Scholarly article on topic 'Acacia: An exclusive survey on in vitro propagation'

Acacia: An exclusive survey on in vitro propagation Academic research paper on "Biological sciences"

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{Callogenesis / Explant / Organogenesis / "Plant growth regulators" / "Somatic embryogenesis" / "Woody plant"}

Abstract of research paper on Biological sciences, author of scientific article — Saikat Gantait, Suprabuddha Kundu, Prakash Kanti Das

Abstract The current survey exemplifies the achievements on experimental results of production of planting materials through in vitro direct or indirect organogenesis of genus Acacia. Several species of Acacia have been given due importance in tree tissue culture owing to their proven wasteland reclamation ability, ecological and economical significance. Plant cell, tissue and organ culture-based techniques have been employed in forest tree research for successful reforestation and forest management programs. The relevance of tissue culture methods has gained impetus to meet the growing demands for biomass and forest products. Ever since the last four decades, in vitro protocols are being developed with the aim to regenerate several woody species. This survey strives to serve as a compendium of various routine processes involving organogenesis of Acacia via in vitro; which would encouragingly be worthwhile for researchers to exploit this perennial woody legume with enormous multidimensional value, via more innovative approaches, in order to promote the cause for its improvement.

Academic research paper on topic "Acacia: An exclusive survey on in vitro propagation"

King Saud University Journal of the Saudi Society of Agricultural Sciences

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Acacia: An exclusive survey on in vitro propagation

Saikat Gantaita*, Suprabuddha Kundub, Prakash Kanti Dasc

a AICRP on Groundnut, Directorate of Research, Bidhan Chandra Krishi Viswavidyalaya, Kalyani, Nadia, West Bengal 741235, India b Department of Agricultural Biotechnology, Faculty of Agriculture, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, Nadia, West Bengal 741252, India

c Department of Agricultural Biotechnology, Faculty Centre for Integrated Rural Development and Management, School of Agriculture and Rural Development, Ramakrishna Mission Vivekananda University, Ramakrishna Mission Ashrama, Narendrapur, Kolkata 700103, India

Received 12 December 2015; revised 14 March 2016; accepted 20 March 2016


Callogenesis; Explant; Organogenesis; Plant growth regulators; Somatic embryogenesis; Woody plant

Abstract The current survey exemplifies the achievements on experimental results of production of planting materials through in vitro direct or indirect organogenesis of genus Acacia. Several species of Acacia have been given due importance in tree tissue culture owing to their proven wasteland reclamation ability, ecological and economical significance. Plant cell, tissue and organ culture-based techniques have been employed in forest tree research for successful reforestation and forest management programs. The relevance of tissue culture methods has gained impetus to meet the growing demands for biomass and forest products. Ever since the last four decades, in vitro protocols are being developed with the aim to regenerate several woody species. This survey strives to serve as a compendium of various routine processes involving organogenesis of Acacia via in vitro; which would encouragingly be worthwhile for researchers to exploit this perennial woody legume with enormous multidimensional value, via more innovative approaches, in order to promote the cause for its improvement.

© 2016 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY-NC-ND license (

Abbreviations: 2,4-D, 2,4-dichlorophenoxy acetic acid; AdS, adenine sulfate; B5, Gamborg et al. (1968); BA, N6-benzyladenine; BAP, N6-benzylaminopurine; BD, Bonner-Devirian medium (Bonner and Devirian, 1939); Ca, callus; CW, coconut water; DKW, Driver Kuniyuki medium (Driver and Kuniyuki, 1984); GA3, gibberellin A3; IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; KB, Knop and Ball medium (Hustache et al., 1986) Kinetin, 6-furfurylaminopurine; KT, Kathju Tewari medium (Kathju and Tewari, 1973); MSt, multiple shoot; MS, Murashige and Skoog medium (Murashige and Skoog, 1962); NAA, a-naphthalene acetic acid; PGR, plant growth regulator; Q-LP, Quoirin Lepoivre medium (Quoirin and Lepoivre, 1977); Rt, root; SH, Schenk and Hildebrandt medium (Schenk and Hildebrandt, 1972); SR, adventitious shoot regeneration; TDZ, N-phenyl-N'-(1,2,3-thiadiazol-5-yl) urea or Thidiazuron; WPM, Woody Plant Medium (Lloyd and McCown, 1981); Zeatin, 4-hydroxy-3-methyl-terms-2-butenyl aminopurine. * Corresponding author.

E-mail address: (S. Gantait). Peer review under responsibility of King Saud University.


1658-077X © 2016 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY-NC-ND license (


1. Introduction........................................................................................................................................................00

2. In vitro organogenesis..........................................................................................................................................00

2.1. Role of explant source..................................................................................................................................00

2.2. Role of surface disinfection procedure............................................................................................................00

2.3. Role of basal media......................................................................................................................................00

2.4. Role of carbohydrate source..........................................................................................................................00

2.5. Role of plant growth regulators on direct organogenesis..................................................................................00

2.6. Role of plant growth regulators on callogenesis................................................................................................00

2.7. Role of plant growth regulators on somatic embryogenesis................................................................................00

2.8. Role of plant growth regulators on rooting in vitro..........................................................................................00

3. Substrate-based acclimatization............................................................................................................................00

4. Marker-assisted genetic fidelity assay......................................................................................................................00

5. Future outlook....................................................................................................................................................00

Authors' contribution..............................................................................................................................................00

Conflict of interest..................................................................................................................................................00

Acknowledgments ..................................................................................................................................................00

References ..............................................................................................................................................................00

1. Introduction

Since last three decades, the population in tropical countries has been rising at an annual rate of 2.8% and as a result the overall forest area in those countries has been declining at 0.8% per year. The year-wise afforestation and reforestation area in those countries was projected to be 1.8 million ha during the period 1981-1990, ensuing in an annual net reduction in forest area of 13.6 (15.4-1.8) million ha (Kozai et al., 2000). Moreover, the decline in biomass of woody plants owing to desertification in arid regions is remarkable as it acts as a precursor of recent climate changes on several geographic zones. It has been foreseen that a demand for woody transplants will rise considerably in future decades for paper, timber, plantation, horticulture and furniture industries, as well as, in environment conservation (Kozai et al., 1997). The usage of plant biomass can be an alternative to the overconsumption of fossil fuels and thus lowers the atmospheric CO2 levels which ultimately assuages climate changes. A steady supply of quality planting materials becomes increasingly important to satisfy the ever increasing growing demand that conventional propagation based plantlet production fails. In vitro propagation system holds its merits over that of the conventional propagation since the in vitro system ascertains the phe-notypically and genotypically uniform disease-free propagules in a sustainable manner (Aitken-Christie et al., 1995). In this review we demonstrate the achievements made (based on experimental results) on in vitro propagation system of an important tropical tree legume genus Acacia, along with ex vitro acclimatization and clonal fidelity assessment.

Comprising around 1200 species, Acacia (family Fabaceae and sub family Mimosaceae) is ample in Australia, Africa, India and America (Simmons, 1987). Typically, to reforest and reclaim the wastelands (Skolmen, 1986) and to improve soil health, as well as to serve as the rich source of fuel wood, timber, and shelter belts (Palmberg, 1981) the genus Acacia plays an enormously essential role. Majority of its species generates exceptional firewood and a few are the source of an affluent supply of tannin, protein, ink, paint, pulpwood,

flavoring agents, and gum. From the environmental perspective, Acacia can acclimatize to extreme atmospheric conditions and consequently, can adapt to both arid and moist areas of tropical soils. Various species are capable of increasing soil fertility by undergoing in a symbiotic association with Rhizobium and Mycorrhizal fungi. Moreover, it minimizes soil erosion and assists in sand dunes stabilization (Skolmen, 1986).

2. In vitro organogenesis

In vitro organogenesis, particularly for tricky and recalcitrant species is chiefly reliant on the type of explants and manipulations of several plant growth regulators (PGRs) in culture media. Accelerated in vitro propagation is the unique feature of plant tissue culture that has been credibly acknowledged with respect to its practicability in bulk and commercial-scale multiplication of propagules. Successful in vitro regeneration of the plant material depends on numerous aspects such as genetic makeup, explant type, media composition, PGRs as well as the culture conditions. Direct regeneration and indirect regeneration via an intermediary callus phase are the two chief fundamental approaches engaged as an efficient in vitro regeneration of forest trees. Among these two approaches indirect organogenesis is less enviable for clonal multiplication due to its reported cases of somaclonal variability. Hence, direct regeneration (devoid of callus-stage) is considered as a consistent approach for clonal propagation. A variety of in vitro culture approaches, for instance de novo organogenesis, callogenesis, and somatic embryogenesis have been used comprehensively for large-scale micropropagation and the production of genetically true clones in bulk quantities. Vigilant selection and collection of explants, with apposite use of basal media, PGRs, antioxidants and additives are the fundamental criteria for standardizing consistent and reproducible micropropagation protocols. There have been scores of reports on in vitro growth and multiplication of Acacia attained through embryogenesis or organogenesis. Nevertheless, explant source and their disinfection process along with the media formulations, culture conditions, accumulation of phenolics in media

Table 1 Achievements on in vitro direct organogenesis of Acacia (arranged in chronological order).

Species Expiant Basal medium PGR (mg/l) Result Reference

Acacia saligna Cotyledon KT 2 NAA + 2 2,4-D Rt Kathju and Tewari (1973)

A. senegal Stem MS BA MSt Dave et al. (1980)

A. nilotica Stem MS 0.5-1 IAA MSt R t Rt Marthur and Chandra (1983)

A. albida Cotyledon MS 0.5 NAA + 3 BA MSt Duhoux and Davies (1985)

0.1 NAA Rt

A. ligulata Shoot MS Rt Williams et al. (1985)

A. melanoxylon Embryo Q-LP AC MSt Jones (1986)

5 IBA + 2.5 NAA + 0.2 BA Rt

A. melanoxylon Node Q-LP 1 BA + 0.5 NAA MSt Jones and Smith (1988)

A. mangium Stem MS PGR-free Rt Darus (1989)

A. albida Node from sucker MS 20 BA MSt Gassama (1989)

0.5 BA + 0.01 NAA Rt

A. auriculiformis Axillary bud B5 0.2 BA + 10% coconut milk MSt Mittal et al. (1989)

0.02 NAA Rt

A. auriculiformis Shoot tip MS kinetin, IAA MSt Ranga Rao et al. (1989)

A. melanoxylon Embryo Q-LP PGR-free MSt Jones et al. (1990)

5 IBA + 2.5 NAA + 0.2 BA Rt

A. mangium Node MS 0.5 BA MSt Darus (1991)

Seradix 3 Rt

A. mangium Node MS 1-2 BA MSt Galiana et al. (1991)

'MS 0.05 IBA Rt

A. auriculiformis Hypocotyl 'MS 1 BA + 0.5 NAA + glutamine MSt Ranga Rao and Prasad (1991)

1 IBA or 1 IBA + 0.5 NAA Rt

A. auriculiformis Shoot White 0.4 IBA + 0.2 IAA or 0.2 IBA Rt Semsuntud and

+ 0.4 NAA Nitiwattanachai (1991)

A. saligna Shoot tip MS 5-9 BA MSt Barakat and El-Lakany (1992)

2 IBA Rt

A. nilotica Cotyledon B5 1.5 BA MSt Dewan et al. (1992)

2 IAA Rt

A. auriculiformis, Axillary bud B5 CW + BA MSt Gupta and Agrawal (1992)

A. nilotica CW + NAA/IAA Rt

A. saligna Shoot tip 'MS 11 zeatin MSt Badji et al. (1993)

' Jordan's 9.0 NAA Rt

A. auriculiformis Shoot MS BA MSt Das et al. (1993)

1-1.5 IBA Rt

A. albida Shoot MS 0.02 NAA MSt Ruredzo and Hanson (1993)

PGR-free Rt

A. mangium Node MS 10 iM BAP MSt Saito et al. (1993)

'MS 10 iM IAA Rt

A. nilotica Node MS, SH, B5, WPM BA, AdS MSt Singh et al. (1993)

A. albida Excised root '/5MS or 9 m-inositol MSt Ahee and Duhoux (1994)

BD or 0.1 NAA Rt

A. tortilis Axillary bud MS 0.5 BAP MSt Detrez (1994)

A. senegal Axillary bud, Node MS BA, NAA MSt Gupta et al. (1994)

A. mearnsii In vitro shoot tip MS 2 BA MSt Huang et al. (1994)

0.6 NAA Rt

A. auriculiformis Cotyledon axillae 'MS 2 BA MSt Ide et al. (1994)

No PGR or 0.02 NAA Rt

A. tortilis Cotyledon node MS 0.1 NAA + 5 BA MSt Macrae (1994)

A. auriculiformis Shoot, axillary bud 'MS 0.02 NAA + 1 BA + GA3 MSt Wantanabe et al. (1994)

PGR-free or 0.02 NAA Rt

A. auriculiformis Axillary bud MS GA3 + NAA + IBA MSt Reddy et al. (1995)

A. auriculiformis, Hypocotyl 'MS 1-2 BA MSt Toda et al. (1995)

A. mangium IBA and NAA Rt

A. auriculiformis Shoot bud MS PGR-free MSt Rt Zhang et al. (1995)

A. meamsii Node MS 3 BA + 0.05 IBA MSt Correia and Graca (1995)

1 IBA Rt

(continued on next page)

Table 1 (continued)

Species Expiant Basal medium PGR (mg/l) Result Reference

A. tortilis Cotyledon node MS 0.1 NAA + 5 BA MSt Nandwani (1995)

¿MS 3 IBA Rt

A. tortilis Cotyledon node MS kinetin, BA, IBA MSt Nangia and Singh (1996)

A. mangium Node MS 3 BA + 0.1 NAA + 100 ascorbic MSt Bhaskar and Subhash (1996)

1 IBA + 0.5 IAA Rt

A. catechu Immature cotyledon WPM, MS kinetin, NAA MSt Das et al. (1996)

A. mearnsii Node MS 2 BA MSt Beck et al. (1998a)

1 IBA Rt

A. mearnsii Coppice MS 2 BA MSt Beck et al. (1998b)

A. mangium Node MS, B5, 4.4 iM BA + 2.5 iM IBA MSt Bon et al. (1998)


A. catechu Node MS 4 BA + 0.5 NAA + 25 AdS + 20 MSt Kaur et al. (1998)

ascorbic acid +150 glutamine

¿MS 3 IAA Rt

A. seyal Shoot tip MS 0.5 NAA + 4 BA MSt Al-Wasel (2000)

4 IBA Rt

A. mearnsii Meristem 'MS; 2 BA or PGR-free MSt Beck et al. (2000)


A. mangium Shoot MS 4 iM IAA Rt Monteuuis and Bon (2000)

A. mangium Seedling MS 4.4 iM BA MSt Monteuuis and Bon (2000)

SH 4 iM IAA Rt

A. catechu Shoot tip MS 1.5 BAP + 1.5 kinetin MSt Kaur and Kant (2000)

¿MS 3 IAA Rt

A. mangium Cotyledon node DKW, B5 2.2 BA MSt Douglas and McNamara


A. mearnsii Node %MS BA, GA3 MSt Quoirin et al. (2001)

A. tortilis, Node MS 2.5 BA MSt Aziz et al. (2002)

A. nilotica ¿MS 4 IBA Rt

A. sinuata Cotyledon node MS 6.66 iM BAP + 4.65 iM kinetin MSt Vengadesan et al. (2002b)

¿MS 7.36 iM IBA Rt

A. .sinuata Node MS 8.9 iM BA + 2.5 iM TDZ + 135.7 MSt Vengadesan et al. (2003b)

iM AdS

¿MS 7.4 iM IBA Rt

A. mangium Node MS 1.5 BAP + 0.05 IAA + 100 AdS MSt Nanda et al. (2004)

¿MS 0.5 IAA Rt

A. mangium Shoot SH 8 iM NAA Rt Monteuuis (2004a)

A. mangium Shoot SH 4 iM IAA Rt Monteuuis (2004b)

A. senegal Node MS 1 BA MSt Khalafalla and Daffalla (2008)

1 IBA Rt

A. chundra Shoot tip, Node MS 1.5 BA + 0.01-0.05 IAA + 50 AdS MSt Rout et al. (2008)

0.25 IBA Rt

A. nilotica Seed MS or B5 2 BAP + 0.5 NAA MSt Abbas et al. (2010)

3 IAA Rt

A. nilotica Node MS 0.6 NAA MSt Dhabhai et al. (2010)

¿MS 0.5 IBA Rt

A. auriculiformis Node MS 2 BAP + 0.1 NAA MSt Girijashankar (2011)

¿MS PGR-free Rt

A. farnesiana Node MS 1 BA MSt Khalisi and Al-Joboury (2012)

¿MS 0.5 IBA + 0.05 NAA Rt

A. auriculiformis Cotyledon MS 2 2iP MSt Banerjee (2013)

1 NAA Rt

A. ehrenbergiana Cotyledon node MS 10 iM BA + 0.1 iM NAA MSt Javed et al. (2013)

5 iM IBA Rt

A. mangium Cotyledon node MS 4 iM BA MSt Shahinozzaman et al. (2012)

8 iM IBA Rt

A. mangium Cotyledon MS 2 iM BA + 1 iM NAA MSt Shahinozzaman et al. (2013)

8 iM IBA Rt

Table 1 (continued)



Basal medium

PGR (mg/l)

Result Reference

A. nilotica

A. mangium x A. auriculiformis A. auriculiformis Shoot

Nodal segment

MS (liquid)


4.4 iM BAP 2.46 mM IBA

1.5 BA + 0.1 NAA 600 IBA

2 kinetin + 0.5 IAA 0.1 IAA

MSt Rathore et al. (2014) Rt

MSt Qiong et al. (2015) Rt

MSt Yadav et al. (2015) Rt

2,4-D - 2,4-dichlorophenoxy acetic acid, AdS - Adenine sulfate, B5 - (Gamborg et al., 1968), BA - N6-benzyladenine, BAP - N6-benzylaminopurine, BD - Bonner Devirian medium (Bonner and Devirian, 1939), CW - Coconut water, DKW - Driver Kuniyuki medium (Driver and Kuniyuki, 1984), GA3 - Gibberellin A3, IAA - Indole-3-acetic acid, IBA - Indole-3-butyric acid, Kinetin 6-furfurylaminopurine, KT - Kathju Tewari medium (Kathju and Tewari, 1973), MS - Murashige Skoog medium (Murashige and Skoog, 1962), MSt - Multiple shoot; NAA - a-naphthalene acetic acid, PGR - Plant growth regulator, Q-LP - Quoirin Lepoivre medium (Quoirin and Lepoivre, 1977), Rt - Root, SH - Schenk and Hildebrandt medium (Schenk and Hildebrandt, 1972), TDZ - N-phenyl-N'-(1,2,3-thiadiazol-5-yl)urea or Thidiazuron, WPM - Woody Plant Medium (Lloyd and McCown, 1981).

and media discoloration considerably influence shoot regeneration even from different species of the same genus (Gantait et al., 2014). Tables 1 and 2 synopsize the in vitro propagation related research achievements on genus Acacia, exclusively on how several factors influence the regeneration of different species of this genus that was not adequately discussed in the other reports (Beck and Dunlop, 2001; Quoirin, 2003) with the only exception (Vengadesan et al., 2002a).

2.1. Role of explant source

In the cases of in vitro propagation, the nature of the plant material exploited considerably influences its multiplication and proliferation. It is necessary for any study to choose a suitable explant prior to tissue culture. The growth rate of explant of various organs varies while some do not grow at all. The frequently exploited explants are the meristematic portions for instance the root tip, stem tip and axillary bud tip. Rates of cell division are higher in these tissues and presumably produce the much-needed growth-regulating substances such as auxins and cytokinins (Akin-Idowu et al., 2009). Although, an array of explants, for example leaves, shoot tips, axillary buds, cotyledon (Fig. 1) and nodal segments have been widely utilized for successful initiation of in vitro direct organogenesis in genus Acacia (Table 1), nodes were more thriving to stimulate multiple shoots per explant (Kaur et al., 1998; Quoirin et al., 2001; Aziz et al., 2002; Vengadesan et al., 2002b, 2003a; Rout et al., 2008; Nanda et al., 2004; Khalafalla and Daffalla, 2008; Dhabhai et al., 2010; Girijashankar, 2011; Khalisi and Al-Joboury, 2012; Rathore et al., 2014). Other explants were not as effective as nodes particularly for in vitro direct multiplication. Apart from several plant tissues or organs mentioned (Table 1), employment of seeds for multiple shoot proliferation was reported by Abbas et al. (2010) in Acacia nilotica subsp. hemispherica. Impact of choice of explants on in vitro indirect organogenesis of Acacia was also evident (described in Table 2). Induction and proliferation of calli from a variety of explants, involving leaf (Fig. 1f) (Tanabe and Honda, 1999; Xie and Hong, 2001a; Vengadesan et al., 2002c; Yang et al., 2006; Arumugam et al., 2009; Thambiraj and Paulsamy, 2012), stem (Hustache

et al., 1986), cotyledons (Fig. 1d) (Rout et al., 1995; Das et al., 1996; Vengadesan et al., 2003b; Rathore et al., 2012), immature zygotic embryo (Xie and Hong, 2001b; Nanda and Rout, 2003), and hypocotyls (Fig. 1e) (Vengadesan et al., 2000) were achieved fruitfully. Leaf explants had shown to put on highest frequency of indirect regeneration in comparison with other explants used for organogenesis through callus culture as the mesophyll cells present in the leaf tissues are generally undifferentiated and might be more totipotent to undergo dedifferentiation.

2.2. Role of surface disinfection procedure

The surface sterilization practice holds a vital importance in plant tissue culture techniques. A superior surface sterilant should undergo least plant damage, while diminishing micro-bial contamination to a much tolerable level. Initiation of in vitro aseptic culture depends on the developmental status of the explant as well as the vulnerability of the plant species to numerous pathogenic contaminants (Gantait et al., 2014). Application of 70% (v/v) ethanol for 10 s, followed by dipping in 1.5% (v/v) sodium hypochlorite (NaOCl) solution was the preliminary approach of surface disinfection for Acacia as reported by Tamura et al. (1984). Sterilization using NaOCl solution following 70% ethanol (5-7 min) has been successful in many of the cases (Dewan et al., 1992; Arumugam et al., 2009; Girijashankar, 2011). Abbas et al. (2010) employed 95% ethanol for 20 s and subsequently 10% NaOCl solution containing 3-6 drops of Tween 20. For most of the Acacia explants, the commonly accepted technique entails surface sterilization with 70% ethanol for 30-90 s trailed by fresh-made 0.1% (w/v) HgCl2 for 5-10 min and repetitive washing in sterilized water (Rout et al., 2008; Dhabhai and Batra, 2010; Rathore et al., 2012; Banerjee, 2013; Monteuuis et al., 2013; Nagashree et al., 2015; Shahinozzaman et al., 2013; Javed et al., 2013). Vengadesan et al. (2000) soaked the seed explants for 15 min in concentrated H2SO4 to provide consistent regeneration as well as to disinfect them prior to treatment with 0.1% HgCl2. But, HgCl2 has been reported to be an environmentally hazardous chemical (Saha, 1972). In addition, plant growth and propagation are negatively affected by heavy

Table 2 Achievements on indirect in vitro organogenesis/embryogenesis of Acacia (arranged : in chronological order).

Species Explant Medium PGR (mg/l) Result Reference

Acacia koa Shoot tip SH 0.2 2,4-D Ca Skolmen and Mapes (1976)


0.2 IBA Rt

A. senegal Stem KB 2 IAA Ca Hustache et al. (1986)

A. melanoxylon Shoot MS 0.2 BA + 0.2 IAA Ca Meyer and Van Staden (1987)

A. salicina/A. saligna/ Node, internode, phyllode IAA or IBA + BA Ca Jones et al. (1990)

A. sclerosperma SR

1.8 IBA Rt

A. mangium Hypocotyl MS 1000 casein hydrolysate + 3 Ca Gong et al. (1991)

NAA + 1 BA

A. catechu Immature cotyledon WPM 3 kinetin + 0.5 NAA Ca Rout et al. (1995)

3 kinetin + 0.5 NAA + 104- SE



A. catechu Immature cotyledon WPM, MS Kinetin, NAA SE Das et al. (1996)

A. nilotica Endosperm culture MS 2,4-D, BA SE Garg et al. (1996)

Acacia mangium 'MS 0.5 kinetin or 0.4 TDZ + 0.5 Ca Quoirin et al. (1998)

A. koa Leaf MS 4.4 iM BA Ca Tanabe and Honda (1999)

A. afarnesiana, Immature zygotic embryo MS 9.05 iM 2,4-D + 4.65 iM kinetin Ca Ortiz et al. (2000)

A. schaffneri No PGR SE

217 iM AdS SR

A. sinuata Hypocotyl MS 6.78 iM 2,4-D + 2.22 iM BAP Ca Vengadesan et al. (2000)

13.2 iM BAP + 3.42 iM IAA SR

'MS 7.36 iM IBA Rt

A. mangium Cotyledon, zygotic MS 9.05 iM 2,4-D + 13.95 iM Ca Xie and Hong (2001a)

embryo, kinetin

leaf, petiole 4.55 iM TDZ + 1.43 iM IAA SR

0.75 iM NAA + 2.33 iM kinetin Rt

A. mangium Immature zygotic embryo 'MS 1-2 TDZ + 0.25-2 IAA SE Xie and Hong (2001b)

5 GA3 SR

A. sinuata Leaf MS 4.52 iM 2,4-D + 2.22 iM BAP Ca Vengadesan et al. (2002c)

MS 4.52 iM 2,4-D + 10% CW SE

(liquid) PGR-free SR

A. arabica Immature zygotic embryo MS 8.88 iM BA + 6.78 iM 2,4-D Ca Nanda and Rout (2003)

6.66 iM BA + 6.78 iM 2,4-D SR

'MS 0.04 iM BA + 0.94 iM IBA Rt

A. sinuata Cotyledon MS 8.1 iM NAA + 2.2 iM BAP Ca Vengadesan et al. (2003a)

'MS 13.3 iM BA + 2.5 iM zeatin SR

'MS 7.4 iM IBA Rt

A. crassicarpa Leaf MS 0.5 TDZ + 0.5 NAA Ca Yang et al. (2006)

'MS 0.5 IBA Rt

Table 2 (continued)

Species Expiant Medium PGR (mg/l) Result Reference

A. confusa Leaf MS 3 2,4-D + 0.01 NAA + 0.05 Ca Arumugam et al. (2009)


WPM 3 BA + 0.05 NAA + 0.1 zeatin SR

+ 5 AdS

MS 4 IBA + 0.05 kinetin Rt

A. nilotica Cotyledon MS 0.4 2,4-D + 0.2 BAP Ca Dhabhai and Batra

0.4 2,4-D + 0.2 BAP + 200 AC SR (2010)

'MS 0.5 IBA Rt

A. senegal Cotyledon MS 0.45 цМ 2,4-D + 2.32 цМ kinetin SE Rathore et al. (2012)

0.22 цМ BAP SR

A. caesia Leaf MS 1.5 TDZ + 0.3 NAA Ca Thambiraj and Paulsamy

2 IBA + 0.5 TDZ SR (2012)

2 IBA + 0.5 kinetin Rt

A. auriculiformis Cotyledon MS 0.2 2iP + 4 NAA Ca Banerjee (2013)

2 2iP + 0.2 NAA SR

1 NAA Rt

2,4-D - 2,4-dichlorophenoxy acetic acid, 2iP - N6-(2-isopentenyl) adenine, AC - Activated charcoal, AdS - Adenine sulfate, BA - N6-benzyladenine, BAP - N6-benzylaminopurine, Ca - Callus; CW - Coconut water, IAA - Indole-3-acetic acid, IBA - Indole-3-butyric acid, KB -Knop and Ball medium (Hustache et al., 1986), Kinetin 6-furfurylaminopurine, MS - Murashige Skoog medium (Murashige and Skoog, 1962), NAA - a-naphthalene acetic acid, PGR - Plant growth regulator, SE - Somatic embryogenesis, SH - Schenk and Hildebrandt medium (Schenk and Hildebrandt, 1972), SR - Adventitious shoot regeneration, Rt - Root, TDZ - N-phenyl-N'-(1,2,3-thiadiazol-5-yl) urea or Thidiazuron, WPM - Woody Plant Medium (Lloyd and McCown, 1981), Zeatin - 4-hydroxy-3-methyl-terms-2-butenyl aminopurine.

metals. In an experiment conducted by Thompson et al. (2009), it has been accounted that 25% (w/v) Jik is a preferable substitute to other sterilants and much safer than HgCl2. He also noted that when explants were exposed to sterilants for 10min they responded significantly better in terms of shoot initiation, than other exposure times tested. Explants exposed for 30 min resulted in an effect that was detrimental to shoot proliferation. The type and exposure duration both manipulate the response of the plant material to organogenesis. These factors influence individually as there is no known interaction. Thambiraj and Paulsamy (2012) utilized various antibiotics before using any surface sterilants. To eliminate fungal contamination he employed carbendazim (50%, w/v) and fungicide (10%) for 15 min and to eliminate bacterial contamination he treated the explants with 5% (w/v) antibiotics (ampicillin and rifampicin) for 30 min followed by a rinsing with sterilized double distilled water. Interestingly, Salehi and Khosh-Khui (1997) in a study with rose, used antibiotics (gentamycin, ampicillin, tetracycline or amoxicillin) at different concentrations and durations for the purpose of disinfection from internal contaminants, noticed that use of an antibiotic solution before surface sterilization was unsuccessful but found highest percentage of disinfected explants when 100mg/l solution of gentamycin or ampicillin was used after surface sterilization. The difference prevailed, since, during surface sterilization the fresh conducting tissue gets exposed by cutting the ends of the explants through which the antibiotic solution percolates down inside the tissue that results in higher frequency of disinfection. The issue of endogenous contamination cannot be totally inhibited by the use of surface sterilants so, a search for more prominent systemic sterilant that spreads efficiently throughout the plant material, remains a strong possibility for research in case of Acacia.

2.3. Role of basal media

The pace of tissue proliferation and the quality of morpho-genetic responses depend upon the type and concentration of mineral nutrients supplied in different types of media. Majority of the scientists recommended semi-solid full strength Murashige and Skoog (1962) (MS) medium for shoot initiation in Acacia (Barakat and El-Lakany, 1992; Das et al., 1993; Zhang et al., 1995; Monteuuis and Bon, 2000; Khalafalla and Daffalla, 2008; Shahinozzaman et al., 2012, 2013; Banerjee, 2013; Yadav et al., 2015). Adjustment in the MS medium for example reduction of MS salts to one half, one third or three fourth was also successful in various species. Ide et al. (1994), Wantanabe et al. (1994), Toda et al. (1995) employed %MS basal medium for initiation of multiple shoot. Also, Dhabhai et al. (2010), Girijashankar (2011), Khalisi and Al-Joboury (2012), Yadav et al. (2015) achieved better root induction in y2MS. Even, %MS confirmed to be adequate for multiple shoot induction (Kaur et al., 1998). Utilization of liquid MS medium is reported by Rathore et al. (2014), since the cost of plant production in commercial scale is much less in liquid medium. Moreover, MS basal medium supported callus induction, subsequently shoot and root formation (Garg et al., 1996; Ortiz et al., 2000; Rathore et al., 2012; Banerjee, 2013). On a contrary note, other media types were rarely reported like the B5 (Gamborg et al., 1968) (reported by Dewan et al., 1992; Gupta and Agrawal, 1992; Douglas and

McNamara, 2000), White (White, 1938), SH (Schenk and Hildebrandt, 1972), KT (Kathju and Tewari, 1973), and Woody Plant Medium (WPM) (Lloyd and McCown, 1981) (reported by Skolmen and Mapes, 1976; Mittal et al., 1989; Semsuntud and Nitiwattanachai, 1991; Rout et al., 1995). Badji et al. (1993) initially used MS medium for the initiation of shoots but later on the roots were successfully induced in a Jordan's medium (Jordan et al., 1978) containing high concentration of auxin that produced 100% roots. Monteuuis (2004a) in an experiment for rooting found y2MS unfavorable, whereas higher responsiveness was observed for the same material when 73SH macronutrients were employed. Ahee and Duhoux (1994) compared three basal media for root culture in vitro. They employed White, BDM, 2xBDM (Bonner and Devirian, 1939 modified by Goforth and Torrey, 1977) and V5MS medium. Contrastingly, they observed that the root growth was significantly higher in BDM and 2xBDM media than in White or 1/5MS media. Hustache et al. (1986) accounted that the best mineral medium for callus induction was the Knop and Ball (KB) medium. In a comparative experiment of organogenesis using MS and B5 by Abbas et al. (2010) it was observed that the MS media was more apposite than the B5 medium, resulting in higher shoot regeneration frequency. Similar comparative experiment was also performed by Rout et al. (1995) where the relative performance of MS was assessed alongside WPM for somatic embryogenesis. He accounted that somatic embryogenesis occurred only on WPM. Nevertheless, to promote germination, the somatic embryos were required to be inoculated onto y2MS basal medium without any PGR. Shahinozzaman et al. (2012) further analyzed the differential effect of basal media on shoot proliferation utilizing MS and WPM as experimental media. Maximum explants produced highest number of shoots on MS medium; however, explants produced longest shoots in WPM medium.

2.4. Role of carbohydrate source

Carbohydrates are one of the most indispensable substances required for growth and organized development (Gamborg et al., 1976), and are essential as an energy source, providing carbon skeletons for biosynthetic processes as well. Presence of sucrose in the culture medium is essential for different metabolic activities. It is necessary for differentiation of xylem and phloem elements in cultured cells (Aloni, 1980). The nutritional necessities and the capacity of plant tissues to absorb sucrose differ from species to species. Murashige and Skoog (1962) recommended the application of 3% (w/v) sucrose since it possesses added proficiency for regeneration of in vitro explants in comparison with the other concentrations. In the first report of in vitro culture of Acacia, Hustache et al. (1986) recommended the use of 3% (w/v) glucose for optimized callus induction and cell suspension culture. Later, most of the researchers confirming the use of MS medium successfully cultured Acacia in vitro by the utilization of 3% sucrose both for direct (Ahmad, 1989; Abbas et al., 2010; Girijashankar, 2011; Javed et al., 2013; Yadav et al., 2015) and indirect organogenesis (Vengadesan et al., 2000; Nanda and Rout, 2003; Yang et al., 2006; Dhabhai and Batra, 2010; Rathore et al., 2012; Banerjee, 2013). Earlier, Badji et al. (1993) reported 2% (w/v) saccharose to be adequate for optimal shoot

multiplication and rhizogenesis. Ahee and Duhoux (1994) compared the two carbohydrate sources (glucose and sucrose) and concluded that the use of 59 mM sucrose proved better in terms of rooting. In an experiment carried out by varying concentrations of sucrose, Beck et al. (1998b) noted greater shoot production with 2% and 3% sucrose. On the contrary, there has been a wide range of carbohydrate sources in different concentrations. There are fewer reports where 2% sucrose showed promising results in bud initiation and multiplication (Monteuuis and Bon, 2000; Monteuuis et al., 2013) also in somatic embryogenesis (Rout et al., 1995; Rout and nanda, 2005). Even a lesser concentration (1.5%) of sucrose was employed in rooting media by Kaur and Kant (2000). Nevertheless, Rout et al. (1995) noted 2% sucrose to be more efficient for somatic embryogenesis induction. Douglas and Mcnamara (2000) in an experiment employed as high as 6% sucrose and noted frequent initiation of adventitious shoots and buds by cotyledon explants. From these studies it was further concluded that plant can readily utilize carbohydrate in the form of sucrose. However, it has been observed that there is variation in effect of carbohydrate on plant depending on its source and concentration. Furthermore, species specificity and formulation of maintenance medium might have additional influence on performance of carbohydrate. Even though scores of literatures were published regarding the uptake and consumption of exogenous carbohydrates by explants cultured in vitro, yet, data on the associations between the experimentation of source of carbon in the culture medium and the modification of sugar composition in in vitro cultured tissues are exiguous.

2.5. Role of plant growth regulators on direct organogenesis

Initiation of adventitious shoot directly from explants is a superior and positive approach for clonal propagation of plant. Asynchronous plants generally result from callus whereas homogeneous diploid individuals are formed from adventitious shoots (Bhojwani and Razdan, 1996). It is an efficient method to produce large-scale true-to-type plants. Variety of explants had been employed and inoculated in number of media formulations fortified with variable sources and measures of plant growth regulators for shoot regeneration in Acacia so far, which has been summarized in Table 1. Tamura et al. (1984) were the preliminary researchers to commence a technique for direct in vitro multiple shoots initiation and propagation in Acacia using high level of kinetin. The occurrence of cytokinin predominantly as PGR, in the growth medium is significant for shoot proliferation (Dave et al., 1980; Gassama, 1989; Darus, 1991; Galiana et al., 1991; Dewan et al., 1992; Huang et al., 1994; Monteuuis and Bon, 2000; Khalafalla and Daffalla, 2008; Khalisi and Al-Joboury, 2012; Shahinozzaman et al., 2013; Rathore et al., 2014). A variety of cytokinins such as N6-benzyladenine (BA), N6-(2-isopentenyl) adenine (2iP), 6-furfurylaminopurine (kinetin), and 4-hydroxy-3-methyl-terms-2-butenyl aminopurine (zeatin) has been used for Acacia micropropagation. Shahinozzaman et al. (2013) in an experiment reported that the highest number of shoots was obtained by utilizing a 4.0 iM BA containing medium which was much superior to kinetin for shoot multiplication of Acacia mangium. The superior effect of BA over kinetin in in vitro organogenesis has been accounted in

many species of Acacia (Mittal et al., 1989; Galiana et al., 1991; Dewan et al., 1992; Badji et al., 1993; Beck et al., 1998a,b; Vengadesan et al., 2002b; Khalafalla and Daffalla, 2008; Rout et al., 2008; Khalisi and Al-Joboury, 2012). Interestingly, although Dhabhai et al. (2010) observed direct regeneration on MS medium having only kinetin (1 mg/l) yet the proliferation remained undifferentiated for one month, until a-naphthalene acetic acid (NAA) (0.6 mg/l) was used, that in turn induced the multiplication of shoots almost instantly. The report displays the probable high endogenous cytokinin concentration. Badji et al. (1993) reported that zeatin which is a natural cytokinin, produced better induction to multiple shoot formation. There are various instances where utilization of a single source of PGR did not give much effect but a combination of the same promoted direct organogenesis much efficiently. Al-Wasel (2000) tested BA or N-phenyl-N0-(1,2,3-thiadiazol-5-yl) urea (Thidiazuron or TDZ) in association with NAA for their influence on shoot proliferation of Acacia seyal. It was observed that NAA could not induce shoot development when employed alone, and BA unaided produced very few shoots; although, a combination of BA and NAA underwent profuse regeneration. Rout et al. (2008) tried twenty different combinations of PGRs and found that incorporation of BA (1.5 mg/l), indole-3-acetic acid (IAA) (0.05 mg/l) along with adenine sulfate (AdS) (50 mg/l) to be the most efficient treatment to encourage shoot regeneration and multiplication. Abbas et al. (2010) found highest number of shoots and shoot regeneration frequency in the presence of 2.0 mg/l BAP and 0.5 mg/l NAA. Shahinozzaman et al. (2013) also reported that incorporation of auxin along with BA to the medium enhanced the frequency of shoot bud differentiation rather than using BA individually in the medium. In that study it was concluded that 2.0 iM BA plus 1.0 iM NAA was the most favorable PGR combination for direct shoot organogenesis. Recently, MS medium containing 2mg/lkinetin and 0.5 mg/l IAA exhibited maximum frequency of shoot regeneration (Yadav et al., 2015). Thus the relevance of cytokinin to auxin in a ratio confirmed to be efficient in high shoot regeneration instead of using cytokinin alone.

2.6. Role of plant growth regulators on callogenesis

Plant cells that proliferate in a disordered way and turn into amorphous mass of tissue are termed as callus (George et al., 2008). On the other hand, when callus is cultured in apposite conditions, it can experience differentiation and transforms into a whole new plant. Table 2 presents a compilation of research works that have been carried out to examine the efficiency of explants on callus induction with or without the use of different PGRs in Acacia. Callus induction from shoot tip occurred for the first time with only SH plus 0.2 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D) without any other PGRs or additives (Skolmen and Mapes, 1976). On the other hand, Tanabe and Honda (1999) induced callus from leaf explant with the supplementation of 4.4 iM BA only in the culture medium. Interestingly they found that the combination of BA and NAA had an antagonistic effect on callusing. However, combinations of auxin and cytokinin were found more effective for callus induction by the majority of the researchers. For instance, of 2,4-D:kinetin (Ortiz et al., 2000; Xie and Hong, 2001a; Rathore et al., 2012) or 2,4-D:N6-

benzylaminopurine (BAP) (Vengadesan et al., 2000, 2002c; Dhabhai and Batra, 2010) induced callus in a more skillful mode in contrast to a single use of PGR (either of auxin or cytokinin). Nanda and Rout (2003) tested various concentrations of BA, 2,4-D and kinetin alone or in combinations and accounted that the intensity of embryogenic callus proliferation was greatest in the media with 8.88 mM BA in association with 6.78 mM 2,4-D. There are also many instances where TDZ (a cytokinin like substance) in combination with an auxin happened to induce callogenesis more efficiently. Quoirin et al. (1998) demonstrated callus induction from explants at high rates for all combinations of NAA and TDZ than utilizing NAA alone. Xie and Hong (2001b) cultured the immature zygotic embryo in the medium containing 2.0 mg/l TDZ and 0.25 mg/l IAA and noted that it was very well capable of inducing embryogenic calli. Similarly, Yang et al. (2006) and Thambiraj and Paulsamy (2012) observed efficient callus formation as well as adventitious shoots on the medium containing a combination of TDZ and NAA. Banerjee (2013) tested various PGRs and found 2iP in association with NAA to be the best in terms of callus initiation and shoot bud regeneration from de-embryonated cotyledon. Importantly, the endogenous hormone levels of Acacia tissues play an important role and this is the main reason behind the variation of different types of explants on exposure to PGRs.

2.7. Role of plant growth regulators on somatic embryogenesis

Somatic embryogenesis conveys enormous potential to accelerate the propagation of woody species (Attree and Fowke, 1993). Somatic embryogenesis is generally used for large-scale production and genetic transformation. The insufficiency of knowledge in the fields of somatic embryogenesis, asynchronous production of somatic embryo and low frequency true to type embryonic competence, leads to the same being held responsible for its shortened commercial application in woody forest species. Accounting two reasons, somatic embryogenesis plays a significant role in forest biotechnology. First, this technique generates countless number of propagules for somatic embryo (Attree et al., 1994). Secondly, genetic transformation research could easily be carried out. Optimum nutrition and culture conditions are crucial for the conversion of somatic embryos into complete plantlets. Somatic embryogenesis encounters some practical applications in woody species, which has been successfully established. In comparison with other plant species, dynamic research on forest trees for somatic embryogenesis has been quite slow-moving. Hence, there are only a few reports on somatic embryogenesis in Acacia (Table 2). Induction of somatic embryogenesis is usually constrained to certain responsive cells of explants and largely determined by a specific developmental stage of the tissue (von Arnold et al., 2002; Rai et al., 2007). Rout et al. (1995) achieved somatic embryogenesis from callus, derived from immature cotyledons of Acacia catechu Willd. On WPM supplemented with 13.9 iM kinetin and 2.7 iM NAA. Moreover, the addition of 0.9-3.5 mM L-proline to the medium induced the somatic embryos to develop. On the other hand, Ortiz et al. (2000) obtained the highest number of somatic embryos in Acacia farnesiana and Acacia schaffneri, in the media devoid of any PGRs but with the addition of ABA the percentage of embryos that reached more advanced

differentiation stages increased. But, Xie and Hong (2001b) found the medium containing 2.0 mg/l TDZ and 0.25 mg/l IAA to be the most efficient for inducing embryogenic calli followed by somatic embryos. The combinations of 2,4-D and kinetin or 2,4-D and BA did not induce somatic embryogenic calli in A. mangium. To induce somatic embryo maturation, a two-step procedure involving gibberellin A3 (GA3) and high concentrations of sucrose was found to be effective indicating the importance of GA3 in promoting somatic embryo maturation in this species. Interestingly, Vengadesan et al. (2002c) studied exclusively the different effects of auxins, cytokinins, carbohydrates, amino acids and casein hydrolysate on production frequency of somatic embryogenesis in suspension culture. Among the auxins (IAA, NAA and 2,4-D) and cytokinins (BA and kinetin) tested, only 2,4-D was effective in inducing and producing somatic embryos. Cytokinins, individually or in combination with any auxin, did not produce somatic embryos. But addition of glutamine enhanced the production of somatic embryos. The findings were in relevance with Vengadesan et al. (2002c) that particularly suggested that 2,4-D is required as vital supplement for the induction of somatic embryogenesis. Casein hydrolyzate was essential for somatic embryogenesis in Phaseolus vulgaris (Martnus and Sondahl, 1984) and Nigella sativa (Banerjee and Gupta, 1976) but it was not effective in case of Acacia sinuata. Rathore et al. (2012) investigated that cotyledons isolated from immature seeds were able to produce somatic embryos on induction medium, whereas cotyledons obtained from mature seeds failed to induce somatic embryo. The differential responses to combination of PGRs are presumably due to the difference in genotype and endogenous hormones present within the type of explant used. Further, the frequency and intensity of somatic embryogenesis was enhanced significantly by the addition of amino acids as reported earlier by other researchers (Rout et al., 1995; Ortiz et al., 2000; Vengadesan et al., 2002c). The medium supplemented with 15 mM L-glutamine increased the production frequency of somatic embryos. L-asparagine and L-arginine did not have a positive effect on the induction of somatic embryogenesis. It was reported that, L-glutamine was frequently used as a source of organic nitrogen in plant tissue culture which provides reduced nitrogen to plant tissues (Barrett et al., 1997) and enhances the synthesis of certain metabolites (Deo et al., 2010). On a contrary note, most of the embryos showed a tendency to lose their germination potential and perish if continued to be on the same development and maturation medium for longer duration. Therefore, the embryos had to be removed from this medium and transferred to growth regulator free or BAP containing medium for their germination. The maximum percentage of germination of somatic embryos was recorded on medium containing 0.22 iM BAP (Rathore et al., 2012).

2.8. Role of plant growth regulators on rooting in vitro

In vitro root induction varied usually with species, explant source and the supplied PGR. Generally explants utilizing juvenile plant parts root more effectively and easily than the mature parts, due to the presence of meristematic tissue. Several researchers deliberated the impact of PGRs in in vitro root initiation of Acacia (Fig. 1c, h). Initially, Williams et al. (1985), Darus (1989) noticed effective root

induction in MS medium from in vitro multiple shoots devoid of PGR. Girijashankar (2011) also noted rooting in %MS without addition of any PGR. Nonetheless, IAA, indole-3-butyric acid (IBA), or NAA is vastly and individually used as the sole PGR source for root induction and has proven competent enough to be used by most of the researchers (Mittal et al., 1989; Dewan et al., 1992; Ahee and Duhoux, 1994; Beck et al., 1998a; Al-Wasel, 2000; Khalafalla and Daffalla, 2008; Shahinozzaman et al., 2012, 2013; Banerjee, 2013; Rathore et al., 2014; Yadav et al., 2015). Among these three auxin sources employed in variable concentrations, suitability of IAA or IBA or NAA was reported to be different in different literatures. Superiority of IBA over IAA or NAA was substantially observed by the majority of researchers (Vengadesan et al., 2000; Khalafalla and Daffalla, 2008; Arumugam et al., 2009; Dhabhai et al., 2010; Shahinozzaman et al., 2012; Javed et al., 2013). Nevertheless, suitability of IAA over IBA was also evident in a number of instances (Kaur et al., 1998; Kaur and Kant, 2000; Abbas et al., 2010).There are fewer casein Acacia where IBA was used in fortification with IAA (Semsuntud and Nitiwattanachai, 1991; Bhaskar and Subhash, 1996) or NAA (Khalisi and Al-Joboury, 2012; Bhaskar and Subhash, 1996) to suffice each other to overcome the problem associated with poor root initiation. Interestingly, the use of PGRs in any of the combinations, for instance IAA plus IBA, IAA plus NAA or IBA plus NAA failed to induce rooting, rather they caused callusing and yellowing of shoots (Rout et al., 2008; Nanda et al., 2004). Engagement of NAA, a source of auxin during successful root induction in Acacia was reported by several researchers (Duhoux and Davies, 1985; Mittal et al., 1989; Ruredzo and Hanson, 1993; Huang et al., 1994; Wantanabe et al., 1994; Monteuuis, 2004a; Banerjee, 2013). There are few instances where use of only NAA was not sufficient for root initiation; rather combinations of NAA plus IBA were required (Jones et al., 1990; Semsuntud and Nitiwattanachai, 1991; Khalisi and Al-Joboury, 2012). Reports on usage of PGR in combinations of auxin and cytokinin are less, though Xie and Hong (2001a), Nanda and Rout (2003), Arumugam et al. (2009), Khalisi and Al-Joboury (2012) along with Thambiraj and Paulsamy (2012) examined the complementary effect of the combined usage of auxin:cytokinin on root induction in Acacia and observed root development to be successful in the existence of BA or kinetin (as cytokinin) in association with NAA or IBA (as auxin). The fact that root induction and successive elongation is accelerated by the utilization of activated charcoal (AC) in the culture medium combined with an auxin has been surveyed by Gantait et al. (2011). It was stated further that AC enhances rooting as it eradicates light and offers a practical atmosphere for the rhizosphere (Gantait and Mandal, 2010). Hence, the application of AC can also be tested in Acacia rooting efficiency.

3. Substrate-based acclimatization

For successful micropropagation of Acacia, acclimatization of in vitro plantlets is a significant phase. Influence of substrate media, temperature, light, and humidity was generally assessed during acclimatization of in vitro regenerated Acacia plantlets. Rapid desiccation of plantlets and its susceptibility to bacterial

and fungal diseases makes the acclimatization procedure more difficult. Rout et al. (2008) reported acclimatization and subsequent greenhouse establishment of in vitro plantlets by relocating them to a mixture of garden soil and sand at a ratio of 1:1 (v/v). Survival rate of 100% was obtained at the hardening phase when a substrate cocopeat was used up (Girijashankar, 2011). Later on, Javed et al. (2013) used sterile soilrite in plastic pots and covered the plantlets with polythene bags to maintain relative humidity. Further, %MS solution was sprayed every three days for two weeks. Later on acclimatized plants were shifted to normal garden soil in greenhouse under natural light. Various researchers added a range of organic substance in the substrate. For instance, Nanda and Rout (2003) mixed sand, cow-dung, soil together at a ratio of 1:1:1 (v/v) and the plantlets were placed inside a greenhouse. Likewise, Shahinozzaman et al. (2013) transferred the plantlets to a mixture of sand, garden soil and compost in 1:1:1 (v/v) ratio which gave high survival frequency during acclimatization of Acacia plantlets in the greenhouse. On the other hand, vermiculite in the substrate ameliorated the survival rate. Dhabhai et al. (2010) transferred the plantlets to polycups containing vermicompost and autoclaved soil (1:3; v/v). A mixture of sand, vermiculite, and garden soil at a ratio of 1:1:2 (v/v) revealed high rates of survival and displayed vigorous growth (Arumugam et al., 2009; Thambiraj and Paulsamy, 2012). A range of 70-85% relative humidity was maintained in the growth chamber (Nanda and Rout, 2003; Rout et al., 2008; Thambiraj and Paulsamy, 2012).

4. Marker-assisted genetic fidelity assay

Micropropagation of a species ensures true to type genotype by easy means for afforestation, biomass production and preservation of valuable and rare germplasm. At the moment, clonal forestry is a key interest in the modern research since the demand for wood is ever-increasing and it will continue throughout the next few decades (Fenning and Gershenzon, 2002). Usually, timbered plants are problematic to regenerate in in vitro environment. Nevertheless, a small number of procedures are there to confirm the genetic fidelity involving forest tree species for commercial purpose. Genetic clonality is a key concern in commercial micropropagation via in vitro tissue culture approach since true-to-type clones are the most critical prerequisites. A key setback confronted with the in vitro culture is the occurrence of somaclonal variation in the midst of sub-clones of one parental line, arising as a result of in vitro culture. DNA methylation, point mutations and chromosome rearrangements are the major causes of somaclonal variation, which arises due to in vitro stresses (Phillips et al., 1994). Accordingly, an appraisal to confirm true-to-type propagules at an early stage of development is considered to be crucial in Acacia in vitro culture. Molecular, cytological or biochemical assays are the key approaches to determine clonal fidelity of in vitro generated plantlets. A superior approach for genetic stability assay can be made by employing an assay of molecular markers that could amplify manifold regions of the genome (Martins et al., 2004; Gantait et al., 2012). PCR-based molecular markers such as RAPD, ISSR, and SSR have been found to be enormously helpful in ascertaining the genetic fidelity of in vivo cultivated as well as in vitro regenerated plants with

medicinal importance, such as aloe (Gantait et al., 2010a, 2011) and allium (Gantait et al., 2010b). For Acacia genus, there is only one report present on the assessment of clonal fidelity published by Nanda et al. (2004) in A. mangium Willd. Where RAPD as molecular marker was employed. A total of 20 arbitrary 10-base primers were utilized for Polymerase Chain Reaction. Out of the different primers tested, only three (0PC-04, OPD-14 and OPC-19) were successful in amplifying the products that were monomorphic across all the micropropagated plants. Other primers produced limited number of monomorphic bands. This technology needs to be exploited more in order to assess the genetic variation if occurred.

5. Future outlook

The reports accessible so far on in vitro intervention in Acacia, are predominantly focused on the development of regeneration protocol, somaclonal variations and its physiological as well as morphological aspects. A competent plant regeneration protocol is a must for the utilization of a range of biotechnological techniques. It can serve as a platform to transmit economically imperative traits through genetic engineering, cryoconserva-tion, inducing somaclonal variations, in vitro mutations, double haploids induction, development and utilization of somatic hybrids in Acacia. A remarkable progress can be achieved in biotechnological improvement on Acacia through the tissue culture-based advanced approaches. The present review endows with a wide-ranging assessment of the in vitro literature of Acacia to date, which will aid in the advance research of Acacia biotechnology.

Authors' contribution

SG and PKD conceived the idea of the review; SG and SK surveyed the literature and wrote the draft manuscript; and SG and PKD scrutinized and corrected final version of the manuscript. All the three authors approved the final version of the manuscript prior to submission.

Conflict of interest

We, the authors of this article, declare that there is no conflict of interest and we do not have any financial gain from it.


Authors acknowledge the library and laboratory assistance from the Department of Genetics and Plant Breeding, Faculty of Agriculture, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, Nadia, West Bengal, India. Authors further are thankful to the anonymous reviewers and the editor of this article for their critical comments and suggestions on the manuscript.


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