acacia: an exclusive survey on in vitro propagation · somatic embryogenesis; woody plant abstract...

Journal of the Saudi Society of Agricultural Sciences (2016) xxx, xxx–xxx

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REVIEW ARTICLE

Acacia: An exclusive survey on in vitro propagation

Abbreviations: 2,4-D, 2,4-dichlorophenoxy acetic acid; AdS, adenine sulfate; B5, Gamborg et al. (1968); BA, N6-benzyladenine

N6-benzylaminopurine; BD, Bonner–Devirian medium (Bonner and Devirian, 1939); Ca, callus; CW, coconut water; DKW, Driver K

medium (Driver and Kuniyuki, 1984); GA3, gibberellin A3; IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; KB, Knop and Ball

(Hustache et al., 1986) Kinetin, 6-furfurylaminopurine; KT, Kathju Tewari medium (Kathju and Tewari, 1973); MSt, multiple sho

Murashige and Skoog medium (Murashige and Skoog, 1962); NAA, a-naphthalene acetic acid; PGR, plant growth regulator; Q-LP,

Lepoivre medium (Quoirin and Lepoivre, 1977); Rt, root; SH, Schenk and Hildebrandt medium (Schenk and Hildebrandt, 1972); SR, adve

shoot regeneration; TDZ, N-phenyl-N0-(1,2,3-thiadiazol-5-yl) urea or Thidiazuron; WPM, Woody Plant Medium (Lloyd and McCown

Zeatin, 4-hydroxy-3-methyl-terms-2-butenyl aminopurine.* Corresponding author.

E-mail address: [email protected] (S. Gantait).

Peer review under responsibility of King Saud University.

Production and hosting by Elsevier

http://dx.doi.org/10.1016/j.jssas.2016.03.0041658-077X � 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 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: Gantait, S. et al., Acacia: An exclusive survey on in vitro propagation. Journal of the Saudi Society of Agricultural Sciencehttp://dx.doi.org/10.1016/j.jssas.2016.03.004

Saikat Gantait a,*, Suprabuddha Kundu b, Prakash Kanti Das c

aAICRP on Groundnut, Directorate of Research, Bidhan Chandra Krishi Viswavidyalaya, Kalyani, Nadia, West Bengal 741235, IndiabDepartment of Agricultural Biotechnology, Faculty of Agriculture, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, Nadia,West Bengal 741252, IndiacDepartment of Agricultural Biotechnology, Faculty Centre for Integrated Rural Development and Management, School ofAgriculture 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

KEYWORDS

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 proto-

cols 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 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

; BAP,

uniyuki

medium

ot; MS,

Quoirin

ntitious

, 1981);

s (2016),

http://creativecommons.org/licenses/by-nc-nd/4.0/

mailto:[email protected]

http://dx.doi.org/10.1016/j.jssas.2016.03.004


http://www.sciencedirect.com/science/journal/1658077X


http://creativecommons.org/licenses/by-nc-nd/4.0/


2 S. Gantait et al.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002. In vitro organogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

2.1. Role of explant source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.2. Role of surface disinfection procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.3. Role of basal media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.4. Role of carbohydrate source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

2.5. Role of plant growth regulators on direct organogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.6. Role of plant growth regulators on callogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.7. Role of plant growth regulators on somatic embryogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

2.8. Role of plant growth regulators on rooting in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003. Substrate-based acclimatization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004. Marker-assisted genetic fidelity assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

5. Future outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Authors’ contribution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

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

1. Introduction

Since last three decades, the population in tropical countrieshas been rising at an annual rate of 2.8% and as a result the

overall forest area in those countries has been declining at0.8% per year. The year-wise afforestation and reforestationarea in those countries was projected to be 1.8 million haduring the period 1981–1990, ensuing in an annual net reduc-

tion in forest area of 13.6 (15.4–1.8) million ha (Kozai et al.,2000). Moreover, the decline in biomass of woody plantsowing to desertification in arid regions is remarkable as it acts

as a precursor of recent climate changes on several geographiczones. It has been foreseen that a demand for woody trans-plants will rise considerably in future decades for paper, tim-

ber, plantation, horticulture and furniture industries, as wellas, in environment conservation (Kozai et al., 1997). The usageof plant biomass can be an alternative to the overconsumptionof fossil fuels and thus lowers the atmospheric CO2 levels

which ultimately assuages climate changes. A steady supplyof quality planting materials becomes increasingly importantto satisfy the ever increasing growing demand that conven-

tional propagation based plantlet production fails. In vitropropagation system holds its merits over that of the conven-tional propagation since the in vitro system ascertains the phe-

notypically and genotypically uniform disease-free propagulesin a sustainable manner (Aitken-Christie et al., 1995). In thisreview we demonstrate the achievements made (based on

experimental results) on in vitro propagation system of animportant tropical tree legume genus Acacia, along withex 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 reforestand 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 Acaciaplays an enormously essential role. Majority of its species

generates exceptional firewood and a few are the source ofan affluent supply of tannin, protein, ink, paint, pulpwood,

Please cite this article in press as: Gantait, S. et al., Acacia: An exclusive survey on inhttp://dx.doi.org/10.1016/j.jssas.2016.03.004

flavoring agents, and gum. From the environmental perspec-

tive, Acacia can acclimatize to extreme atmospheric conditionsand consequently, can adapt to both arid and moist areas oftropical soils. Various species are capable of increasing soil

fertility by undergoing in a symbiotic association withRhizobium and Mycorrhizal fungi. Moreover, it minimizes soilerosion 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 manipula-tions of several plant growth regulators (PGRs) in culturemedia. Accelerated in vitro propagation is the unique feature

of plant tissue culture that has been credibly acknowledgedwith respect to its practicability in bulk and commercial-scalemultiplication of propagules. Successful in vitro regenerationof the plant material depends on numerous aspects such as

genetic makeup, explant type, media composition, PGRs aswell as the culture conditions. Direct regeneration and indirectregeneration via an intermediary callus phase are the two chief

fundamental approaches engaged as an efficient in vitro regen-eration of forest trees. Among these two approaches indirectorganogenesis is less enviable for clonal multiplication due to

its reported cases of somaclonal variability. Hence, directregeneration (devoid of callus-stage) is considered as a consis-tent approach for clonal propagation. A variety of in vitro

culture approaches, for instance de novo organogenesis,callogenesis, and somatic embryogenesis have been used com-prehensively for large-scale micropropagation and the produc-tion of genetically true clones in bulk quantities. Vigilant

selection and collection of explants, with apposite use of basalmedia, PGRs, antioxidants and additives are the fundamentalcriteria for standardizing consistent and reproducible micro-

propagation protocols. There have been scores of reports onin vitro growth and multiplication of Acacia attained throughembryogenesis or organogenesis. Nevertheless, explant source

and their disinfection process along with the media formula-tions, culture conditions, accumulation of phenolics in media

vitro propagation. Journal of the Saudi Society of Agricultural Sciences (2016),


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

Species Explant 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 Marthur and Chandra (1983)

Rt

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)

½MS 0.05 IBA Rt

A. auriculiformis Hypocotyl ½MS 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

+ 0.4 NAA

Rt Semsuntud and

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,

A. nilotica

Axillary bud B5 CW+ BA MSt Gupta and Agrawal (1992)

CW+NAA/IAA Rt

A. saligna Shoot tip ½MS 11 zeatin MSt Badji et al. (1993)

½ 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 lM BAP MSt Saito et al. (1993)

½MS 10 lM IAA Rt

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

A. albida Excised root 1/5MS or 9 m-inositol MSt Ahee and Duhoux (1994)

BD or 0.1 NAA Rt

White

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 ½MS 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 ½MS 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,

A. mangium

Hypocotyl ½MS 1–2 BA MSt Toda et al. (1995)

IBA and NAA Rt

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

Rt

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

1 IBA Rt

(continued on next page)

In vitro propagation of Acacia 3

Please cite this article in press as: Gantait, S. et al., Acacia: An exclusive survey on in vitro propagation. Journal of the Saudi Society of Agricultural Sciences (2016),http://dx.doi.org/10.1016/j.jssas.2016.03.004


Table 1 (continued)


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

½MS 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

acid

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 lM BA+ 2.5 lM IBA MSt Bon et al. (1998)

SH Rt

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

ascorbic acid + 150 glutamine

¼MS 3 IAA Rt

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

4 IBA Rt

½MS

A. mearnsii Meristem ½MS; 2 BA or PGR-free MSt Beck et al. (2000)

MS; WPM

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

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

SH 4 lM IAA Rt

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

¼MS 3 IAA Rt

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

(2000)

A. mearnsii Node ¾MS BA, GA3 MSt Quoirin et al. (2001)

A. tortilis,

A. nilotica

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

½MS 4 IBA Rt

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

½MS 7.36 lM IBA Rt

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

lM AdS

½MS 7.4 lM IBA Rt

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

½MS 0.5 IAA Rt

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

A. mangium Shoot SH 4 lM 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

½MS

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)

½MS 0.5 IBA Rt

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

½MS PGR-free Rt

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

½MS 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 lM BA+ 0.1 lM NAA MSt Javed et al. (2013)

5 lM IBA Rt

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

8 lM IBA Rt

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

8 lM IBA Rt

4 S. Gantait et al.



Table 1 (continued)


A. nilotica Node MS (liquid) 4.4 lM BAP MSt Rathore et al. (2014)

2.46 mM IBA Rt

A. mangium �A. auriculiformis

Nodal segment MS 1.5 BA+ 0.1 NAA MSt Qiong et al. (2015)

½MS 600 IBA Rt

A. auriculiformis Shoot MS 2 kinetin + 0.5 IAA MSt Yadav et al. (2015)

½MS 0.1 IAA 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-N0-(1,2,3-thiadiazol-5-yl)urea or Thidiazuron, WPM

– Woody Plant Medium (Lloyd and McCown, 1981).


and media discoloration considerably influence shoot regener-ation even from different species of the same genus (Gantait

et al., 2014). Tables 1 and 2 synopsize the in vitro propagationrelated research achievements on genus Acacia, exclusively onhow several factors influence the regeneration of different

species of this genus that was not adequately discussed in theother reports (Beck and Dunlop, 2001; Quoirin, 2003) withthe only exception (Vengadesan et al., 2002a).

2.1. Role of explant source

In the cases of in vitro propagation, the nature of the plantmaterial exploited considerably influences its multiplication

and proliferation. It is necessary for any study to choose a suit-able explant prior to tissue culture. The growth rate of explantof various organs varies while some do not grow at all. The fre-

quently exploited explants are the meristematic portions forinstance the root tip, stem tip and axillary bud tip. Rates of celldivision are higher in these tissues and presumably produce the

much-needed growth-regulating substances such as auxins andcytokinins (Akin-Idowu et al., 2009). Although, an array ofexplants, for example leaves, shoot tips, axillary buds, cotyle-

don (Fig. 1) and nodal segments have been widely utilizedfor successful initiation of in vitro direct organogenesis ingenus Acacia (Table 1), nodes were more thriving to stimulatemultiple 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 andDaffalla, 2008; Dhabhai et al., 2010; Girijashankar, 2011;

Khalisi and Al-Joboury, 2012; Rathore et al., 2014). Otherexplants were not as effective as nodes particularly forin vitro direct multiplication. Apart from several plant tissues

or organs mentioned (Table 1), employment of seeds for mul-tiple shoot proliferation was reported by Abbas et al. (2010) inAcacia nilotica subsp. hemispherica. Impact of choice ofexplants on in vitro indirect organogenesis of Acacia was also

evident (described in Table 2). Induction and proliferation ofcalli 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; Arumugamet al., 2009; Thambiraj and Paulsamy, 2012), stem (Hustache


et al., 1986), cotyledons (Fig. 1d) (Rout et al., 1995; Daset al., 1996; Vengadesan et al., 2003b; Rathore et al., 2012),

immature zygotic embryo (Xie and Hong, 2001b; Nanda andRout, 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 compar-ison with other explants used for organogenesis through callusculture as the mesophyll cells present in the leaf tissues are

generally undifferentiated and might be more totipotent toundergo dedifferentiation.

2.2. Role of surface disinfection procedure

The surface sterilization practice holds a vital importance inplant tissue culture techniques. A superior surface sterilantshould undergo least plant damage, while diminishing micro-

bial contamination to a much tolerable level. Initiation ofin vitro aseptic culture depends on the developmental statusof 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 dippingin 1.5% (v/v) sodium hypochlorite (NaOCl) solution was the

preliminary approach of surface disinfection for Acacia asreported by Tamura et al. (1984). Sterilization using NaOClsolution following 70% ethanol (5–7 min) has been successfulin many of the cases (Dewan et al., 1992; Arumugam et al.,

2009; Girijashankar, 2011). Abbas et al. (2010) employed95% ethanol for 20 s and subsequently 10% NaOCl solutioncontaining 3–6 drops of Tween 20. For most of the Acacia

explants, the commonly accepted technique entails surfacesterilization 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 consis-tent regeneration as well as to disinfect them prior to treatmentwith 0.1% HgCl2. But, HgCl2 has been reported to be an envi-

ronmentally 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)

5 BA SR

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)

SR

A. salicina/A. saligna/

A. sclerosperma

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

SR

1.8 IBA Rt

A. mangium Hypocotyl MS 1000 casein hydrolysate + 3

NAA+ 1 BA

Ca Gong et al. (1991)

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

3 kinetin + 0.5 NAA+ 104–

4029

SE

L-proline

½MS SR

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)

SR

Acacia mangium ½MS 0.5 kinetin or 0.4 TDZ + 0.5

NAA

Ca Quoirin et al. (1998)

SR

Rt

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

A. afarnesiana,

A. schaffneri

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

No PGR SE

217 lM AdS SR

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

13.2 lM BAP + 3.42 lM IAA SR

½MS 7.36 lM IBA Rt

A. mangium Cotyledon, zygotic

embryo,

leaf, petiole

MS 9.05 lM 2,4-D + 13.95 lM Ca Xie and Hong (2001a)

kinetin

4.55 lM TDZ + 1.43 lM IAA SR

0.75 lM NAA+ 2.33 lM kinetin Rt

A. mangium Immature zygotic embryo ½MS 1–2 TDZ + 0.25–2 IAA SE Xie and Hong (2001b)

5 GA3 SR

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

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

(liquid) PGR-free SR

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

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

½MS 0.04 lM BA+ 0.94 lM IBA Rt

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

½MS 13.3 lM BA+ 2.5 lM zeatin SR

½MS 7.4 lM IBA Rt

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

SR

½MS 0.5 IBA Rt

6 S. Gantait et al.



Table 2 (continued)

Species Explant Medium PGR (mg/l) Result Reference

A. confusa Leaf MS 3 2,4-D + 0.01 NAA+ 0.05

kinetin

Ca Arumugam et al. (2009)

WPM 3 BA+ 0.05 NAA+ 0.1 zeatin

+ 5 AdS

SR

MS 4 IBA+ 0.05 kinetin Rt

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

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

½MS 0.5 IBA Rt

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

0.22 lM BAP SR

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

(2012)2 IBA + 0.5 TDZ SR

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-N0-(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.

Figure 1 In vitro regeneration of Acacia auriculiformis. (a) Indirect shoot formation from cotyledonary callus (Bar, 5 mm), (b) multiple

shoot proliferation from cotyledonary callus (Bar, 4 mm), (c) complete micro-plantlet with in vitro shoot and root derived from indirect

regeneration (Bar, 5 mm), (d and f) induction and proliferation of calli from cotyledon, hypocotyls and leaf explants, respectively

(Bar, 4 mm), (g) direct regeneration of multiple shoots from cotyledon (Bar, 3 mm), (h) complete plantlet with shoot and root regenerated

from cotyledon (Bar, 5 mm) (unpublished photographs of PKD).




8 S. Gantait et al.

metals. In an experiment conducted by Thompson et al.(2009), it has been accounted that 25% (w/v) Jik is a preferablesubstitute to other sterilants and much safer than HgCl2. He

also noted that when explants were exposed to sterilants for10 min they responded significantly better in terms of shootinitiation, than other exposure times tested. Explants exposed

for 30 min resulted in an effect that was detrimental to shootproliferation. The type and exposure duration both manipulatethe response of the plant material to organogenesis. These

factors influence individually as there is no known interaction.Thambiraj and Paulsamy (2012) utilized various antibioticsbefore using any surface sterilants. To eliminate fungal con-tamination he employed carbendazim (50%, w/v) and fungi-

cide (10%) for 15 min and to eliminate bacterialcontamination he treated the explants with 5% (w/v) antibi-otics (ampicillin and rifampicin) for 30 min followed by a rins-

ing with sterilized double distilled water. Interestingly, Salehiand Khosh-Khui (1997) in a study with rose, used antibiotics(gentamycin, ampicillin, tetracycline or amoxicillin) at differ-

ent concentrations and durations for the purpose of disinfec-tion from internal contaminants, noticed that use of anantibiotic solution before surface sterilization was unsuccessful

but found highest percentage of disinfected explants when100 mg/l solution of gentamycin or ampicillin was used aftersurface sterilization. The difference prevailed, since, duringsurface sterilization the fresh conducting tissue gets exposed

by cutting the ends of the explants through which the antibi-otic solution percolates down inside the tissue that results inhigher frequency of disinfection. The issue of endogenous con-

tamination cannot be totally inhibited by the use of surfacesterilants so, a search for more prominent systemic sterilantthat 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 ofmineral nutrients supplied in different types of media. Major-ity of the scientists recommended semi-solid full strength

Murashige and Skoog (1962) (MS) medium for shoot initiationin 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 MSmedium 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 ½MS 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 rootinduction in ½MS. Even, ¼MS confirmed to be adequate formultiple shoot induction (Kaur et al., 1998). Utilization of liq-

uid MS medium is reported by Rathore et al. (2014), since thecost of plant production in commercial scale is much less in liq-uid medium. Moreover, MS basal medium supported callus

induction, subsequently shoot and root formation (Garget 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 byDewan et al., 1992; Gupta and Agrawal, 1992; Douglas and


McNamara, 2000), White (White, 1938), SH (Schenk andHildebrandt, 1972), KT (Kathju and Tewari, 1973), andWoody 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 aJordan’s medium (Jordan et al., 1978) containing high concen-tration of auxin that produced 100% roots. Monteuuis (2004a)

in an experiment for rooting found ½MS unfavorable, whereashigher responsiveness was observed for the same materialwhen 1/3SH macronutrients were employed. Ahee andDuhoux (1994) compared three basal media for root culture

in vitro. They employed White, BDM, 2xBDM (Bonner andDevirian, 1939 modified by Goforth and Torrey, 1977) and1/5MS medium. Contrastingly, they observed that the root

growth was significantly higher in BDM and 2xBDM mediathan 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 exper-iment 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 regenerationfrequency. Similar comparative experiment was also per-formed by Rout et al. (1995) where the relative performanceof MS was assessed alongside WPM for somatic embryogene-

sis. He accounted that somatic embryogenesis occurred onlyon WPM. Nevertheless, to promote germination, the somaticembryos were required to be inoculated onto ½MS basal med-

ium without any PGR. Shahinozzaman et al. (2012) furtheranalyzed the differential effect of basal media on shoot prolif-eration utilizing MS and WPM as experimental media. Maxi-

mum explants produced highest number of shoots on MSmedium; however, explants produced longest shoots in WPMmedium.

2.4. Role of carbohydrate source

Carbohydrates are one of the most indispensable substancesrequired for growth and organized development (Gamborg

et al., 1976), and are essential as an energy source, providingcarbon skeletons for biosynthetic processes as well. Presenceof sucrose in the culture medium is essential for different meta-

bolic activities. It is necessary for differentiation of xylem andphloem elements in cultured cells (Aloni, 1980). The nutri-tional 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 sinceit possesses added proficiency for regeneration of in vitroexplants 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 optimizedcallus induction and cell suspension culture. Later, most of the

researchers confirming the use of MS medium successfullycultured Acacia in vitro by the utilization of 3% sucrose bothfor direct (Ahmad, 1989; Abbas et al., 2010; Girijashankar,

2011; Javed et al., 2013; Yadav et al., 2015) and indirectorganogenesis (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 con-centrations of sucrose, Beck et al. (1998b) noted greater shootproduction with 2% and 3% sucrose. On the contrary, there

has been a wide range of carbohydrate sources in different con-centrations. There are fewer reports where 2% sucrose showedpromising results in bud initiation and multiplication

(Monteuuis and Bon, 2000; Monteuuis et al., 2013) also insomatic embryogenesis (Rout et al., 1995; Rout and nanda,2005). Even a lesser concentration (1.5%) of sucrose wasemployed in rooting media by Kaur and Kant (2000). Never-

theless, Rout et al. (1995) noted 2% sucrose to be more effi-cient for somatic embryogenesis induction. Douglas andMcnamara (2000) in an experiment employed as high as 6%

sucrose and noted frequent initiation of adventitious shootsand buds by cotyledon explants. From these studies it wasfurther concluded that plant can readily utilize carbohydrate

in the form of sucrose. However, it has been observed thatthere is variation in effect of carbohydrate on plant dependingon its source and concentration. Furthermore, species speci-

ficity and formulation of maintenance medium might haveadditional influence on performance of carbohydrate. Eventhough scores of literatures were published regarding theuptake and consumption of exogenous carbohydrates by

explants cultured in vitro, yet, data on the associations betweenthe experimentation of source of carbon in the culture mediumand 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 asuperior and positive approach for clonal propagation ofplant. Asynchronous plants generally result from callus

whereas homogeneous diploid individuals are formed fromadventitious shoots (Bhojwani and Razdan, 1996). It is anefficient method to produce large-scale true-to-type plants.Variety of explants had been employed and inoculated in num-

ber of media formulations fortified with variable sources andmeasures of plant growth regulators for shoot regenerationin Acacia so far, which has been summarized in Table 1.

Tamura et al. (1984) were the preliminary researchers to com-mence a technique for direct in vitro multiple shoots initiationand propagation in Acacia using high level of kinetin. The

occurrence of cytokinin predominantly as PGR, in the growthmedium is significant for shoot proliferation (Dave et al., 1980;Gassama, 1989; Darus, 1991; Galiana et al., 1991; Dewanet 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 varietyof 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 numberof shoots was obtained by utilizing a 4.0 lM BA containingmedium which was much superior to kinetin for shoot

multiplication of Acacia mangium. The superior effect of BAover 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). Inter-estingly, although Dhabhai et al. (2010) observed direct regen-eration on MS medium having only kinetin (1 mg/l) yet the

proliferation remained undifferentiated for one month, untila-naphthalene acetic acid (NAA) (0.6 mg/l) was used, that inturn induced the multiplication of shoots almost instantly.

The report displays the probable high endogenous cytokininconcentration. Badji et al. (1993) reported that zeatin whichis a natural cytokinin, produced better induction to multipleshoot formation. There are various instances where utilization

of a single source of PGR did not give much effect but acombination of the same promoted direct organogenesis muchefficiently. Al-Wasel (2000) tested BA or N-phenyl-N0-(1,2,3-thiadiazol-5-yl) urea (Thidiazuron or TDZ) in association withNAA for their influence on shoot proliferation of Acacia seyal.It was observed that NAA could not induce shoot develop-

ment when employed alone, and BA unaided produced veryfew shoots; although, a combination of BA and NAA under-went profuse regeneration. Rout et al. (2008) tried twenty

different combinations of PGRs and found that incorporationof BA (1.5 mg/l), indole-3-acetic acid (IAA) (0.05 mg/l) alongwith adenine sulfate (AdS) (50 mg/l) to be the most efficienttreatment to encourage shoot regeneration and multiplication.

Abbas et al. (2010) found highest number of shoots and shootregeneration frequency in the presence of 2.0 mg/l BAP and0.5 mg/l NAA. Shahinozzaman et al. (2013) also reported that

incorporation of auxin along with BA to the medium enhancedthe frequency of shoot bud differentiation rather than usingBA individually in the medium. In that study it was concluded

that 2.0 lM BA plus 1.0 lM NAA was the most favorablePGR combination for direct shoot organogenesis. Recently,MS medium containing 2 mg/l kinetin and 0.5 mg/l IAA

exhibited maximum frequency of shoot regeneration (Yadavet al., 2015). Thus the relevance of cytokinin to auxin in a ratioconfirmed to be efficient in high shoot regeneration instead ofusing 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 (Georgeet al., 2008). On the other hand, when callus is cultured inapposite conditions, it can experience differentiation and

transforms into a whole new plant. Table 2 presents a compi-lation of research works that have been carried out to examinethe efficiency of explants on callus induction with or withoutthe use of different PGRs in Acacia. Callus induction from

shoot tip occurred for the first time with only SH plus0.2 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D) without anyother PGRs or additives (Skolmen and Mapes, 1976). On the

other hand, Tanabe and Honda (1999) induced callus from leafexplant with the supplementation of 4.4 lM BA only in theculture medium. Interestingly they found that the combination

of BA and NAA had an antagonistic effect on callusing. How-ever, combinations of auxin and cytokinin were found moreeffective for callus induction by the majority of the researchers.

For instance, of 2,4-D:kinetin (Ortiz et al., 2000; Xie andHong, 2001a; Rathore et al., 2012) or 2,4-D:N6-



10 S. Gantait et al.

benzylaminopurine (BAP) (Vengadesan et al., 2000, 2002c;Dhabhai and Batra, 2010) induced callus in a more skillfulmode in contrast to a single use of PGR (either of auxin or

cytokinin). Nanda and Rout (2003) tested various concentra-tions of BA, 2,4-D and kinetin alone or in combinations andaccounted that the intensity of embryogenic callus prolifera-

tion was greatest in the media with 8.88 mM BA in associationwith 6.78 mM 2,4-D. There are also many instances whereTDZ (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 highrates for all combinations of NAA and TDZ than utilizingNAA alone. Xie and Hong (2001b) cultured the immature

zygotic embryo in the medium containing 2.0 mg/l TDZ and0.25 mg/l IAA and noted that it was very well capable ofinducing embryogenic calli. Similarly, Yang et al. (2006) and

Thambiraj and Paulsamy (2012) observed efficient callusformation as well as adventitious shoots on the medium con-taining a combination of TDZ and NAA. Banerjee (2013)

tested various PGRs and found 2iP in association with NAAto be the best in terms of callus initiation and shoot bud regen-eration from de-embryonated cotyledon. Importantly, the

endogenous hormone levels of Acacia tissues play an impor-tant role and this is the main reason behind the variation ofdifferent types of explants on exposure to PGRs.

2.7. Role of plant growth regulators on somatic embryogenesis

Somatic embryogenesis conveys enormous potential to acceler-ate the propagation of woody species (Attree and Fowke,

1993). Somatic embryogenesis is generally used for large-scale production and genetic transformation. The insufficiencyof knowledge in the fields of somatic embryogenesis, asyn-

chronous production of somatic embryo and low frequencytrue to type embryonic competence, leads to the same beingheld responsible for its shortened commercial application in

woody forest species. Accounting two reasons, somaticembryogenesis plays a significant role in forest biotechnology.First, this technique generates countless number of propagulesfor somatic embryo (Attree et al., 1994). Secondly, genetic

transformation research could easily be carried out. Optimumnutrition and culture conditions are crucial for the conversionof somatic embryos into complete plantlets. Somatic embryo-

genesis encounters some practical applications in woody spe-cies, which has been successfully established. In comparisonwith 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 inAcacia (Table 2). Induction of somatic embryogenesis isusually constrained to certain responsive cells of explants

and largely determined by a specific developmental stage ofthe tissue (von Arnold et al., 2002; Rai et al., 2007). Routet al. (1995) achieved somatic embryogenesis from callus,

derived from immature cotyledons of Acacia catechu Willd.On WPM supplemented with 13.9 lM kinetin and 2.7 lMNAA. Moreover, the addition of 0.9–3.5 mM L-proline to

the medium induced the somatic embryos to develop. On theother hand, Ortiz et al. (2000) obtained the highest numberof somatic embryos in Acacia farnesiana and Acacia schaffneri,

in the media devoid of any PGRs but with the addition ofABA 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/lIAA to be the most efficient for inducing embryogenic calli fol-

lowed by somatic embryos. The combinations of 2,4-D andkinetin or 2,4-D and BA did not induce somatic embryogeniccalli in A. mangium. To induce somatic embryo maturation, a

two-step procedure involving gibberellin A3 (GA3) and highconcentrations of sucrose was found to be effective indicatingthe importance of GA3 in promoting somatic embryo matura-

tion in this species. Interestingly, Vengadesan et al. (2002c)studied exclusively the different effects of auxins, cytokinins,carbohydrates, amino acids and casein hydrolysate on produc-tion 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 inducingand producing somatic embryos. Cytokinins, individually or

in combination with any auxin, did not produce somaticembryos. But addition of glutamine enhanced the productionof somatic embryos. The findings were in relevance with

Vengadesan et al. (2002c) that particularly suggested that2,4-D is required as vital supplement for the induction ofsomatic embryogenesis. Casein hydrolyzate was essential for

somatic embryogenesis in Phaseolus vulgaris (Martnus andSondahl, 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 oninduction medium, whereas cotyledons obtained from matureseeds failed to induce somatic embryo. The differential

responses to combination of PGRs are presumably due tothe difference in genotype and endogenous hormones presentwithin the type of explant used. Further, the frequency and

intensity of somatic embryogenesis was enhanced significantlyby the addition of amino acids as reported earlier by otherresearchers (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 positiveeffect on the induction of somatic embryogenesis. It wasreported that, L-glutamine was frequently used as a source oforganic nitrogen in plant tissue culture which provides reduced

nitrogen to plant tissues (Barrett et al., 1997) and enhances thesynthesis of certain metabolites (Deo et al., 2010). On a con-trary note, most of the embryos showed a tendency to lose

their germination potential and perish if continued to be onthe same development and maturation medium for longerduration. Therefore, the embryos had to be removed from this

medium and transferred to growth regulator free or BAP con-taining medium for their germination. The maximum percent-age of germination of somatic embryos was recorded on

medium containing 0.22 lM 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 utilizingjuvenile plant parts root more effectively and easily than themature parts, due to the presence of meristematic tissue.

Several researchers deliberated the impact of PGRs inin 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 devoidof PGR. Girijashankar (2011) also noted rooting in ½MSwithout addition of any PGR. Nonetheless, IAA, indole-3-

butyric acid (IBA), or NAA is vastly and individually usedas the sole PGR source for root induction and has provencompetent 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 andDaffalla, 2008; Shahinozzaman et al., 2012, 2013; Banerjee,

2013; Rathore et al., 2014; Yadav et al., 2015). Among thesethree auxin sources employed in variable concentrations, suit-ability of IAA or IBA or NAA was reported to be different indifferent 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 ofinstances (Kaur et al., 1998; Kaur and Kant, 2000; Abbas

et al., 2010).There are fewer casein Acacia where IBA was usedin fortification with IAA (Semsuntud and Nitiwattanachai,1991; Bhaskar and Subhash, 1996) or NAA (Khalisi and Al-

Joboury, 2012; Bhaskar and Subhash, 1996) to suffice eachother to overcome the problem associated with poor root ini-tiation. Interestingly, the use of PGRs in any of the combina-tions, for instance IAA plus IBA, IAA plus NAA or IBA plus

NAA failed to induce rooting, rather they caused callusing andyellowing 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 andHanson, 1993; Huang et al., 1994; Wantanabe et al., 1994;

Monteuuis, 2004a; Banerjee, 2013). There are few instanceswhere 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; Khalisiand Al-Joboury, 2012). Reports on usage of PGR in combina-tions 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 andPaulsamy (2012) examined the complementary effect of thecombined usage of auxin:cytokinin on root induction in Aca-

cia and observed root development to be successful in the exis-tence of BA or kinetin (as cytokinin) in association with NAAor IBA (as auxin). The fact that root induction and successive

elongation is accelerated by the utilization of activated char-coal (AC) in the culture medium combined with an auxinhas been surveyed by Gantait et al. (2011). It was stated fur-ther that AC enhances rooting as it eradicates light and offers

a practical atmosphere for the rhizosphere (Gantait andMandal, 2010). Hence, the application of AC can also betested in Acacia rooting efficiency.

3. Substrate-based acclimatization

For successful micropropagation of Acacia, acclimatization of

in vitro plantlets is a significant phase. Influence of substratemedia, temperature, light, and humidity was generally assessedduring acclimatization of in vitro regenerated Acacia plantlets.

Rapid desiccation of plantlets and its susceptibility to bacterial


and fungal diseases makes the acclimatization procedure moredifficult. Rout et al. (2008) reported acclimatization andsubsequent greenhouse establishment of in vitro plantlets by

relocating them to a mixture of garden soil and sand at a ratioof 1:1 (v/v). Survival rate of 100% was obtained at the harden-ing phase when a substrate cocopeat was used up

(Girijashankar, 2011). Later on, Javed et al. (2013) used sterilesoilrite in plastic pots and covered the plantlets with polythenebags to maintain relative humidity. Further, ½MS solution

was sprayed every three days for two weeks. Later on acclima-tized plants were shifted to normal garden soil in greenhouseunder natural light. Various researchers added a range oforganic substance in the substrate. For instance, Nanda and

Rout (2003) mixed sand, cow-dung, soil together at a ratioof 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 acclimatiza-tion 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 con-taining 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). Arange 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 genotypeby 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 thedemand for wood is ever-increasing and it will continue

throughout the next few decades (Fenning and Gershenzon,2002). Usually, timbered plants are problematic to regeneratein in vitro environment. Nevertheless, a small number of pro-

cedures are there to confirm the genetic fidelity involving foresttree species for commercial purpose. Genetic clonality is a keyconcern in commercial micropropagation via in vitro tissue cul-ture approach since true-to-type clones are the most critical

prerequisites. A key setback confronted with the in vitro cul-ture is the occurrence of somaclonal variation in the midst ofsub-clones of one parental line, arising as a result of in vitro

culture. DNA methylation, point mutations and chromosomerearrangements 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 propagulesat an early stage of development is considered to be crucialin Acacia in vitro culture. Molecular, cytological or biochemi-cal assays are the key approaches to determine clonal fidelity

of in vitro generated plantlets. A superior approach for geneticstability assay can be made by employing an assay of molecu-lar markers that could amplify manifold regions of the genome

(Martins et al., 2004; Gantait et al., 2012). PCR-based molec-ular markers such as RAPD, ISSR, and SSR have been foundto 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 of20 arbitrary 10-base primers were utilized for Polymerase

Chain Reaction. Out of the different primers tested, only three(OPC-04, OPD-14 and OPC-19) were successful in amplifyingthe products that were monomorphic across all the microprop-

agated plants. Other primers produced limited number ofmonomorphic bands. This technology needs to be exploitedmore 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 regenerationprotocol, somaclonal variations and its physiological as well asmorphological aspects. A competent plant regeneration proto-col is a must for the utilization of a range of biotechnological

techniques. It can serve as a platform to transmit economicallyimperative traits through genetic engineering, cryoconserva-tion, inducing somaclonal variations, in vitro mutations,

double haploids induction, development and utilization ofsomatic hybrids in Acacia. A remarkable progress can beachieved in biotechnological improvement on Acacia through

the tissue culture-based advanced approaches. The presentreview endows with a wide-ranging assessment of the in vitroliterature of Acacia to date, which will aid in the advanceresearch of Acacia biotechnology.

Authors’ contribution

SG and PKD conceived the idea of the review; SG and SKsurveyed the literature and wrote the draft manuscript; andSG and PKD scrutinized and corrected final version of themanuscript. 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 conflictof interest and we do not have any financial gain from it.

Acknowledgments

Authors acknowledge the library and laboratory assistance

from the Department of Genetics and Plant Breeding, Facultyof Agriculture, Bidhan Chandra Krishi Viswavidyalaya,Mohanpur, Nadia, West Bengal, India. Authors further are

thankful to the anonymous reviewers and the editor of this arti-cle for their critical comments and suggestions on themanuscript.

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