh medium of the same composition
were observed per leaf and stem explants respectively. The addition of after 1 day to avoid the accumulation of phenolic compounds. The
2 g/l AC resulted in a 1.96-fold and 2.10-fold increase in number of maturation of cryopreserved M. indica embryogenic cultures occurred
mature embryos in leaf and stem explants respectively as compared to on modified B5 medium supplemented with 2 g/l AC (Wu et al., 2007).
the medium without AC (Zhang et al., 2000). Secondary SEs were successfully induced in banana (Musa spp.) on MS
Plant regeneration from cultured immature inflorescence segments medium supplemented with various PGRs. Approximately 90% of the
of sugarcane (Saccharum sp.) was obtained via somatic embryogenesis. secondary SEs were germinated and complete plantlets were devel-
Regeneration was obtained on MS medium supplemented with NAA, oped when subcultured on MS medium supplemented with 0.01 g/l
CW and 0.1 g/l AC or MS medium with 0.1 g/l AC alone (Blanco et al., AC, BA and IAA (Khalil et al., 2002). In G. max embryo differentiation
1997). In grapes (Vitis sp.) SE regeneration and maturation have been occurred on modified MS medium supplemented with maltose and
successfully achieved after 30 days of cultivating embryogenic cultures 0.05 g/l AC (Klink et al., 2008).
in an embryo regeneration medium (modified NN medium) supple- AC is used only at the last stage for germination of European
mented with AC and polyethylene glycol (PEG) (Motoike et al., 2001). chestnut (Castanea sativa) SEs and was transferred to Magenta vessels
In another report Gambino et al., 2007 induced somatic embryogenesis containing P24 basal medium (Teasdale, 1992) supplemented with
in Vitis spp. when embryogenic calli were transferred to modified NN 0.1 g/l AC (Sauer and Wilhelm, 2005). In another report on somatic
medium supplemented with 2-napthoxyacetic acid (NOA), BA, IAA and embryogenesis in C. sativa, SE germination was occurred on two
0.02 g/l AC. Fan et al. (2008) used MS medium supplemented with media one consisted of 1/2 MS and AC, and another one with WPM
1.5 g/l AC as embryo germination medium for V. vinifera. plus 500 mg/l 2-[N-Morpholino] ethenesulfonic acid (MES), 500 mg/l
Somatic embryogenesis and plant regeneration of saw palmetto PVP, and BA which was previously used in micropropagation of
(Serenoa repens) from immature zygotic embryos was achieved by American chestnut (Xing et al., 1997). Embryo germination was
Gallo-Meagher and Green (2002). Embryos were cultured on MS significantly better on the latter medium than the former in rates of
medium containing 0.015 g/l AC, 452 μM 2, 4-D and 2iP. Clusters of SEs plant regeneration (2 vs 0.6%), shoot regeneration (13.1 vs 4.2%), and
developed from all immature zygotic embryos 5 weeks after culture rooting (13.6 vs 3.6%). Hence AC had a negative role as compared to
initiation. After 12 weeks, explants were transferred to the same other medium in this case (Xing et al., 1999). In Brazilian grape tree
medium with the amount of 2, 4-D reduced to 90.4 μM which resulted (Myrciaria aureana) light, AC, and PEG were tested for the regeneration
in SE proliferation. The authors had noted that without AC in the and maturation of SEs. A combination of light and PEG increased the
medium, oxidation was high with all zygotic embryos becoming brown number of mature embryosm whereas AC was detrimental to
and failing to respond. When AC present in the medium no oxidation or embryogenesis (Motoike et al., 2007). In Canary Island date palm
624 T.D. Thomas / Biotechnology Advances 26 (2008) 618–631

Phoenix canariensis the presence of AC, combined with the highest (Aesculus hippocastanum) embryogenic calli were induced from stamen
auxin concentration, completely inhibited the induction of embryo- filament on modified MS medium supplemented with 2, 4-D. Such
genic callus (Huong et al., 1999). embryogenic calli were subcultured on different combinations of PGRs
for SE induction. When embryogenic calli were transferred to MS
4. Protoplast culture medium fortified with 50 g/l PEG and 1 g/l AC, more than 18 SEs at
cotyledonary stage were observed per culture after 4 weeks and 70%
In protoplast culture AC has been used to enhance the colony normal mature embryos were recorded after 8 weeks (Capuana and
formation, prevent browning and promote regeneration of protoplast Debergh, 1997). During A. hippocastanum secondary embryogenesis in
derived calli. In lisianthus (Eustoma grandiflorum) the timing of androgenic embryos originating from uninucleared microspores and
addition of AC play crucial role in getting successful growth of anther cultures, medium with AC (1–10 g/l) stimulated the production of
protoplast colonies. Maximum colony formation (approximately 700 secondary embryos originating from suspension culture and maximum
colonies/dish) was observed when AC was added 0–7 days after 83% secondary embryo induction was observed on MS medium
culture. However, the addition of AC after 14 days of culture did not supplemented with glutamine and 1 g/l AC (Calic et al., 2005).
yield any significant variation on the colony formation. The amount of The effect of addition of 0.1 ml drop of AC on microspore
AC also influenced the colony formation with 0.2 g/l AC gave highest embryogenesis was studied in nine morphotypes of Brassica oleracea.
colony formation in two cultivars and above 0.2 g/l AC resulted in a Embryo yields were significantly increased in all the nine morpho-
lower colony formation. Hence it is clear that by using the optimum types studied. The magnitude of the response varied with the plants
conditions with respect to timing and weight of AC incorporated in and morphotypes. The presence of AC never produced any detri-
medium gave rise to optimum results (Kunitake et al., 1995). mental effect. A qualitative improvement of the subsequent develop-
In potato (Solanum tuberosum) when the medium was supple- ment of embryos to plants was also improved with the addition of AC
mented with 0.1 g/l AC, there was an increase in colony formation. The (De Silva Dias, 1999). High frequency (2–5 fold increase) microspore
number of dividing cells showed a significant increase in AC embryogenesis was obtained in B. campestris when microspores were
supplemented medium 12 d after culture. The medium which was cultured on NLN medium (Lichter, 1982) supplemented with 150 g/l
not supplemented with AC showed cell division during the initial AC. The enhancing effect of AC could be due to adsorption of toxic or
stages but gradually they became brown and a few calli were obtained poly-phenolic compound produced by the microspores. Similarly AC
(Garlberg et al., 1983). In plant regeneration from embryogenic induced microspore embryogenesis and embryo germination was
suspension-derived protoplasts of ginger (Zingiber officinale), AC had reported in B. napus (Gland et al., 1988; Gu et al., 2004; Shi et al., 2008;
been used to prevent the browning of the protoplast derived calli. Zhang et al., 2006a,b; Zhou et al., 2002) and B. rapa ssp.chinensis (Gu
Even though AC was not as effective as AgNO3 but it could relieve et al., 2003). AC (5 g/l) had been also employed to induce interspecific
callus browning to some extent (Guo et al., 2007). hybrids through ovary culture in B. rapa (Zhang et al., 2004).
In sugarcane (Saccharum hybrid) the protoplast derived calli were In a perennial herbaceous flowering plant of ranunculaceae, He-
regenerated on MS medium fortified with Kn, NAA and 0.2 g/l AC. A patica nobilis anther culture technique had been successfully applied
maximum 8 out of 10 protocalli regenerated on this medium with an by Nomizu et al. (2004). Androgenic embryos were induced by anthers
average of 3 plants per callus mass (Aftab and Iqbal, 1999). In cotton on NN medium supplemented with 0.1 g/l AC at 5 or 35 °C for a few
(Gossypium sp.) plant regeneration from protoplast derived calli was days and by then incubating them in the dark at 25 °C. Potato
carried out on MSB medium (Wu et al., 2004) with 0.05 g/l AC (Sun et al., (Solanum phureja) androgenic embryos were induced on liquid LS
2005). In another report on somatic hybrid production in G. hirsutum × G. (Linsmaier and Skoog, 1965) medium supplemented with AC (2.5 g/l),
davidsonii, calli derived from fusion mixture was regenerated on BA and IAA (Boluarte-Medina and Veilleux, 2002). In potato (S. phur-
modified MS medium with maltose and 0.05 g/l AC (Sun et al., 2007). eja) androgenesis, AC was removed after autoclaving the medium
The addition of 0.01 g/l AC prevented the necrosis of shoots derived from followed by addition of PGRs (BA and/or IAA) by filter sterilization.
protoplast colonies of Phalaenopsis sp. (Shrestha et al., 2007). Xiao et al. This experiment was conducted to determine the positive role of PGRs
(2007) reported rooting of protoplast derived SEs of Musa acuminate on if added to the medium after the removal of AC. Removal of AC 24 h
MS basal medium supplemented with 0.01 g/l AC. after autoclaving the medium did not have any effect on anther culture
response. The removal of AC was beneficial only during the embryo
5. Anther and microspore culture harvest as the small globular embryos are hardly visible in presence of
AC (Chani et al., 2000).
It is believed that AC is capable of trapping gases especially In Avena sativa and A. sterilis cold pretreated anthers were isolated
ethylene, released from cultured tissues and there by increasing the and cultured on MS basal medium containing 2.5 g/l AC. For A. sterilis
embryo yield (Johansson et al., 1982). It may also adsorb the toxic or 13 green and three albino regenerants were produced, of which five
poly-phenolic compound produced by the microspores. The addition plants (haploids) survived transfer to the greenhouse. However for
of AC in the medium increased the androgenic response in Nicotiana A. sativa, various differentiation media combinations did not yield any
tabacum. AC is supposed to remove ethylene in the culture vials, which results (Kiviharju and Pehu, 1998). In Anemone coronaria androgenic
is produced by anthers during anther culture. However, it was found haploids were induced on a double layer medium. The lower layer
that the hike in androgenesis in AC containing medium is not due to consisted of 5 ml of NN medium with 10 g/l AC and agar; the upper
ethylene absorption but the removal of certain unidentified inhibitory layer (4 ml of the same medium lacking both AC and agar) was added
substances (Horner et al., 1977). Positive effect of AC on androgenesis immediately prior to the floating of the anthers. Some cultures were
in N. tabacum were also reported by Anagnostakis (1974). The supplemented with IAA also. Cultures were maintained in the dark for
ethylene absorption by AC is highly influenced by culture conditions 7 d. By using this technique up to 16.9 regenerants per 100 cultured
like culture vial volume, shape, volume of the medium, surface anthers were obtained (Laura et al., 2006). During anther culture
exposed to the inner atmosphere and explant position with respect to leading to double haploid plant production in cork oak (Quercus suber),
the medium (Mensuali-Sodi et al., 1993). the induction medium consists of modified MS supplemented with
In wheat (Triticum aestivum) anther culture, pretreatment of anthers 0.1 g/l AC. This medium coupled with a heat shock of 33 °C for 5 days
with 0.8 M sucrose and 0.3 g/l AC for 1–2 h prior to culture enhanced both enhanced the anther embryogenesis to 7.12% (Pintos et al., 2005).
frequency of induction of microspore-derived calli as well as regenerated The addition of various concentrations (0.025 to 0.2 g/l) of AC on
green plants in winter genotypes. However the spring genotypes did not shed-microspore cultureof Indonesian hot pepper (Capsicum annuum)
have any effect on this pretreatment (Zhang et al.,1987). In horse chestnut cultivar ‘Tombak’ confirmed the importance of AC as embryo formation
T.D. Thomas / Biotechnology Advances 26 (2008) 618–631 625

almost completely failed without its addition. Both the total embryo hypogea derived from regenerated virus free ELISA tested calli. About
yield as well as the yield of normal looking embryos increased with 60–80% of the shoots rooted when cultured on MS medium containing
increasing concentrations of AC from 0 to 0.2 g/l. However, 2% AC 0.04 g/l AC and 0.2 g/l CH, another extra additive commonly used in plant
impaired embryonic shoot development. Consequently, AC at a tissue culture (Radhakrishnan et al., 1999).
concentration of 0.1 g/l was chosen as standard for further experiments In Dutch elm hybrid (Ulmus hybrid) the rooting was promoted by
(Supena et al., 2006). AC (2 g/l) when used in modified MS medium alone or in combination
In some cases the application of AC in the medium had an inhibitory with IBA. However the maximum response was observed when 5 μM
role also. In B. napus, secondary embryogenesis (i.e. embryos developing IBA alone was used (Jouira et al., 1998). In Thapsia garganica (apiaceae)
from the surface of androgenic embryos) had been significantly inhibited rooting of the stock cultures kept on MS medium supplemented with
by the inclusion of AC (0.1–1.0 g/l) in the medium. Further the embryos in BA and NAA were rooted on 1/2 MS medium fortified with IBA prior to
AC containing medium showed stunted growth (Shu and Loh, 1987). In B. 0.5 g/l AC treatment. 50% of the plantlets rooted and an average
juncea, addition of AC in medium both at initiation of culture or in Petri number of 6 roots per shoot were observed in each shoot (Makunga
dishes containing globular embryos hindered the growth and develop- et al., 2006). Micropropagated shoots of enset (Ensete ventricosum)
ment of microspores as compared to control medium. On microscopic were rooted on 1/2 MS medium supplemented with IBA, BA and 1 g/l
examination, the charcoal particles were found sticking to the dividing AC (Negash et al., 2000). In Prosopis laevigata in vitro root formation in
microspores or developing embryos thus hindering their growth and shoots took twice as long when AC was added to the induction
development (Pream et al., 2005). AC had a negative role during the medium instead of PVP (Buendia-Gonzalez et al., 2007).
anther culture of Japanese pear (Pyrus pyrifolia). Callus induction was In vitro rooting of avocado (Persea americana) was induced by a two
comparatively less and no embryos were regenerated from callus on AC step protocol. 1.5–2 cm long shoots were incubated for 3 days in solid
containing medium (Kadota and Niimi, 2004). MS medium with macroelements at one-third and IBA. After this quick
treatment, the shoots were subcultured on the same medium without
6. Synthetic seed auxin but with 1 g/l AC. By using this technique 90% of the shoots
rooted and 70% of the plants survived after field transfer (Barcelo-
In rice (Oryza sativa) synthetic seeds incorporated with synthetic Munoz et al., 1999). Successful rooting of camphor tree (Cinnamomum
endosperm exhibited higher % of germination and conversion than camphora) shoots derived from shoot tips and nodal segments were
without the synthetic endosperm. Two types of endosperms were reported on WPM medium supplemented with AC, IBA and NAA. 100%
employed and one with MS medium nutrients, vitamins, sucrose, of the shoots rooted on IBA and 2 g/l AC containing medium (Nirmal
PGRs namely IAA + NAA + BA and AC (0.13 g/l) gave the maximum Babu et al., 2003). For in vitro rooting, the H. abyssinica shoots were
germination of 30% and the conversion of 27%, whereas the second kept in the dark for 4 days followed by transfer of shoots to WPM basal
one with only MS medium nutrients, vitamins and sucrose gave 12% medium with 0.03 g/l AC. 100% rooting was induced by using this
germination and 8% conversion. The presence of AC in the synthetic method depending on genotype (Feyissa et al., 2005).
endosperm enhanced the germination and conversion of synthetic Micropropagated sugar apple (Annona squamosa) shoots were
seeds to the maximum extent by increasing the diffusion of gases, rooted on WPM medium fortified with 10 g/l AC before treatment with
diffusion of nutrients and respiration of embryoids (Arun Kumar e al., NAA or IBA (Lemos and Blake, 1996). In sycamore maple (Acer
2005). During the germination of encapsulated embryos of interior pseudoplatanus) rooting of the in vitro propagated shoots derived from
spruce (Picea glauca engelmannii complex) and black spruce (Picea plumule, hypocotyl and radicle explants occurred on MS medium
mariana Mill.) Lulsdorf et al. (1993) noted that the addition of 0.05 g/l fortified with IBA for 24 h followed by transfer to PGR-free MS
AC to the alginate capsule significantly enhanced root development medium with 0.1 g/l AC, or placed directly on AC containing MS
and germination for SEs but not for zygotic embryos. They had also medium. The rooting response of shoots without IBA pretreatment
tried to develope an artificial endosperm with or without sucrose to was 40%. Application of IBA for 24 h resulted in an increase in root
the alginate-AC capsule. formation to 65% (Wilhelm, 1999).
In silk tassel bush, Garrya elliptica transferring the in vitro shoots
7. Rooting directly from multiplication medium to rooting medium containing auxin
resulted in extensive callusing, with roots developing on the callus.
AC alone or in combination with an auxin induced rooting of Subculturing shoots on 1/2 WPM containing 0.02 g/l AC prior to transfer to
micropropagated shoots were reported in several plants. During the auxin containing medium prevented the callusing. (Woodward and
development of a successful transformation technique for pineapple Thomson,1996). In Salvia africana-lutea plantlets rooted without additives,
(Ananas comosus), the transformed and multiplied shoots were rooted produced calli at the shoot base and this problem was solved by using
on 1/2 MS medium supplemented with chlorsulfuron and 0.5–3 g/l AC. 0.5 g/l AC (Makunga and Van Staden, 2008). The promotery role of AC on
The authors noted that the addition of AC considerably enhanced the root induction of in vitro shoots of zedoary (Curcuma zedoaria) had been
rooting ability of transgenic shoots (Firoozabady et al., 2006). 0.5 g/l AC reported by Loc et al. (2005). About 17 roots per shoot were observed
had been employed along with 1/2 MS medium supplemented with when 1 g/l AC was employed along with CW and IBA in MS medium.
NAA for rooting in Swertia chirayita micropropagation (Joshi and In Garcinia mangostana the callus derived shoots were rooted on
Dhawan, 2007). In a rutaceae shrub Fortunella crassifolia, the shoots WPM medium supplemented with BA, 0.025 g/l AC and phloroglucinol
formed from epicotyl explants exhibited maximum rooting (75%) on 1/2 (PG; Techato and Lim, 1999). In C. annuum the callus regenerated
MS medium supplemented with NAA, Kn, and 0.5 g/l AC (Yang et al., plantlets were rooted on MS medium supplemented with 0.002 g/l AC
2006). 85% shoots derived from nodal segments were rooted on MS (Anu et al., 2004). In strawberry (Fragaria × ananassa) rooting of the
medium supplemented with IBA, BA and 0.05 g/l AC in Simmondsia micropropagated shoots observed on MS medium supplemented with
chinensis (Jojoba), a dioecious shrub seen in arid zones. AC not only IBA and 0.5 g/l AC (Jemmali et al., 2002). During globe artichoke
increased the percentage response but also reduced the callusing of the (Cynara scolymus) micropropagation, rooting of the shoot occurred on
explant (Agrawal et al., 2002). In another report on S. chinensis, of the 5 modified 1/2 MS medium supplemented 2 g/l AC and NAA (Le Saos and
male and female genotypes used for in vitro micropropagation Hourmant, 2001). The shoots of somatic hybrids derived from fusion
experiments through nodal cuttings culture, irrespective of genotypes, between navel orange (Citrus sinensis) and grape fruit (C. paradisi) were
sex and duration of pulse treatment, shoots of all genotypes developed rooted on 1/2 MT basal medium fortified with NAA and 0.05 g/l AC (Guo
roots directly (without callus) within 30 days on MS + IBA + 0.05 g/l AC et al., 2000). In A. hippocastanum root induction of in vitro multiplied
(Tyagi and Prakash, 2004). Roots were induced on shoots of Arachis shoots occurred on 1/2 MS basal medium containing 0.002 g/l AC. The
626 T.D. Thomas / Biotechnology Advances 26 (2008) 618–631

rooted shoots were immediately transferred to MS medium supple- hormone free H3 medium supplemented with 0.5 g/l AC. 100% rooting
mented with PVP and BA to prevent shoot tip necrosis (Zdravkovic- was also observed on the same media. No PGRs were employed in the
Korac et al., 2004). In mulberry (Morus alba) about 72% of the in vitro final steps of shoot growth and rooting (Ket et al., 2004). In Dendro-
shoots produced well-developed roots on MS basal medium fortified bium nobile the asymbiotic seed germination was observed on 1/2 MS
with 0.005 g/l AC after 6 weeks (Agarwal and Kanwar, 2007). Mulwa medium supplemented with 0.05 g/l AC. When the cotyledons
and Bhalla (2006) obtained roots in macadamia (Macadamia tetra- appeared, the seedlings were transferred to 1/2 MS supplemented
phylla) micropropagated shoots on 1.5 MS medium supplemented with with BA (Men et al., 2003). In Catasetum fimbriatum the asymbiotic
IBA and 0.1 g/l AC. In Musa acuminata the rooting of the SEs were seed germination occurred on modified Vacin–Went medium (Vacin
obtained on MS basal medium supplemented with 0.1 g/l AC (Jalil et al., and Went, 1949) supplemented with BP and 1 g/l AC (Vaz et al., 1998).
2003). Rooting in common lavender (Lavandula vera) was induced In red vanda Renanthera imschootiana root initiation was achieved
either by the application of AC alone in 1/2 MS medium or with IBA or in leaf base derived shoots within one week after transferring the
NAA. However, the response was better when IBA or NAA were shoots to Mitra medium (Mitra et al., 1976) containing NAA and 0.1 g/l
employed with out AC (Andrade et al., 1999). In Anacardium occidentale AC (Seeni and Latha, 1992). In the terrestrial orchid Cymbedium faberi
AC at 0.0005, 0.001 or 0.005 g/l during or after 5 days root induction for rooting of the shoots AC was employed along with NAA in modified
period by IBA, did not affect rooting in any way (Boggetti et al., 2001). MS medium and 90% rooting was observed on this medium. The
acclimatized plants were transplanted to pots containing pearlite,
8. Orchid tissue culture sands, AC and soil (3:2:2:3) (Chen et al., 2005). During Geodorum
densiflorum micropropagation, rhizome sections cultured on MS
In a terrestrial orchid Haemaria discolor the germinated seeds were medium fortified with BA and 0.01 g/l AC produced multiple shoots
transferred from 1/2 MS basal medium (half-strength macronutrients) and 100% cultures responded with an average number of 8.2 shoots
to growth medium containing 0.02 g/l AC, banana pulp (BP), TDZ per explant (Sheelavantmath et al., 2000). Protocorm like bodies (PLB)
(thidiazuron-N-phenyl N1 1,2,3-thidiazol-5-yl urea) alone or in were induced in Phalaenopsis hybrid from leaf segments in vitro for
combination with NAA for further growth (Shiau et al., 2005). bioreactor cultures supplemented with AC. Continuous immersion
Similarly, in Paphiopedilum ciliolare AC and BP were not necessary cultures (air lift column and air lift-balloon bioreactor), and temporary
during initial stage of seed germination but had positive influence on immersion cultures (with or without AC filter attached) were used for
further development of the seedlings. (Pierik et al., 1988). In lady's
slipper orchid Cypripedium flavum the mature seeds were induced to
germinate and multiple shoot formation on Harvais (1973) medium
supplemented with two cytokinins i.e. BA or Kinetin (Kn). Maximum
rooting (100%) of shoots was observed on 1/2 Harvais medium
supplemented with AC alone. Highest level of AC (0.6 g/l) was more
suitable for root induction than the other two levels. The authors
concluded that the AC created partial darkness similar to the
underground environment of C. flavum habitats, was most likely the
cause of the increased rooting. (Yan et al., 2006).
The promotive role of AC on seed germination of South African orchid
disa had been reported by Thompson et al. (2006). A dual-phase culture
medium was used to induce seed germination and seedling establish-
ment from immature embryos of nine species of Disa (Disa spp.). The
dual-phase cultures were initiated from seeds and comprised of a solid
modified MS medium containing 2 g/l AC, overlaid with a reduced
strength, liquid medium fraction of the same type. The presence of AC
negate the influence of leached phenols and allowed protocorms to
establish polarity, while the liquid medium increased water availability
(Thompson et al., 2007). A two-layered culture system was also used for
high frequency in vitro flowering in Dendrobium sp. The two-layered
medium consisted of gelrite-solidified medium with addition of 0.003 g/l
AC overlaid with 20 ml of basal liquid medium supplemented with BA.
When AC was added to the Gelrite-solidified layer, the frequency of
flower buds was 65% and addition of BA in the liquid layer increased the
flowering frequency to 100% (Sim et al., 2007). In vitro germination and
plantlet development was achieved in several Ophrys spp. by using
modified basal medium (Malmgren, 1996) containing 0.5 g/l AC and CM
or pineapple juice (Kitsaki et al., 2004).
During Cymbidium forrestii micropropagation through seed
derived rhizome, BA was effective in shoot induction. However, due
to the browning of media containing BA, AC was added as a
supplement to test its effect on rhizome growth and shoot formation.
The most effective concentration in terms of rhizome production and
fresh weight gain was 0.01 g/l AC, but shoot formation was inhibited
(Paek and Yeung, 1991).
In Anoectochilus formosanus shoot tips were cultured on Hyponex
medium (H3; Kano, 1965) fortified with BA and TDZ. The addition of Fig. 1. Asymbiotic seed germination and multiple shoot formation in orchid Rhynchos-
tylis retusa. A. A seedling on MS medium supplemented with BA (6 µM), NAA (0.2 µM,
0.001 g/l AC significantly increased the shoot formation (11.2 shoots and AC (1 g/l) one month after subculture. B. Seedling growing on the same medium but
per explant). However, the regenerating shoots showed slow growth with only 0.5 g/l AC. C. Emergene of multiple shoots from seedlings on MS medium
and this difficulty was prevail over by transferring the shoots to supplemented with TDZ (4 µM) and 1 g/l AC. Scale bars represent 1 cm.
T.D. Thomas / Biotechnology Advances 26 (2008) 618–631 627

the culture of PLB sections. A temporary immersion culture with AC supplemented with 2 g/l AC (Sul and Korban, 2004). AC induced shoot
filter attached was most suitable for PLB culture (Young et al., 2000). growth and rooting had also been reported in short leaf pine (Pinus
In Rhynchostylis retusa the in vitro seedling growth was promoted spp.) by Jang and Tainter (1991).
by the addition of AC in the medium. Seedling growth was maximum In Pinus sylvestris AC had an important role to play during the
on MS medium supplemented with BA, NAA and 1 g/l AC (Fig. 1A–C). induction of somatic embryogenesis. The 8 or 24-week-old embryonal
The multiple shoot induction from seedlings was also promoted by AC masses were cultured on maturation medium containing 10 g/l AC. AC
and a maximum 8.2 shoots were observed on MS medium fortified exerted a beneficial effect on mature SE production of 24-week-old
with TDZ and 1 g/l AC (Thomas and Michael, 2007). cultures; there was no evidence of such an effect in 8-week-old
cultures (Lelu-Walter et al., 2008). In another species P. pinaster
9. Lower plants relatively high SE maturation yield was observed when cells were
coated with particles of AC. SEs were produced abundantly in
AC promotes morphogenic response in several pteridophytes in vitro. presence of AC whatever the phytagel concentration of the medium.
The addition of AC in leaf cell suspension culture medium (MS) of a fern, This is related to the hygroscopic property of AC and SEs that matured
Platycerium bifurcatum significantly improved the number of regener- in presence of AC developed faster and in greater numbers, clearly
ated sporophytes even in basal medium. The presence of AC in medium improving the productivity of the culture (Lelu-Walter et al., 2006).
resulted in regeneration of single sporophytes, which was not possible in During somatic embryogenesis in a conifer Abies numidica, the addition
non AC medium. Further more prevention of formation of gametophyte of 10 g/l AC along with IBA on MS as well as Schenk and Hildebrandt (SH;
clusters; bud clusters and prevention of hyperhydricity of regenerated Schenk and Hildebrandt, 1972) media had positive influence on SE
sporophytes were the additional advantages of employing AC in germination. Embryo germination frequency was more on AC supple-
medium. AC also interacted with PGRs and prevented abnormal mented medium than IBA medium when used individually. However a
morphogenesis, normalizing the development of singly existing combination of AC and IBA gave rise to maximum embryo germination
sporophytes and increasing regeneration efficiency in P. bifurcatum. (Vookova and Kormutak, 2001). AC supported SE maturation had been
(Teng, 1997). In Equisetum arvense the protoplasts were isolated from reported in A. alba and embryogenic suspensor mass were conditioned 1
subcultured gametophytes and cultured in vitro on MS medium fortified week on basal medium with 10 g/l AC for maturation of SEs (Hristoforoglu
with mannitol. Addition of AC to medium enhanced the rate of cell et al., 1995). Similarly in Douglas fir (Pseudotsuga menziesii), SE maturation
division, as well as the survival of regenerating protoplasts. However, medium was supplemented with 1 g/l AC (Zhang et al., 1999).
subsequent division of cells was not favoured by the addition of AC in the Chang et al. (2001) reported micropropagation of Taxus mairei
medium and hence AC was omitted from the medium for further culture from bud explants. Browning of tissues was one of the major problems
(Kuriyama et al., 1990). While studying the role of gibberellins in faced during culture and was rectified by supplementing the basal
determining sex in the gametophyte of fern Blechnum spicant, Menendez medium with 1 g/l AC and 0.1 g/l silver nitrate. In T. wallichiana plant
et al. (2006) noted that the addition of AC influenced gametophyte regeneration and elongation of regenerated shoots occurred on
development. The formation of antheridia was significantly diminished modified 1/2 Lloyd and Mc Cown's basal (Lloyd and Mc Cown, 1980)
to almost zero by addition of AC with the highest concentration (1 g/l) medium fortified with 0.1 g/l AC. On this medium the maximum shoot
tested. In an edible fern Matteuccia struthiopteris (Ostrich fern) a rapid elongation (2.15 cm) was observed (Datta et al., 2006).
micropropagation protocol had been standardized by Thakur et al. For stepwise SE maturation in hybrid larch (Larix) the embryonal
(1998) by using side shoots. It was observed that rhizogenesis and masses were transferred for one week to PGR-free MS medium
growth of regenerants were best achieved on one-quarter-strength containing 10 g/l AC, after which they were transferred to MS medium
basal MS medium amended with 0.1 g/l AC. The protoplasts of a supplemented with abscisic acid (ABA; Lelu-Walter et al., 1994; Aderkas
liverwort Marchantia polymorpha regenerated new cell walls normally et al., 2002). Same procedure was adopted for maturation of secondary SE
during the in initial culture. But after the cell wall regeneration, the of Larix originated from cotyledons (Saly et al., 2002). Lin et al. (2004)
regenerated cells started to divide actively only after being transferred to noted that AC promoted callus proliferation and bud formation in Larix
a medium with 0.1 g/l AC. Protoplasts were suspended in MS medium sp. owing to its adsorption of phytohormones and other phenolic
with β-glucosyl Yariv reagent (bglcY) and 0.1 g/l AC at a density of substances. While trying to get stablet transgenic lines of L. gmelinii,
1 × 104/ml to 4 × 104/ml. On this medium the cells divided vigorously, and organogenesis in hygromicin resistant tissues occurred on 1/2 DCR
the cell division rate was remarkably increased to about 80% (Shibaya medium (Lin et al., 2004) with 0.01 g/l AC. Adventitious buds formed from
and Sugawara, 2007). In a moss Hyophila involuta AC (0.05–0.2 g/l) do transformed calli in large numbers when the cultures were subcultured
not induce bud induction from protonema. AC also had an inhibitory role to a medium containing AC (Lin et al., 2005). In Hinoki cypress
in protonemal growth in this plant (Rahbar and Chopra, 1982). (Chamaecyparis obtuse) germination of mature SEs were carried out on
In gymnosperms AC promoted the in vtro response by preventing the WPM medium containing 2 g/l AC. The SEs germinated at a frequency of
phenol exudation. It also enhanced the SE germination and maturation. 60% and the addition of AC in the medium was effective in stimulating
In Pinus radiate tracheary element (TE) differentiation from xylem strips plantlet growth (Taniguchi et al., 2004; Taniguchi et al., 2005).
derived callus cultures were occurred on modified EDM medium (Walter In sitka spruce (Picea sitchensis) for SE maturation, liquid suspension
and Grace, 2000) supplemented with 0–20 g/l AC. The differentiation cultures from shake flasks and bioreactors containing SEs were
rate increased from 20% when callus was grown in darkness, to 45% transferred to Petri dishes containing 1/2 LM (Litvay et al., 1985)
when grown with a 16 h or 24 h photoperiod. In addition to light, the medium fortified with ABA, IBA, 1 g/l AC. The medium was either
differentiation rate was influenced by the concentration of AC added to solidified with agar or submerged culture consisted of a thin layer of agar
the induction medium, the optimum concentration being 5 g/l. solidified medium covered with 7 ml liquid medium (Ingram and
Exclusion of AC from the induction medium decreased the differentia- Mavituna, 2000). In Picea abies (Norway spruce) extensive studies by
tion rate to 2% (Moller et al., 2003; Moller et al., 2006). Pullman et al. (2005) revealed that AC in combination with ABA
In Pinus heldreichii the pretreated cotyledon, hypocotyl and stem increased the cotyledonary SE yield. It also enhanced the number of
apex were cultured on Gresshoff and Doy (1972) medium (GD), as genotypes forming cotyledonary embryos. The embryos exhibited
modified by Sommer et al. (1975), supplemented with 0.05 g/l AC to improved maturation characteristics, size and reduced embryo produc-
prevent exudation from the explant (Stojicic and Budimir, 2004). AC tion costs. The medium showed a rapid decline in ABA in presence of AC
induced shoot elongation during micropropagation had been reported within a few hours followed by a gradual decrease over the next few
in P. heldreichii on 1/2 MS basal medium supplemented with 0.05 g/l days with little change from 2 to 6 weeks. Agar decreased the adsorption
AC (Stojicic et al., 1999) and Pinus pinea using 1/2 MS basal medium of ABA compared to Gelrite and Phytagel.
628 T.D. Thomas / Biotechnology Advances 26 (2008) 618–631

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