ò á ï â ó ~ Michael Crichton -...

“If you do not know the history, you don’t know anything; you are a leaf

that doesn’t know it’s part of a tree”

~ Michael Crichton

Micropropagation of Saraca asoca (Roxb.) De Wilde in response to biochemical and seasonal variations Page | 8

CHAPTER-2

REVIEW OF LITERATURE

Use of in vitro techniques for micropropagation of woody trees has opened

new possibilities for rapid and mass multiplication of the elite germplasm (Domecq,

1988 and Vila et al., 2003). To overcome the problems encountered in traditional

methods of propagation, micropropagation through axillary shoot proliferation is the

most reliable approach. Studies on effect of media, phytohormones, seasonal response

in bud proliferation, changes in biochemicals and antioxidant enzymes during in vitro

propagation is an additional advantage to understand the overall effects and

mechanism. Assessment of genetic fidelity of micropropagated plants to ensure safe

mode of regeneration is essential before mass production of clonal planting material

for operational planting of woody perennials. The relevant literature on

“Micropropagation of Saraca asoca (Roxb.) De wilde in response to biochemical and

seasonal variation” is cited in the following sections:-

2.1. In vitro propagation

Conventional breeding is rather slow and less productive and cannot be used

efficiently for the mass multiplication. Advanced biotechnological methods of

culturing plant cells and tissues provide new means for conserving and rapidly

propagating valuable, rare and endangered tree species. The advantages of tissue

culture over conventional methods of propagation are rapid and large scale

multiplication of important plants under in vitro conditions irrespective of season with

limited space and time (Nehra and Kartha, 1994). During the past decade, major

advances have been made in the field of plant tissue culture and now it has become an

industrial technology. Schaeffer (1990) defined micropropagation as the in vitro

clonal propagation of plants from shoot tips or nodal explants, usually with an

accelerated proliferation of shoots during subcultures.

Micropropagation is usually described as having four distinct stages:

Stage “0” Pre-preparation of in situ donor material (fungicide and/or PGR treatments,

hedging, etiolation etc.), Stage “I” initiation (including surface sterilization) of

explants, Stage “II” shoots multiplication (optimization of proliferation media),

Review of literature …


Stage “III” root induction on micro-cuttings (in vitro or ex vitro) and Stage “IV”

acclimatization of rooted shoots (or unrooted microcuttings, to ex vitro conditions)

Various degrees of difficulty can be encountered in stages I through IV

between species and even between genotypes within a species (Naik et al., 2003).

Inability to achieve sterile explants, poor explant performance due to oxidation,

phenolic leakage and premature death of explants can be encountered in stage I

(Lynch, 1999). Lack of response to cytokinins, slow growth, abnormal growth, e.g.

hyperhydric transformation, shoot miniaturization or stunting, prolonged phenolic

exudation, shoot necrosis or excessive callusing may impede optimization of shoot

multiplication media in stage II, especially with woody species (Benson, 2000). Lack

of response (poor or no root induction) to auxin(s), excessive callusing or

deterioration in overall shoot quality can be encountered in stage III, especially with

woody plants (Lynch, 1999).

In stage IV, transfer to soil, plants need physiological adjustment to the ex

vitro environment. This entails physiological adjustment to reduced mineral nutrient

loading, more variable temperatures, higher lighting levels, reduced humidity, re-

instatement of waxy leaf coatings to prevent desiccation and resumption of stomatal

function and regaining root function to allow mass flow transpiration (Preece and

Sutter, 1991). In addition, plants must successfully survive the transition from primary

dependence on medium sugar as the carbon source (photoheterotrophic or

photomixotrophic) and become photoautotrophic again (Pospisilova et al., 1999a and

b). All these changes need to occur relatively quickly for the plant to regain

physiological competence and avoid a prolonged transition period in stage IV with

subsequent risk of senescence resulting from oxidative trauma (Batkova et al., 2008)

and pathogen infection (Williamson et al., 1998).

For successful in vitro cloning of plants, it is utmost important first to have

contamination free cultures. Proper sterilization and bioassay of systemic fungus, type

of explant used, plant growth regulators, type of nutrient media, season, and agar

concentrations, all effect the very success of the technique.

2.1.1. Aseptic Culture Establishment

Many sterilization techniques and treatments are in vogue. Ramanayake and

Yakandwala (1997) surface sterilization the nodes of D. giganeus with NaOCl and

10000 ppm Benlate® solution. Again treated with 0.3 % solution of HgCl2. Mishra et



al. (2001) sterilized nodal explants by immersion for 30 seconds in 70 % ethanol and

transferred to 0.1x (10 %) dilute solution of Cetramide®. Surface sterilization was

done for 8 – 10 minutes with aqueous 0.1 % HgCl2 solution. Godbole et al. (2002)

dipped nodal segments of D. hamiltoni in 70 % alcohol for 5 minutes before treating

them with 4 % NaOCl solution on shaker while Babaei et al. (2012) treated

Curculigo latifolia explants with 70 % ethanol for 90 s, 30 % Clorox® and Tween 20

for 15 minutes, mercuric chloride (0.1 %) for 5 minutes, and 10 % Clorox® for 10

minutes for eliminating contaminants. Bakshi (2012) presterilized explants of T.

falconeri with blue copper followed by surface sterilization with HgCl2. In all the

studies cited above, the researchers found HgCl2 as the best for sterilization of

explants before inoculation. On the other hand, Mishra et al. (2008) rinsed explants of

Bambusa tulda Roxb. with 0.1 % diluted aqueous solution of Dettol for aseptic

culture establishment.

A wide range of microrganisms (filamentous fungi, yeasts, bacteria, viruses

and viroids) and micro-arthropods (mites and thrips) have been identified as

contaminants in plant tissue cultures. Contaminants may be introduced with the

explant, during manipulations in the laboratory, by micro-arthropod vectors

(Tanprasert and Reed, 1997; Leifert and Cassells, 2001) or endophytic bacteria (Reed

et al., 1995; Pereira et al., 2003). Fungus may arrive with an explant, or airborne, or

enter a culture (Babaoglu et al., 2001). Frequently encountered bacterial and fungal

contaminations especially in laboratories of commercial micropropagation pose a

considerable problem (Reed et al., 1998). Various antibiotics and fungicides were

used to eliminate contaminants (George, 1993). Reed et al. (1998) observed internal

bacterial contamination in hazelnut shoot cultures which were evident at culture

establishment, or became apparent after several subcultures. Bürün et al. (2001)

identified contaminants in in vitro culture of Lilium candidum based on their

morphological and cultural characteristics by cultivation which comprised of

Fusarium, Penicillium, Alternaria, Rhizopus, Cylindrocarpon and Aspergillus species.

The most effective treatment against fungal contaminations was achieved by utilizing

a combination of Benomyl and Nystatin.

2.1.2. Bud proliferation

Success of in vitro bud proliferation depends on various factors. Some of the

important factors are age of explant, type of nutrient medium used, plant growth



regulators & their combinations, seasonal effect and sub-culturing micro shoots. The

literature more relevant to this subject matter is presented in following section.

2.1.2.1. Effect of explant age on culture establishment

The explant type viz. apical shoot and nodal shoot segments have been most

commonly used for in vitro cloning through axillary shoot proliferation

(Muthukrishnan et al., 2013). Response of these explants may depend on the age,

juvenility of explants and plant species. The nodal shoot segment as an explant has

been proved better than shoot tip in Meliaceae tree species such as Azadirachta indica

(Drew, 1993; Yasseen, 1994; Venkateswarlu et al., 1998; Chaturvedi et al., 2004;

Arora et al., 2010), Melia azedarach (Thakur et al., 1998; Husain and Anis, 2009) and

Toona ciliata (Mroginski et al.,2003). The nodal explants proved better than apical

shoot in other woody plant species viz. Tecomella undulata (Rathore et al., 1991),

Maytenus emarginata (Rathore et al., 1992), Madhuca longifolia (Rout and Das,

1993), Ochreinauclea missionis (Dalal and Rai, 2001), Santalum album (Sanjaya et

al., 2006; Goyal, 2007), Pongamia pinnata (Sugla et al., 2007), Jatropa curcus

(Dubey, 2009), Embelia ribes (Annapurna and Rathore, 2010) and Dalbergia sissoo

(Thirunavoukkarasu et al., 2010).

2.1.2.2. Effect of nutrient media

The nutrient media provides essential elements which are required for the cell

growth and development under in vitro conditions. Lack of nutrient availability in the

medium leads to poor cell growth and development of shoots. Nutritional

requirements of cultures may vary with the plant species for shoot induction,

multiplication and root induction (Chang and Ho, 1997). The main components of

most plant tissue culture media are mineral salts, sugar as carbon source and water.

Other components may include organic supplements, growth regulators and a gelling

agent (Gamborg et al., 1968; Gamborg and Phillips, 1995). Although, the amounts of

the various ingredients in the medium vary for different stages of culture and plant

species but the basic MS (Murashige and Skoog, 1962) and LS (Linsmaier and Skoog,

1965) are most widely used media. During the past decades, many types of media

have been developed in plant tissue culture (Street and Shillito, 1977; Pierik, 1989).

Media compositions have been formulated for the specific plants and tissues (Nitsch

and Nitsch, 1969). Some tissues respond much better on solid media while others on



liquid media. As such, no single medium can be suggested as being entirely

satisfactory for all types of plant tissues and organs. Some of the earliest plant tissue

culture media were developed by White (1943) and Gautheret (1939). All subsequent

media formulations are based on White’s and Gautheret’s media.

Some common media used to fulfill the requirements of cultured tissue are MS

(Murashige and Skoog, 1962), Gamborg (1968), Nitsch and Nitsch (1969), Gressholf

and Doy (1972), Eeuwens (1976), Llyod and McCown (1980), Branton and Blake

(1983). Murashige and Skoog (1962) is the most widely used medium, especially in

procedures where plant regeneration is the main objective. There are some examples

where modified MS medium have also been used viz. Moringa pterygosperma

(Mohan et al., 1995), Hovenia dulcis (Echeverrigaray et al., 1998), Lagerstroemia

reginae (Sumana and Kaveriappa, 2000), Bambusa vulgaris (Ndiaye et al., 2006).

Adventitious shoots were induced from the hypocotyl explants of Sesbania rostrata

on Nitsch’s medium (Nitsch, 1969). Mukhopadhyay and MohanRam (1981) used

Gamborg's B5 medium for the multiplication of Dalbergia sissoo whereas Datta and

Datta (1983) obtained multiple shoots from nodal explants of Dalbergia sissoo on MS

medium supplemented with vitamins of Gamborg's B5 medium and NAA. In Acacia

nilotica, Dewan et al. (1992) observed the highest number of shoots (6.3) on B5

medium. Purohit et al. (1994) reported that among the different media, MS, SH,

WPM and B5 tested in Wrightia tomentosa, bud break frequency from nodal explant

was highest (90 %) in MS medium supplemented with BAP (2.0 mg/l) as compared to

SH (60 %), WPM (54 %) and B5 (40 %). Venkateswarlu et al. (1998) also tested MS

and WBM supplemented with various concentrations of BAP (0.5-2.5 mg/l) for shoot

induction in mature neem tree and found that MS medium supplemented with BAP

(2.0 mg/l) was most effective for high rate (80 %) of shoot induction. Bhatt and Dhar

(2004) established higher efficiency of WPM over B5 (Gamborg et al., 1968) and MS

and their different strengths were tried for shoot proliferation in Myrica esculenta.

They observed that neither MS nor B5 have satisfactory response even when the salt

concentration is reduced to half and shoot response was severely inhibited.

Feyissa et al. (2005) compared MS and WPM with BAP and with IBA for

shoot multiplication in Hagenia abyssinica. They recorded maximum shoot

multiplication rate (2.0 folds) in MS medium and 2.1 folds in WPM with BAP (4.4

µM) + IBA (0.49 µM). Thakur and Shukla (2006) compared MS medium and

modification of NH4NO3 (1750 and 1000 mg/l) and KH2 PO4 ( 500 and 340 mg//l) of



MS medium and revealed that MS medium without modification proved better for

shoot multiplication and shoot growth in J. curcas.

Kumar et al. (2005) compared different media viz. B5, Nitsch, WPM, MS and

Knop‘s for shoot induction from nodal explants from 18-20 year old trees of

Holarrhena antidysenterica. They revealed that MS medium was found to be the most

effective with 83 % response and 1.66 shoots per nodal explant with an average shoot

length of 2.64 cm within 30 days.

Sivanesan (2007) compared different media (MS, SH and B5) for the shoot

multiplication from the shoot tip explants of mature plants of W. somnifera. MS

medium was found superior to SH and B5 medium. Goyal (2007) evaluated various

media such as MS, modification of MS (MS3/4, MS/2, MS/4 and MS1/2 N2 source),

SH, B5, WPM and HE on shoot multiplication, and reported that MS/2 medium was

the most effective for high (3.11 fold) rate of shoot multiplication in red sanders.

Husain and Anis (2009) used different media viz; MS, MS/2, B5 and WPM

supplemented with optimum concentration of BAP (5 µM) for shoot induction from

nodal stem segment of M. azedarach. Among the different media, MS medium was

found as the best for shoot induction. Similarly, Sen et al. (2010) also observed that

MS medium with BAP (1.2 mg/l) and IAA (0.1 mg/l) and AS (9.0 mg/l) was best for

shoot multiplication in M. azedarach.

Thirunavoukkarasu et al. (2010) compared effects of MS and WPM on the

shoot initiation from axillary buds of epicormic and coppice shoots of D. sissoo. They

found maximum (84.6 %) response on shoot induction in MS medium supplemented

with BAP (6.6 µM) and IAA (1.14µM). Annapurana and Rathore (2010) compared,

different media viz; B5, Heller, WPM and MS supplemented with BAP (22.20 µM) or

TDZ (1.13 µM) with IAA (0.57 µM) for shoot initiation in E. ribes. Among the

different media tested, MS medium supplemented with TDZ (1.13 µM) favored the

highest percentage (61.11 %) of bud break, followed by a significantly low bud break

on B5 and WPM (30.55 %) while, Heller medium exhibited the lowest frequency of

bud break (19.45 %).

Arora et al. (2010) compared modified compositions of MS medium and

designated such medium as BM1, BM2 and BM3 on shoot proliferation from nodal

segment of a 40 year old tree of A. indica. They found that inorganic and organic

constituents of the medium influenced growth and general condition of proliferating

shoot. BM1 medium supplemented with BAP (1.11 µM), IAA (1.43 µM) and adenine



hemisulphite (81.43 µM) was found to be the most effective for shoot induction while

BM2 medium with BAP (1.11 µM), IAA (1.43 µM) and adenine hemisulphite (81.43

µM) was most effective for shoot multiplication and elongation. Khan et al. (2011a)

demonstrated the best shoot induction response in nodal explants of Salix tetrasperma

in WPM medium supplemented with different plant growth regulators.

2.1.2.3. Effect of Plant growth regulators (PGR’s)

The success of a culture is affected by the type and concentration of applied

cytokinins because their uptake, transport, and metabolism differ among varieties and

interaction with endogenous cytokinins of an explant (Werbrouck et al., 1996; Strnad

et al., 1997; Van Staden et al., 2008).

Cytokinins like BAP, Kinetin, Zeatin, etc. are responsible for cell division and

shoot differentiation. BAP has been the most effective cytokinin for shoot tip

meristem and bud cultures followed by Kinetin (Murashige, 1974). Cytokinin has

been regularly incorporated into tissue culture for shoot regeneration (George and

Sherrington, 1984). Kopp and Nataraja (1990) regenerated plantlets of Tamarindus

indica on MS medium supplemented with 2.0 mg/l BAP. Multiple shoots were

obtained from cotyledonary nodes of Dalbergia latifolia on MS medium fortified with

BAP (Sita and Swamy, 1992). Ajithkumar and Seeni (1998) also found BAP more

effective for producing longer shoot than Kinetin in Aegle marmelos.

In woody plants, TDZ has been shown to be suitable for micropropagation and

regeneration of recalcitrant species or genotypes (Huetteman and Preece, 1993;

reviewed in Durkovic and Misalova, 2008). Quraishi et al. (2004) also observed that

BAP alone was effective for shoot multiplication and further maintenance of shoot

culture of A. indica. A perusal of literature reveals that it has successfully been used to

induce axillary or adventitious shoot proliferation in a number of plant species

including herbaceous, perennials and tree species such as Cassia angustifolia

(Siddique and Anis, 2007a & b; Parveen and Shahzad, 2011), Pterocarpus marsupium

(Husain et al., 2007), Pongamia pinnata (Sujatha and Hazra, 2007), Cardiospermum

halicacabum (Jahan and Anis, 2009) and Bacopa monnieri (Ceaser et al., 2010).

2.1.2.4. Effect of combination of Plant growth regulators (PGR’s)

The plant growth regulators particularly cytokinins and auxins have specific

roles in cell elongation, cell division and differentiation. Endogenous levels of PGRs

vary with the species and therefore, exogenous requirement may also vary with plant



species and sometimes between different types of explants. Skoog and Miller (1957)

demonstrated the hormonal regulation of morphogenesis in plants, which allowed the

controlled formation of shoots/roots in culture from cells and tissues.

Kathiravan and Ignacimuthu (1999) reported that the combination of BAP and

Kinetin produced maximum number of shoots in Canavalia virosa. Mroginski et al.

(2003) tested different cytokinins viz. BAP (0.05-5.0 mg/l), Kn (0.5 mg/l), zeatin (0.5

mg/l) and TDZ (0.5 mg/l) along with or without auxin viz. IBA (0.01-1.0 mg/l), NAA

(0.1 mg/l), 2,4-D (0.1 mg/l) and IAA (0.1 mg/l) for shoot induction from nodal

segment of 2-years old plants of Toona ciliata (Meliaceae family. They observed that

combined use of BAP (0.5 mg/l) and IBA (0.1 mg/l) was found to be the best

treatment for shoot induction whereas, Scocchi and Mroginski (2004a) used MS

medium with BAP 2 µM + IBA 0.5 µM + GA3 0.3 µM for the establishment of the

culture of M. azedarach. Chaturvedi et al. (2004) reported that combination of BAP 1

µM + GA3 0.5 µM in MS/2 (major inorganic salts reduced to half strength) proved

suitable for the multiple shoot induction in A. indica.

Gopi and Vatsala (2006) reported the potential of 2,4-D (0.1-5.0 mg/l) + NAA

on callus induction in Gymnema sylvestre. Chabukswar and Deodhar (2006) reported

multiple shoot induction in Garcinia indica in WPM medium supplemented with 8.9

μM BA and 0.5 μM thidiazuron (TDZ). Singh and Lal (2007) reported that media

supplemented with BAP (1.0 mg/l) in combination with NAA (2.0 mg/l) supported

hundred per cent callus induction from hypocotyl and cotyledonary leaf segments of

Leucaena leucocephala. An efficient, rapid and reproducible plant regeneration

protocol using nodal explants of Cassia angustifolia cultured on MS medium

supplemented with BAP and TDZ was developed by Siddique and Anis, (2007b). The

maximum per cent regeneration in Olea europaea was achieved on the medium

supplemented with 2.22 μM BAP (Peixe et al., 2007). Kalimuthu et al. (2007)

developed a regeneration protocol for Jatropha curcas using nodal explants on MS

supplemented with BAP (1.5 mg/l), Kn (0.5 mg/l) and IAA (0.1 mg/l). The maximum

number of shoots in Aegle marmelos was obtained on the medium supplemented with

8.84 μM BAP in combination with 5.7 μM IAA (Pati et al., 2008).

Husain and Anis (2009) tested different concentrations of cytokinins viz. BAP,

Kn and 2iP (1.0-10.0 µM) for shoot induction from nodal segment of M. azedarach.

They observed 90 % shoot induction from nodal segment in MS medium with BAP

5.0 µM alone. Similarly, Sen et al. (2010) also tested different concentrations of



cytokinins viz. BAP and Kn (1.0-2.0 mg/l) for shoot initiation from nodal shoot

segment as an explant from in vitro raised seedling in M. azedarach. They observed

maximum (90 %) shoot initiation on MS medium supplemented with BAP (1.2 mg/l)

alone.

Arora et al. (2010) used different concentrations of BAP (0.44 - 4.44 µM), 2,

iP (0.61-1.23 µM), zeatin (0.57-1.14 µM) and TDZ (0.57-1.14 µM) either alone or in

combinations with IAA (1.43 µM) and adenine hemisulphate (27.14-543 µM) in the

medium for shoot induction from nodal segment of A. indica. They observed that

maximum (81.2 %) bud break was in MS medium supplemented with combined use

of BAP 1.11 µM + IAA 1.43µM and 81.43 µM adenine hemisulphate.

Effect of plant growth regulators in other tree species have been reported by

several workers. Rathore et al. (1992) tested two cytokinins BAP and Kn (0.5-5.0

mg/l) and auxins IAA, IBA, NAA (0.05-2.5 mg/l) in MS medium in Maytenus

emarginata for their effect on bud break frequency. Maximum (84 %) explants

responded with 10-12 shoot per nodal explant in MS medium with combined use of

IAA (0.1 mg/l) + BAP (2.5 mg/l) + AS (25.0 mg/l) in 4 weeks. At higher

concentrations of BAP, the rate of response and shoot length was reduced. Medium

with BAP was more effective than Kn for all the parameters at all the concentrations

tested.

Dhar and Upreti (1999) tested cytokinins viz. BAP, Zeatin, Kn and TDZ (1.0-

5.0 µM) alone in the MS medium to compare their effects on shoot induction and

number of multiple shoots produced in Bauhinia vahlii. They reported that maximum

shoot initiation (41.6 %) with maximum number of shoots (2.3 shoots per nodal

explant) was in the medium containing Kn (2.5 µM) alone. Response was further

increased to 58 % with 4.5 shoots per explant, when AS (100 mg/l) was also

incorporated in the medium. They also revealed that medium consisted of BAP

resulted in callus induction at the base of explants and suppressed shoot growth,

whereas when TDZ alone was used in MS medium, it stimulated development of

adventitious shoot buds, which failed to elongate. A very low rate of response was

observed with zeatin in the medium.

Deora and Shekhawat (1995) used BAP and Kn 1.0-25.0 mg/l and IAA, IBA

and NAA (0.1-2.0 mg/l) either alone or in combinations in Capparis decidua. They

observed maximum bud break (76 %) frequency with 4-7 shoots per explant with a

shoot length of 2.6 cm on MS medium with NAA (0.1 mg/l) and BAP (5.0 mg/l) with



additives. Higher concentration of BAP (10 mg/l) resulted in dwarf (0.5 cm) shoots.

Similarly, combined use of BAP with NAA proved most effective for shoot induction

in various tree species such as Aegle marmelose (Hossain et al., 1995; Nayak et al.,

2007), Grevillea robusta (Rajasekaran, 1994), Ficus benghalensis (Munshi et al.,

2004), Santalum album (Sanjaya et al., 2006 and Goyal, 2007), Gmelina arborea

(Thirunavoukkarasu and Debata, 1998; Behera et al., 2008), Acacia senegal (Kaur et

al., 1998) and Catharanthus roseus (Faheem et al., 2011). Romano et al. (2002)

compared effect of BAP, Kn, Zeatin and 2iP (0.5 and 1.0 mg/l) alone on in vitro shoot

proliferation of two genotypes (Mulata and Galhosa) of Ceratonia siliqua. Maximum

(1.5 folds) shoot multiplication rate was obtained on MS medium with zeatin (1.0

mg/l) in Mulata and 1.3 folds in Galhosa. Whereas, Johnson and Manickam (2003)

reported that BAP (3.10 µM) was the best cytokinin to obtain maximum (3.4) number

of shoot in Baliospermum montanum. Prakash et al. (2006) reported that MS medium

with combined use of BA (4.4 µM) and TDZ (2.2 µM) favored high (3.5 folds) rate of

shoot multiplication in Pterocarpus santalinus within 6 weeks.

Annapurana and Rathore (2010a) tested three different cytokinins viz. BAP

(2.22-44.44 µM), Kn (11.60 µM) and TDZ (0.45-2.27 µM) in MS medium for shoot

initiation in Embelia ribes. Among the various cytokinins tested, TDZ (1.13 µM)

alone in MS medium proved to be the most effective for high frequency bud break.

2.1.2.5. Effect of different seasons on shoot proliferation

Physiological state of the explant material is influenced by the season, which

also varies with the age of the mother plant. It is a major determinant in the success of

the explant response in culture. Seasonal effect on frequency of shoot induction,

number of shoots and shoot length from the explants have been reported in Meliaceae

such as Melia azedarach (Husain and Anis, 2009), Azadirachta indica (Chaturvedi et

al., 2004; Quraishi et al., 2004; Arora et al., 2010). Thes studies revealed that the best

response on shoot initiation was from the explant obtained during the month of April

(i.e. summer period).

Seasonal effect on establishment of aseptic culture and shoot initiation have

been reported by other workers in various woody plant species such as Prosopis

cineraria (Shekhwat et al., 1993), Tectona grandis (Tiwari et al., 2002), Crataeva

adansonii (Sharma et al., 2003), Holarrrhena antidysenterica (Kumar et al., 2005),

Pterocarpous santalinus (Prakash et al., 2006) and Embelia ribes (Annapurna and



Rathore, 2010). Most of the studies revealed that March-July is the best period for the

best response on shoot initiation whereas, Devi et al. (1994) reported that December

was the best month for the establishment of cultures from field grown trees of Tectona

grandis.

The nodal explants harvested during the months of March-April and August-

October was found to be the best for cultures establishment of Capparis decidua

(Deora and Shekhawat, 1995). Bansal and Chibbar (2000) observed best response

from nodal segments of Madhuca latifolia in the month of May. The collection of

explants during a relatively milder weather condition (December to March) was best

for promoting survival of explants in Acacia sinuata (Vengadesan et al., 2003).

Sharma et al. (2003) also observed best explants response in the month of October

and November with maximum number of shoot buds in Crataeva adansonii. The best

shoot initiation response was reported from November to February when the trees

produced fresh sprouts, the shoot initiation was rare in explants collected during other

periods in Callophyllum apetalum (Nair and Seeni, 2003). Nodal explants of Myrica

esculenta collected during winter (November-December) gave the maximum response

(Bhatt and Dhar, 2004) while the nodal segments of Wrightia tinctoria collected

during March-June from young lateral branches showed maximum bud break

response (Purohit and Kukda, 2004). In Holarrhena antidysenterica, nodal explants

showed maximal morphogenic response from May to July, and declined in subsequent

months till dropping to zero from October to February (Kumar et al., 2005). Singh

and Goyal (2007) observed that the season between August-October was the best for

explant collection in Salvadora oleoides. Pati et al. (2008) observed that nodal

explants of Aegle marmelos excised during September-October was found ideal

because most of the explants showed bud break whereas, bud break frequency

reduced in other months. The cultures of Melia azedarach initiated during March

exhibited the best response not only in terms of the frequency of bud break but also in

shoot vigor (Husain and Anis, 2009). The explants of Maerua oblongifolia collected

during the months of July-August responded best in vitro as compared to explants

harvested in any other months of the year (Rathore and Shekhawat, 2011). The nodal,

inter-nodal segments and shoot apices of Ficus religiosa collected in May and June

gave maximum response (Siwach et al., 2011).



2.1.2.6. Effect of subculturing of micro shoots

During in vitro cultures, delay in subculturing leads to deterioration of cultures

due to depletion of nutrient in the medium, leaching of phenolics and accumulation of

toxic gases. Periodical sub subculturing is required to maintain growth phase and

increase the number of shoots and length.

Islam et al. (1997) reported 4.5 shoots per nodal shoot segment as an explant

up to fourth subculture from the 25-years old tree on MS medium supplemented with

BAP (1.0 mg/l) and Kn (1.0 mg/l). Bajaj et al. (1988) obtained around 2200 plantlets

of Thymus vulgaris from a single shoot grown in vitro in 5 months (four passages).

Ajithkumar and Seeni (1998) reported that repeated subculturing of nodes and leaf

from shoot cultures of Aegle marmelos helped to achieve continuous production of

callus-free healthy shoots at least up to five subculture cycles. Thakur et al. (1998)

tested various concentrations of BA (4.44-17.75 µM) and Kn (4.65-9.29) in MS

medium for in vitro shoot proliferation of M. azedarach. They found that BAP was

better than Kn for shoot multiplication and observed maximum multiple shoots on MS

medium with BAP (17.75 µM) within 4-7 weeks. Borthakur et al. (1999) established a

mass multiplication protocol for Alpinia galanga by subculturing the regenerated

explants to kinetin supplemented medium for more than one year. They obtained an

average of 1000 plantlets with four to five successive subculture cycles i.e. within 40-

45 days. Likewise, Raghu et al. (2007) observed that the micropropagated shoots of

Aegle marmelos could be subcultured up to 20 cycles without loss of vigour to

produce shoots free from morphological and growth abnormalities. Similarly, in

Simmondsia chinensis, around 10-15 shoots were produced by repeated subculturing

upto three successive subcultures (Singh et al., 2008).

The increase in shoot number may be due to suppression of apical dominance

during subculture that induced basal dormant meristematic cells to form new shoots

(Shukla et al., 2009). Hence, by adopting this procedure of shoot excision and re-

culturing of the mother explants to the fresh medium, a large number of shoots could

be obtained per explants within few months (Asthana et al., 2011). This approach of

increasing the yield of shoots at an enhanced pace was adopted earlier for other

woody taxa (Kaveriappa et al., 1997; Jain and Babbar, 2000; Hiregoudar et al., 2005;

Prakash et al., 2006; Anis et al., 2010; Tripathi and Kumar 2010; Shekhawat and

Shekhawat, 2011).



Vengadesan et al. (2003) transferred the initiated shoots in MS medium

supplemented with 8.9, µM BA and 2.5, µM TDZ and found maximum (10.4) number

of shoots per explant after first subculture in A. sinuate. Chaturvedi et al. (2004)

reported shoot multiplication at a rate of five fold in 30 days after fifth passage on MS

medium supplemented with 0.25 mg/l BAP+ 0.25 mg/l IAA + 15 mg/l AS in

Azadirachta indica.

Sujatha et al. (2005) subcultured the original explants with differentiated

shoots in MS medium supplemented with BA (2.2–44.4, µM) and TDZ (2.3–45.4,

µM) for multiple shoot production in J. curcas from 3 months old plant. They found

maximum (24 shoots per nodes) on MS medium with BA (8.9 µM) after 12 weeks (4

subcultures). Dubey (2009) reported four time subculturing of nodal shoot segment

with differentiated shoots and observed production of multiple shoots (13.83

shoots/explant) on MS medium with IBA (0.1 mg/l) and BAP (0.5 mg/l) in 10 weeks

period in J. curcas.

Husain and Anis (2009) in M. azedarach also reported effect of subculturing

to increase the shoot multiplication, the subculturing was continued up to the six

passage of cultures and an increase in number of shoots was observed up to the third

passage and thereafter, the multiplication rate was stabilized at the fourth passage and

subsequently declined. Annapurna and Rathore (2010) reported single shoot

production from the nodal shoot segment of mature plants of E. ribes even on the

medium with higher concentrations of cytokinins (BAP/Kn/TDZ), but repeated

subculturing of differentiated shoots with or without explant on the same fresh

medium at the interval of two weeks favored production of multiple shoots (12-15)

after 3rd passage of subculture.

2.1.3. In vitro callogenesis

Callus is a coherent and unstructured tissue which is formed when plant cells

multiply in an unsystematic manner. It may be induced in or upon parts of an intact

plant by wounding, by the presence of insects or microorganisms, or as a result of

some stress. Callus can be initiated in vitro by placing small pieces of the whole plant

(explants) onto a growth-supporting medium under sterile conditions. Under the

stimulus of endogenous growth regulators or some growth regulating chemicals added

to the medium, the metabolism of cells, which were in a quiescent state, is changed,

and they begin active division. During this process, cell differentiation and



specialization, which may have been occurring in the intact plant, are reversed, and

the explant gives rise to new tissue, which is composed of meristematic and

unspecialized cell types. Two mechanisms appear to be important for in vitro

formation of embryogenic cells; i) asymmetric cell division and ii) control of cell

elongation (De Jong et al., 1993). Asymmetric cell division is promoted by PGRs that

alter cell polarity by interference with the pH gradient or the electrical field around

cells (Smith and Krikorian, 1990). The ability to control cell expansion is associated

with polysaccharides of the cell wall and corresponding hydrolytic enzymes (De Jong

et al., 1993).

2.1.3.1. Effect of explant on callus induction

The type of explant to be used for callus induction is a critical factor for in

vitro callugenesis. In vitro raised cultures as a source of explants have several

advantages over the field grown plants viz. better rejuvenated explants, minimum

accumulation of phenolics and inhibitors, better uniformity of explants and no

carryover effect of sterilant. Nirmalakumari et al. (1993) tested various explants viz.

leaf, stem, bark and cotyledons for callus induction in MS medium with different

levels of auxins and cytokinins in A. indica. They reported friable callus from leaf

segment on MS medium with 2,4-D (2.0 mg/l) and Kn (0.5 mg/l) and proved the best

explant. Su et al. (1997) reported embryogenic callus from cotyledons on MS medium

with NAA (0.5 mg/l) and BAP (1.0 mg/l) in A. indica. Akula et al. (2003) reported

that callus was induced from entire leaf lamina, when cultured on medium with BAP

(22.2 µM) and 2,4-D (2.3 µM) in A. indica.

Chaturvedi et al. (2003) reported callus induction from anther in MS medium

supplemented with 1 µM 2,4-D, 1 µM NAA and 5 µM BAP in Azadirachta indica

while cotyledons were exploited for organogenic callus regeneration by Sharry et al.

(2006) in M. azedarach.

Senerath et al. (2007) tested various explants such as apical buds, nodal

segments, petioles, floral parts (petals) and leaf disc of Munronia pinnata for callus

induction in MS medium. They observed that leaf explant produced high rate of callus

induction with mean fresh weight 0.167 gm as compared to other explant cultured on

MS medium with 2,4-D (1.1 mg/l) and BAP (0.5 mg/l).

Srivastava et al. (2009) used unfertilized ovaries for callus induction on MS

medium with 2,4-D (1 µM) and BAP (5µM) in A. indica whereas, Rafiq and Dahot



(2010) reported highest (78 %) callus induction from immature flowers on MS

medium with 2,4-D, (1.0 mg/l), BAP (1.0 mg/l) and NAA (0.2 mg/l) in A. indica.

In Populus, (Cheema, 1989; Michler and Bauer, 1991) reported embryogenic

callus induction from complete leaf and internode sections of 40 years old trees.

Michler and Bauer (1991) also reported the importance of leaf explant at various

developmental stages on callus induction and found developing leaves prior to full

expansion for efficient embryogenic callus induction.

Morini et al. (2000) used leaf and petioles collected from the second to fourth

nodes from the apical portion of in vitro propagated shoots of Quince (Cydonia

oblonga Mill). After 2, 4 and 6 days of inoculation, leaves showed different responses

with respect to embryogenic callus induction.

Rangaswamy (2007) used various explants viz. leaf, internode and nodal

segments from in vitro culture of S. album on callus induction and reported that the

percentage of explants producing callus was highest (91.67 %) with fresh weight of

1.299 g from the leaf explants obtained in in vitro culture of mature trees.

Dubey (2009) tested leaf and petiole segments as an explant to obtain high

frequency of callus induction in MS medium supplemented with additives and NAA

(1.0 mg/l) in J. curcus and reported maximum (87.71 %) callus formations with more

fresh weight (2.04 g), from leaf explant on MS medium with NAA (1.0 mg/l). Das

(2011) reported friable embryogenic callus from immature zygotic embryo on MS

medium with BAP (1.0 mg/l) and NAA (2.0 mg/l) in S. glauca.

2.1.3.2. Effect of nutrient media on callus induction

Nitrogen, one of the major component of the different nutrient media, has a

key role in plant growth and development affecting the rate of cell growth,

differentiation and totipotency (Kirbey et al., 1987).

Most of the studies are based on single type of medium on embryogenic callus

induction. Reddy et al. (1987) also used MS and B5 media for callus initiation in

Ricinus communis. Muralidharan and Mascarenhas (1987) reported somatic

embryogenesis in Eucalyptus citriodora on semisolid agar based B5 medium

supplemented with NAA and increased sucrose concentration (5 %). In Meliaceae,

MS medium was used widely for callus induction in A. indica (Nirmalakumari et al.,

1993; Su et al., 1997; Wewetzer, 1998; Akula et al., 2003; Chaturvedi et al., 2003;

Srivastava et al., 2009; Rafiq and Dahot, 2010), M. pinnata (Yapabandara et al., 2003;



Senerath et al., 2007) and M. azedarach (Thakur et al., 1998), whereas Sharry et al.

(2006) reported use of MS/2 medium with NAA (3 mg/l), BAP (1.0 mg/l) and GA3

(5.0 mg/l) for callus induction in M. azedarach.

Chalupa (1990) reported that induction of embryogenic callus was the best on

MS and WPM containing BAP (1.0 mg/l) alone or in combination with GA3 (1.0

mg/l) or IBA in Quercus robur L., whereas Gharyal and Maheshwari (1990) in

Albizzia lebbeck, Cassia siamea and C. fistula used B5 basal medium supplemented

with combination of NAA (2.0 mg/l) and BAP (1.0 mg/l) or IAA (0.5 mg/l) and BAP

(0.5 mg/l) for the induction of embryogenic callus.

Nuutila et al. (1991) reported use of N7 basal medium with 2,4 –D (9.05 µM)

and Kn (2.32 µM) and enriched with casein hydrolysate (1 g/l) for embryogenic callus

induction in birch using leaf segments, whereas Kumar et al. (2003) in Commiphora

wightii used B5 medium supplemented with 2,4,5-T and IBA for the induction of

embryogenic callus from zygotic embryo as a explant. They observed that about 15-

20 % of the explants produced embryogenic callus on the above cited medium.

Rangaswamy (2007) reported that MS medium with 2,4-D (1.0 mg/l) induced

high rate (93.33 %) of callus with maximum (1.297 g) fresh weight as compared to

B5, SH and Heller media in S. album. Similarly, Ahmed et al. (2009) reported callus

induction on MS medium with 2,4-D (0.5 mg/l), NAA (1.0 mg/l) and 10 % coconut

water in Gymnema sylvestre.

2.1.3.3. Effect of PGR’s on callus induction

Somatic cells contain all the genetic information necessary to develop

complete and functional plant. It has been reported that, PGRs and stress play a

central role in mediating the signal transduction cascade leading to the reprogramming

of gene expression which results in a series of cell divisions that induce either

unorganized callus growth or polarized growth leading to somatic embryogenesis

(Dudits et al., 1995). Requirement of type of PGRs, their combination and

concentration vary with the plant species. In monocotyledons, 2,4-D alone is most

commonly used auxin for embryogenic callus induction, but in dicotyledons, it varied

with the species and sometimes with explant type and source (seedling /mature plant).

Rangaswamy (2007) reported maximum callus intensity with highest response

(93.33 %) and fresh weight of 1.363 gm on MS medium with 2,4-D (2.0 mg/l) from

leaf segment of in vitro shoot of mature trees of S. album.



Mehta et al. (2011) used different concentrations of 2,4-D (1.0, 1.5, 2.0, 2.5,

5.0 and 7.0 mg/l) in MS medium, out of which 2,4-D (5.0 mg/l) showed highest

response to callus induction in Bambusa nutans. Zhang et al. (2010) suggested that

2,4-D (1.0-3.0 mg/l) was suitable range to induce desirable vigorous, granular and

compact callus but the higher concentration of 2,4-D (10 mg/l) resulted in less

abundant sticky watery mucilage callus from the mature zygotic embryos of D.

hamiltonii. Maximum (91.6 %) callus induction was obtained from nodal shoot

segments of D. asper on MS medium supplemented with 30 μM of 2,4-D (Arya et al.,

2008).

2.1.3.4. Effect of PGR’s combination on callus induction

Sinha and Mallick (1991) reported embryogenic callus on the MS medium

with 2,4-D alone or in combination with BAP in woody legume Sesbania bispinosa

(Jacq.) W. F. Wight. Use of low levels of BAP (0.2-0.5 mg/l) with 2,4-D (2.0 mg/l)

supported better dedifferentiation. Callus was relatively compact and pale yellow in

colour and occurred in less than 3 weeks.

Nirmalakumari et al. (1993) reported nodular and friable callus from the leaf

segment on MS medium with 2,4-D (2.0 mg/l) and Kn (0.5 mg/l) in A. indica. Thakur

et al. (1998) reported different concentrations and combinations of auxin (2,4-D, IAA

and NAA) and cytokinins ( Kn and BAP) in MS medium for callus induction from

different explant viz. leaf, internode, root and cotyledonary leaf lacking any

meristematic buds of in vitro raised seedling of M. azedarach. Among various auxins

tested, they found an actively growing and friable callus from internodal segments on

MS medium fortified with 2,4-D (4.52 µM) and BAP (4.44 µM). Sugathadasa et al.

(1998) reported that combination of BAP (1.25 mg/l) and NAA (4.5 mg/l) was proved

to be the best for callus induction in Munronia pinnata, whereas Yapabandara et al.

(2003) reported the best callus induction on MS medium supplemented with IAA (0.2

mg/l) and BAP (2.0 mg/l) in M. pinnata.

Sharry et al. (2006) reported organogenic callus from cotyledons on MS

medium with 0.5 mg/l NAA and 1.0 mg/l BAP in M. azedarach. Senerath et al.

(2007) reported that the combined use of 2,4-D (1.1 mg/l) and BAP (0.5 mg/l) in MS

medium was the best for callus induction with fresh weight 0.167 gm from leaf

explant of M. pinnata.



Srivastava et al. (2009) reported that combined use of 2,4-D (1 µM) and BAP

(5 µM) in MS medium was the best for callus induction from unpollinated ovary

cultures of A. indica while maximum (92.72 %) response of callus induction from leaf

explants of J. curcus was reported in MS medium with NAA (1.0 mg/l) (Dubey ,

2009).

Rafiq and Dahot (2010) reported highest (78 %) callus induction when

immature flowers were inoculated on MS medium with addition of 2,4-D (1.0 mg/l),

BAP (1.0 mg/l) and NAA (0.2 mg/l) in A. indica. Das (2011) used various

concentrations of BA or Kn (0-2.0 mg/l), NAA or 2,4-D (0-3.0 mg/l) either alone or in

combinations in MS medium for callus induction from immature zygotic embryo of S.

glauca. MS medium with BAP (1.0 mg/l) and NAA (2.0 mg/l) was the most effective

combination to induce friable embryogenic callus.

2.1.3.5. Effect of season on callus induction

Seasonal variation in callus induction and its frequency has been often

reported. Vartak and Shindikar (2008) observed seasonal effect on callus induction in

Bruguiera cylindrical L. and found significant variation among pre and post monsoon

season.

Siwach (2011) performed series of experiments to identify best season for

callusing in F. religiosa L. April to July season was the most favourable months for

callus induction. Karimi et al. (2012) reported significant variation on callus induction

in Cereus peruvianus during different season and found summer and spring season

best for callusing.

2.1.3.6. Callus differentiation to shoots

Callus growth is usually initiated by placing the chosen explant on a semi-

solid medium into which auxin has been incorporated at a relatively high level, with

or without a cytokinin. One or more transfers on the same medium may be necessary

before the callus is separated from the parental tissue for subculture. More than one

kind of callus may arise from a single explant. Therefore, successful propagation

depends on being able to recognize and subculture only the type (or types) which will

eventually be able to give rise to shoots or somatic embryos.

Nirmalakumari et al. (1993) reported maximum (6-7) shoots from one month

old calli of leaf and stem in MS medium supplemented with BAP (2.0 mg/l) and IAA

(0.5 mg/l) in Azadirachta indica while Chaicharoen et al. (1996) reported maximum 9



shoots from callus, when medium was supplemented with BA (1.0 mg/l) in M.

azedarach.

Xie and Hong (2001) observed calli from five types of explants (embryo axes

and cotyledons of mature zygotic embryos, leaflets, petioles and stems of seedlings)

of Acacia mangium on MS medium containing 2,4-D (9.05 μM) and Kn (13.95 μM).

A combination of TDZ (4.55 μM) and IAA (1.43 μM) in the medium promoted the

highest percentage of callus to form nodules, 8–11 % of calli derived from cotyledons,

embryo axes, leaflets or petiole and in 4 % of calli derived from stems.

Chaturvedi et al. (2003) reported that BAP (5 µM) alone was effective in MS

medium for shoot induction from anther derived callus of A. indica. Vila et al. (2003)

reported successful regeneration of plantlets from leaf derived callus by using

combination of BA (4.4 μM) and NAA (0.46 μM) in MS medium in M. azedarach.

Koroch et al. (2003) reported high shoot regeneration frequency (63 %) and number

(2.3 shoots per explant) of shoots per explant from the leaf callus of Echinacea

pallida on MS medium supplemented with combined use of 26.6 mM BAP and 0.11

mM NAA.

Faisal et al. (2005) reported adventitious shoot regeneration from petiole

surface callus of T. indica on MS medium with 2,4-D (10 mM) and TDZ (2.5 mM).

Shen et al. (2007) reported shoot regeneration (7.9 shoots) in Dieffenbachia cv.

camouflage from callus cultured on MS medium with 40 μM N6-(D2-isopentenyl)

adenine and 2 μM IAA. The combined use of NAA (3.0 mg/l) and BAP (3.0 mg/l) in

MS medium promoted highest 32.90 % adventitious shoot induction from callus of M.

pinnata raised from leaf disc (Senerath et al., 2007). Rajore and Batra (2007) used

BAP (1.5, mg/l) with different concentrations of IBA (0.5-5.0 mg/l) for callus

differentiation in MS medium from 2-3 year old plant of J. curcas and observed that

maximum shoot differentiation was observed from callus on MS medium with BAP

(1.5 mg/l) and IBA (0.5 mg/l) in 21days.

Hu and Li (2008) reported adventitious shoots (57.0 ± 8.3 %), when callus was

subcultured on MS medium with BA alone in Amorphophallus albus. Similarly,

Srivastava et al. (2009) reported maximum (78 %) shoot regeneration when callus

was subcultured on MS medium containing 5 µM BAP alone in A. indica. Verma et

al. (2010) reported (90 %) organogenic calli on MS medium supplemented with BAP

(2.0 mg/l) and IBA (0.5 mg/l) from leaf derived callus of Tylophora indica whereas

Raman et al. (2011) reported multiple (87 %) shoot induction on MS medium



amended with BAP (2.5 mg/l), Kn (1.5 mg/l) and NAA (0.1 mg/l) from organogenic

callus of Clerodendron phlomidis.

2.1.4. Root induction

The success of any in vitro plant tissue culture protocol depends on the rate of

shoot multiplication and frequency of rooting. Normally, the nutrient media of small

osmotic potential employed for the induction and growth of roots in micropropagated

shoots. The high salt levels in the media are frequently inhibitory to root initiation. By

testing four concentrations of MS salts (quarter, half, three quarters and full strength)

against four levels of sucrose (1, 2, 3 and 4 %), Harris and Stevenson (1979) found

that correct salt concentration (½ or ¼ MS) was more important than sucrose

concentration for root induction on grapevine cuttings in vitro. The benefit of low salt

levels for root initiation may be due more to the need for a low nitrogen level than for

an increased osmotic potential. Serres (1988) observed best rooting response in

Castanea species when shoots were inoculated in WPM medium containing 4 %

sucrose.

There are reports that an excessive sugar concentration can inhibit root

formation. Green cotyledons of Sinapis alba and Raphanus sativus were found by

Lovell et al. (1972) to form roots in 2 % sucrose in the dark, but not in light of 5500

lux luminous intensity. In the light, rooting did occur if the explants were kept in

water, or (to a lesser extent) if they were treated with DCMU (a chemical inhibitor of

photosynthesis) before culture in 2 % sucrose. The inhibitory effect of high sucrose

could be due to feedback inhibition as it is synthesised in tissues during

photosynthesis as well as present in excess in the medium. Rahman and Blake (1988)

while working with Artocarpus heterophyllus found that shoots when kept on a

rooting medium in the dark increased the number and weight of roots with the

inclusion of up to 80 g/l sucrose while the optimum sucrose concentration was 40 g/l

if the shoots were grown in the light. Although 4 % (occasionally 8 %) sucrose has

been used in media for isolated root culture, 2 % has been used in the great majority

of cases (Butcher and Street, 1964). Street and McGregor (1952) in tomato roots

found sucrose concentrations between 1.5 and 2.5 % increased the root fresh weight

and with optimal 1.5 % sucrose, it produced the best growth of the main root axis and

the greatest number and total length of lateral roots

Rooting in Saraca asoca was performed via in vitro and ex vitro methods.



2.1.4.1. In vitro root induction

Rooting from shoots of seedling origin, is easy but often reduces with increase

in age of the source plants, particularly in tree species. Different plant species vary in

their optimum requirement of auxin and its concentration for adventitious root

formation. Different explants of different species may require different concentrations

of auxins to produce roots. Interaction of a number of exogenous and endogenous

factors influenced the ability of plant to form root. Joarder et al. (1993) tested

different concentrations (0.2-1.5 mg/l) of IBA, NAA and IAA in MS/2 medium for

adventitious root induction from in vitro shoots of A. indica. They found that among

various auxins used, IBA (0.5 mg/l) in MS/2 was the best for maximum (82.2 %)

rooting frequency.

Similarly, Thakur et al. (1998) reported that among various auxins (IAA, IBA

and NAA) used in MS/2 medium for in vitro rooting in M. azedarach, IBA (4.92 µM)

in MS/2 medium was most effective for initiating roots whereas, Venkateswarlu et al.

(1998) found IAA (2.5 mg/l) to be the best for maximum (80 %) rooting in A. indica.

In Celastrus paniculatus, Nair and Seeni (2001) found that among various auxins,

IAA on MS medium under initial dark conditions gave the maximum rooting within a

period of 5 weeks. Mroginski et al. (2003) reported 62 % rooting in MS medium with

IBA (0.1 mg/l) in M. azedarach whereas, Chaturvedi et al. (2004) reported high (82

%) frequency rooting in MS/4 medium containing IBA (0.5 µM) in A. indica. Even

within same auxin, the various concentrations depicted different response (Reddy et

al. 2006) in A. indica with maximum 86.6 % rooting observed with IBA (2.0 µM) in

MS/2 medium. Senerath et al. (2007) on the other hand, used different concentrations

of IBA (0.1-0.3 mg/l) and IAA (0.1-0.5 mg/l) in MS/2 medium in M. pinnata and

reported that IAA (0.2 mg/l) was the most effective treatment for maximum (75 %)

root induction, highest mean root length (15.05) and 3.15 mean number of roots.

Shirin and Rana (2007) tested various auxins; IAA, IBA, IPA, NAA, 2,4-D and

Coumarin (25 μM) alone for root induction in B. glaucescens and found 100 % root

induction with an average of 9.67 roots/shoot propagule in MS liquid medium

supplemented with IBA in 4 weeks.

The effect of different concentrations of auxins on rooting was examined by

various researchers (Arora et al., 2010; Sen et al., 2010; Kaur et al., 1998). Ahn et al.

(2007) reported rooting from in vitro shoots of Ricinus communis on the media



consisted of IBA (84.3 %), NAA (87.4 %) at 5 μM, but overall shoot and root growth

was better on the medium with IBA as compared to NAA while Rout et al. (2008)

recorded 72 % rooting in MS/2 medium with combined use of IAA (0.1 mg/l) and

IBA (0.25 mg/l) in Acacia chundra. Similarly, Annapurna and Rathore (2010)

reported 100 % rooting in MS/2 medium with IBA (3.70 µM) alone in E. ribes.

2.1.4.1.1. Acclamatization and Hardening

The plants produced via any pathway in tissue culture require hardening and

acclimatization prior to transplanting in field to endure the external environmental

stress.

It is a generally observed that the step of transfer from tissue culture vessels to

soil is often very difficult because the in vitro produced plants are not well adapted to

an in vivo climate. Apart from many adaptation problems of the leaf and shoot

systems, the system of root regeneration in vitro in agar-gelled media appears to be

one of the most vulnerable one. In many cases even negatively gravitropic roots

appear in agar gelled media within glass vessels. Furthermore, the in vitro formed

roots do not function properly (fewer root hairs) in vivo, are rather weak, and often die

in soil, in vitro formed roots often have to be replaced by newly formed roots. As a

consequence of the non-functional roots, transpiration outside the glass vessels is too

high and can result in the loss of many plants. The plantlets under in vitro culture are

in high humidity and in heterotrophic mode, hence should be gradually acclimatized

under suitable temperature and humidity. Hardening conditions and duration may

vary with the species for its high rate of survival.

Sudha and Seeni (1994) reported that direct transplanting of tissue culture

raised plantlets of Adhatoda beddomei to nursery resulted in mortality rate of more

than 70 %. On the other hand, hardening of plants in humidity chamber for 4 weeks

prior to their transfer in the nursery, showed 95 % survival rates in potting mixture of

sand, top soil and cattle manure (1:1:1,v/v). Chaicharoen et al. (1996) reported 70 %

survival rate in M. azaedarach when micropropagated plantlets were transplanted into

clay pots which contained autoclaved vermiculite covered with a plastic bag.

Venkateswarlu et al. (1998) reported 85-90 % survival rate, on transfer of rooted

plantlets of A. indica to soil + vermiculite mix (1:1) and hardening for 30 days in a

mist chamber. Eeswara et al. (1998) reported 100 % survival rate when rooted shoots



of A. indica were transferred to pots containing Levington M2 compost (Fisons, UK)

and sand (3:1) and were kept in the growth chamber.

Chaturvedi et al. (2004) reported 87 % survival rate in A. indica when rooted

plants were transplanted to soilrite in hycotrays (Sigma, St. Louis, MO) and placed in

a glasshouse which is furnished with facilities to maintain a gradient of humidity by

the Fan and Pad system and a temperature of 25 ± 2ºC. Reddy et al. (2006) reported

80 % survival of plantlets when plantlets were transplanted to polycup containing soil

and vermicompost (3:1, v/v) and maintained under high humidity in the culture room

for 30 days.

Saxena and Dhawan (2001) transplanted in vitro generated plantlets of

Anogeissus pendula and A. latifolia into polybags filled with soil and maintained in

green house at 28 ± 2° C temperature for 7 days. For the first 2 days, the plants were

placed close to the cooling pad (RH 80-85 %) and then gradually shifted away

towards the exhaust fan for 5 days. The plants were then shifted to polyhouse for 30-

45 d depending upon the season and gradually to open nursery under the shade of tree

or thatch for 2-3 weeks. The plants were retained in the nursery for 6 months before

transferring them to field where they exhibited 85 % survival.

Kalimuthu et al. (2007) used various potting media (i) red soil and sand, (1:1,

v/v), (ii) vermiculate + sand + sphagnum mass (1:1:1, v/v), (iii) commercial media

consisting of decomposed coir waste + perlite + compost (1:1:1, v/v), (iv)

vermicompost + red soil + sand (1:1:1, v/v) for hardening of in vitro rooted plantlets.

Maximum survival rate (85 %) was observed in the potting media containing

decomposed coir waste + perlite + compost, followed by vermi-compost (70 %) in J.

curcas whereas, Samuel et al. (2009) established micropropagated plant in soil with a

survival frequency of 60 % when in vitro micropropagated plants were transferred to

potting mixture and acclimatized initially for 4 weeks in culture and then in

glasshouse for 8 weeks.

2.1.4.2. Ex vitro rooting

To cut down the steps of in vitro micropropagation and to make the technique

cost effective, ex vitro rooting is advisable. Another advantage being the rapidity of

acclimatization improving the survival rate of plantlets.

Ex vitro rooting of in vitro multiplied shoots has been reported for several

species, including Gardenia jasminoides (Economou and Spanoudaki, 1985), apple



(Zimmerman and Fordham, 1985; Stimart and Harbage, 1993), Actinidia deliciosa

(Pedroso et al., 1992), Cornus nuttallii (Edson et al., 1994), blueberry (Isutsa et al.,

1994), hazelnut (Nas and Read, 2004), Nyctanthes arbor-tristis (Siddique et al.,

2006), Vitex negundo (Ahmad and Anis, 2007), Malus zumi (Xu et al., 2008),

Metrosideros excelsa (Iapichino and Airo, 2008), Terminalia bellirica (Phulwaria et

al., 2012). The system is now preferred in woody plant micropropagation to rapidly

produce high-quality plantlets and to avoid the potential of off-types (Suttle, 2000).

Pruski et al. (2005) attempted rooting in Prunus fruticosa and P. tomentosa

using IBA (9.80 µM) alone or in combination with NAA (2.70 µM) and a commercial

rooting powder Rootone F containing IBA (0.057 %) + NAA (0.067 %) mixture. In

vitro regenerated shoots, when treated with combinations of IBA (9.80 µM) + NAA

(2.69 µM) resulted in maximum (79 %) rooting in a mixture of peat and perlite (1:1

v/v). Sanjaya et al. (2006) reported rooting of microshoots of S. album using various

auxins viz; IAA (253.5-2535 µM), IBA (246-2460 µM), NAA (268.5-2685 µM) and

NOA (247-2470 µM) for 30 minutes, followed by transfer in soilrite medium. Pulse

treated shoots with IBA (1230 µM) resulted in maximum 50 % rooting in soilrite

medium whereas, Goyal (2007) tested ex vitro rooting in S. album by pulse treatment

of in vitro regenerated shoots with different concentrations of IBA (500-4000 ppm)

for 30 minutes. A maximum 22.9 % rooting was obtainable with 2500 ppm IBA when

planted in soilrite medium.

Husain and Anis (2009) found that microshoots of Melia azedarach treated

with 250 µM for 15 minutes favored high (90 %) rooting percentage and induced

maximum number (4.7) of root per shoot after 4 weeks of transplantation to potting

mixture. Annapurna and Rathore (2010) reported ex vitro rooting with IBA (4.93 µM)

exhibited the highest rooting (95.2 %) with a maximum shoot length (4.5 cm) in E.

ribes.

2.2. Biochemical dynamics during in vitro propagation

Changes in the levels of metabolites and enzymes during plant tissue culture

may be helpful in understanding the biochemical basis of developmental pathway.

Seasonal fluctuations influence the metabolic pathways of the plants controlling their

enzymatic activities. These fluctuations lead to change in the growth pattern of the

plant cell. The biochemical study is necessary in plant tissue culture for successful

establishment of callus culture. About 40 % of the total phenol content in the plant



material was found to be released by the explants into the medium which interfere

with the growth. Similarly, many other biologically active indicators represent the

growth under in vitro conditions. The evaluation of the metabolic products and anti-

oxidant enzyme levels act as markers and helps us to identify the viability of the plant

cell for in vitro cultures.

The plant tissues are under stress of various salts and chemicals in the medium.

Several changes take place when the explants are transferred to the culture medium

and grown under in vitro conditions. Apart from possible effect on levels of

endogenous auxin, cytokinins appear to be implicated in sugar metabolism. Both

decreases and increases in the specific activity of enzymes of the glycolytic and

oxidative pentose phosphate pathways have been reported. Scott et al. (1964) reported

that medium containing adenine sulphate in addition to kinetin for shooting in

Nicotiana tabacum cause a marked increase in the activities of two enzymes of the

oxidative pentose phosphate pathway (glucose-6-phosphate dehydrogenase and 6-

phosphogluconate dehydrogenase), compared to their activities in a non-shoot

forming medium. Conditions favouring bud formation, including the availability of

cytokinins, seem to enhance starch metabolism in tobacco callus (Thorpe and Meier,

1972). Callus, which produces shoots had high specific activities of enzymes involved

in both starch accumulation and breakdown (Thorpe and Meier, 1974). Cytokinins

reduce the oxygen uptake of cells (Neumann, 1968) and inhibit the alternative

cyanide-resistant respiration pathway, which exists in many plants (Miller, 1979;

1980; 1982: Musgrave and Siedow, 1985). In plants, the phenols were found to react

with hydrogen peroxide produced during IAA degradation, thereby protecting the cell

from its toxic effects.

Most vegetative propagation techniques relaying on morphogenetic process are

conditioned by the season (Hartman and Kester, 1986). Seasonal changes greatly

influence explants establishment (Siril and Dhar, 1997). Gupta et al. (1980) noticed

the seasonal effects on regeneration of Tactona grandis as affected by the time of

explant collection, phenolics exudation and degree of contamination. The nodal

segments of Eucalyptus tereticornis collected during July to September were more

responsive because of negligible phenolic exudation as compared to explants collected

in October-November and May-June due to high amount of phenolic exudation (Das

and Mitra, 1990). Excellent regeneration has been reported in plant species during

spring season (March-May) when reserve food material (carbohydrate) is available in



plenty that helps the plants to sprout and bloom (Bhatt and Todaria, 1990). Some

observations on natural levels of auxin protectors might suggest that their low levels

are coupled with root initiation, but high levels with root growth. For example, shoots

of apple grown in vitro were found to have low phenol contents at the root induction

phase, but high content as roots were growing (Druart et al., 1982). In

Sequoiadendron giganteum, phenolic compounds decreased when shoots were moved

to a root induction medium (Monteuuis et al., 1987).

Singh et al. (2011b) showed that phenol content followed a zig-zag path from

non-differentiated callus to differentiated one, the total phenols increased up to 5th

day, decreased on 10th and 15th day and then again increased on 20th day. They

reported gradual decrease in phenolic content during differentiation. Phenols

participate in formation of cross-linking of cell wall constituents which is catalyzed by

peroxidase (Mader and Fussel, 1982). There is increasing evidence that seasonal

differences influence the regulation of cell cycle and this can affect morphogenetic

processes (Anderson et al., 2001).

In the undifferentiated callus of Chlorophytum borivilianum, Singh et al.

(2006) found that starch content was high initially and on 5th and 10th day after

subculture to rooting medium, the content increased further. The starch content

decreased during root formation (10th and 15th days) but a slight increase was

observed on 20th day. The starch content in the callus during shoot differentiation

showed an ambiguous trend. Thus, it was observed that the undifferentiated calli

contained more starch content than the differentiated ones.

Studies on total sugar and reducing sugar were reported by Madamba et al.

(1977) in the tissue cultures of Banana. Roy (2000) reported higher content of total

and reducing sugar in the callus of several tomato variants while De Greef and Jacab

(1979) found higher percentage of total and reducing sugar in callus culture in

comparison to the plant material of sugar beet.

A steady rise in total soluble sugars was observed by Singh et al. (2011a) from

undifferentiating callus i.e. 0 day to 10th day old calli kept on root differentiation

medium followed by a marked decrease in callus from root initiation to root

appearance i.e. 15th and 20th day. Overall results revealed the differentiated calli had

less soluble sugars than undifferentiated calli. The decline in total soluble sugar

content was associated with utilization of sugars for growth and differentiation

process. Similar trend was also reported in Cardiospermum halicacabum (Jeyaseelan



and Rao, 2005). In root differentiating calli, a steady increase in protein content was

observed up to 15th day followed by a little decrease after 15th day. In contrast, in

case of shoot differentiating calli, the content increased slowly up to 5th day but a

sharp increase was observed by 10th day followed by a decline on 15th day and

increase on 20th day. It was observed that total soluble proteins in root and shoot

forming calli were higher during root and shoot differentiation than in controlled

callus. This is due to synthesizes of new proteins during differentiation. Similar

observation was also reported by Mohapatra and Rath (2005).

Reductants such as ascorbic acid and polyphenols, inhibit the oxidation of

IAA by riboflavin; possibly on account of the natural occurrence of these compounds,

due to which some plant cells are able to grow and divide in the light. The best time to

explant shoot tips from adult chestnut material to a root inducing medium, was during

one of the first two peaks of growth of shoots, which coincided with the occurrence of

maximum quantitites of natural phenolics (Chevre and Salesses, 1987). 4-

Chlororesorcinol (21), a polyphenol oxidase inhibitor (i.e. inhibiting the conversion of

monophenols and dihydric phenols to polyphenols) has been found to improve the

rooting and subsequent growth of cuttings (Gad et al., 1988).

2.3. Enzyme dynamics during in vitro propagation

The oxidative stress of plants under in vitro conditions is linked to the

production of reactive oxygen species (ROS) which cause damage to lipids, proteins

and nucleic acids (Hernandez et al., 2000). Reactive oxygen species (ROS) are highly

reactive because they can control different processes and interact with a number of

other molecules and metabolites such as proteins, lipids, DNA and pigments (Mittler,

2002). In varying degrees, present day plants possess a number of antioxidant

enzymes that protect against potentially cytotoxic effects of ROS. To remove stress

and to increase tolerance to stress, the activity of antioxidant enzymes, such as

guaiacol peroxidase, superoxide dismutase, catalase, ascorbate peroxidase and

glutathione reductase, is generally increased in plants (Foyer et al., 1997). The role of

antioxidant enzymes such as superoxide dismutases, peroxidases, and catalases was

depicted by Landberg and Greger (2002); Meloni et al. (2003) in their work.

Microelements of the medium are also involved in metabolism via control of

enzymatic activities. Manganese (Mn) is generally added in similar concentrations to

those of iron and boron, i.e. between 25-150 mM and has similar chemical properties



as Mg2+. It is apparently able to replace magnesium in some enzyme systems (Hewitt,

1948). The most probable role for Mn is in structure of metalloproteins involved in

respiration and photosynthesis (Clarkson and Hanson, 1980) and is known to be

required for the activity of several enzymes, which include decarboxylases,

dehydrogenases, kinases and oxidases and superoxide dismutase enzymes. Zinc is a

component of stable metallo-enzymes with many diverse functions, making it difficult

to predict the unifying chemical property of the element, which is responsible for its

essentiality (Clarkson and Hanson, 1980). Zinc is required in more than 300 enzymes

including alcohol dehydrogenase, carbonic anhydrase, superoxide dismutase and RNA

polymerase. Copper is also an essential micronutrient, even though plants normally

contain only a few parts per million of the element. The element becomes attached to

enzymes, many of which bind to, and react with oxygen. They include the cytochrome

oxidase enzyme system, responsible for oxidative respiration, and superoxide

dismutase (an enzyme which contains both copper and zinc atoms). Detrimental

superoxide radicals, which are formed from molecular oxygen during electron transfer

reactions, are reacted by superoxide dismutase and thereby converted to water.

Copper atoms occur in plastocyanin, a pigment participating in electron transfer.

In tissue cultures, omission of Mn ions from Doerschug and Miller (1967)

medium reduced the number of buds initiated on lettuce cotyledons. A high level of

manganese could compensate for the lack of molybdenum in the growth of excised

tomato roots and vice versa (Hannay and Street, 1954). Natural auxin levels are

thought to be reduced in the presence of Mn2+ because the activity of IAA-oxidase is

increased. This is possibly due to Mn2+ or Mn containing enzymes inactivating

oxidase inhibitors, or because manganous ions are one of the cofactors for IAA

oxidases in plant cells (Galston and Hillman, 1961). Manganese complexed with

EDTA increased the oxidation of naturally-occurring IAA, but not the synthetic

auxins NAA or 2,4-D (MacLachlan and Waygood, 1956). However, Chée (1986) has

suggested that, at least in blue light, Mn2+ tends to cause the maintenance of, or

increase in, IAA levels within tissues by inactivating a co-factor of IAA oxidase.

When the Mn2+ level in MS medium was reduced from 100 mM to 5 mM, the

production in blue light, of axillary shoots by Vitis shoot cultures was increased.

In cultures of wild cherry, endogenous levels of IAA are also considerably

reduced by the presence of 2,4-D, although the availability of tryptophan (the

precursor of IAA biosynthesis) was increased (Sung, 1979). This suggests that 2,4-D



interfered directly with IAA synthesis or hastened IAA conjugation/degradation. 2,4-

D inhibition of IAA synthesis has been noted in sycamore suspension cultures (Elliott

et al., 1978). Conversely, reducing the external 2,4-D and NAA concentration resulted

in a significant increase in internal free IAA concentration in the auxin-dependent and

cytokinin autonomous tobacco cell strain VBI-0 (Zažímalová et al.,1995). Maeda and

Thorpe (1979) suggested that indole-based synthetic auxins might protect IAA from

natural destruction by competing with it for IAA oxidase enzymes.

Grambow and Langenbeck-Schwich (1983) recorded that the substitution

pattern of phenols affects the rate of IAA oxidation. Some monophenolics increase the

rate, while some 3-substituted phenols depress it. Phenols were found to react with

hydrogen peroxide produced during IAA degradation, thereby protecting the cell from

its toxic effects. Relatively large amounts of natural inhibitors of IAA oxidase have

been reported to be present in meristematic and juvenile tissues, but not in normal

mature differentiated cells until they are wounded (Stonier and Yoneda, 1967; Stonier,

1969). The normal process of auxin (IAA) inactivation has also been reported to be

inhibited in the callus produced following crown-gall infection, which is capable of

autonomous growth in culture (Lipetz and Galston, 1959; Platt, 1954; Bouillenne and

Gaspar, 1970). Some workers (Basu et al., 1969; Hammerschlag, 1982) have

questioned whether the stimulatory effect of phenols in promoting rooting is not due

to some other function than that of preventing IAA destruction. Shoots of a non-

rooting mutant of tobacco race contain high levels of auxin protectors, namely

chlorogenic acid and total soluble phenols (Faivre-Rampant et al., 2000). Lee (1980)

found that, in maize, some phenolic compounds can alter the relative proportions of

free and bound IAA. On the other hand, enhancement of quercetin glucosylation by

2,4-D was described in Vitis cell Scultures (Kokubo et al., 2001).

Rawal and Mehta (1982) found that shoot formation from haploid tobacco

callus occurred as the content of natural phenolic substances declined, while cellular

differentiation occurred with increasing phenolic accumulation. Juvenile tissues

naturally have high levels of auxin protectors (Stonier, 1972). Also, the level of

aromatic amines, like tyramine or phenetylamine seems to be correlated to

morphogenetic events (Martin-Tanguy and Carre, 1993). On the other hand, a

decreased content of phenolic acids (mainly derivatives of cinnamic acid found in

alfalfa (Medicago falcata L.) cell suspension culture after treatment with an inhibitor

of phenylalanine ammonia lyase (PAL), 2-aminoindan-2-phosphonic acid (AIP), was



connected with a decreased level of IAA, lower IAA-oxidase activity in later stages of

the culture and with slower growth of the culture (Hrubcová et al., 2000).

Many natural coumarins are found in plants, but their biochemical or

physiological roles are not well understood. Compounds of this kind have been found

to affect a wide variety of processes, low levels sometimes exerting a stimulatory role,

but higher levels are often inhibitory. This is particularly noticeable in the effect of

coumarins on the activity of many classes of enzymes. Scopoletin has been reported to

either increase or decrease IAA oxidase activity (Imbert and Wilson, 1970) and in

tobacco callus it can inhibit IAA degradation (Skoog and Montaldi, 1961), perhaps by

acting as a substrate for peroxidase enzymes. The synthetic auxins, which are added

exogenously to control the growth and organization of cultured tissues, may affect

endogenous IAA levels. This can be caused by inhibition of IAA oxidase. Callus

cultures of Arabidopsis thaliana can be initiated and maintained on a medium

containing 2,4-D, but progressively lose their morphogenic capacity the longer they

are maintained on it, until after 6-8 months there is no regeneration at all.

Tissues may be damaged by culture in media containing synthetic chelating

agents where the pH approaches neutrality, because at these pH levels, EDTA and

EGTA have been shown to remove calcium ions from the membranes of mitochondria

and this inhibits NAD(P)H oxidation and respiration (Moller and Palmer, 1981).

Chelating agents have been found to inhibit the action of the growth substance

ethylene and are thought to do so by sequestering Cu ions within plant tissues, thereby

interfering with the synthesis or action of a Cu-containing enzyme responsible for

ethylene metabolism. EDTA can also inhibit the activity of plant polyphenol oxidase

enzymes in vitro (Weinstein et al., 1951) and Smith (1968) thought that this might

occur because EDTA made Cu ions less available for enzyme incorporation, when he

found the chelating agent was able to prevent the blackening of freshly isolated Carex

flacca shoot tips.

In chestnut, rhizogenesis occurred during an increase in the level of auxin

protectors, whose basipetal transport was inhibited by applied IBA (Mato and Vieitez,

1986). The best time to explant shoot tips from adult chestnut material to a root

inducing medium, was during one of the first two peaks of growth of shoots, which

coincided with the occurrence of maximum quantities of natural phenolics (Chevre

and Salesses, 1987). 4-chlororesorcinol, a polyphenol oxidase inhibitor (i.e. inhibiting



the conversion of monophenols and dihydric phenols to polyphenols) has been found

to improve the rooting and subsequent growth of cuttings (Gad et al., 1988).

The increase in the peroxidase activity can be correlated with the fact that

when plants are grown under in vitro conditions with exogenous growth regulators

(auxins and cytokinins) are also present in growth medium, calli formation exhibit

high ethylene production (Csiszar et al., 2003). As a result of ethylene production,

defense mechanisms at a transcriptional level and generation of active oxygen species

including H2O2 are activated, which result in increased peroxidase activities (Levins et

al., 1995). Peroxidase isoenzymes are widely distributed among higher plants and are

frequently organ or tissue specific and due to these characteristics, different organs

from same plant may show different peroxidase patterns (Thorpe et al., 1978; Asins et

al., 1982). Appearance of transient and persistent isoperoxidases during part of growth

cycle has been reported by Balasimha and Subramanian (1983), Swarnkar et al.

(1987) and Meena and Patni (2007).

Gaspar et al. (1992) observed a direct relationship between the time of

maximum PO activity and that of the formation of the meristematic structures. A

typical minimum of PO activity appears at the root inductive phase, while the peak of

activity within the subsequent increase marks the end of the root initiation phase and

the beginning of the protrusion phase (Gaspar et al., 1992). Chen et al. (2002)

indicated that a decline in PO activity in NAA-treated tissues was accompanied by a

decrease in lignin content during the induction of adventitious roots in soybean

hypocotyls. Chao et al. (2001) reported that a decrease in PO activity corresponded

with a rise in endogenous IAA levels in IBA-treated tissues during the formation of

roots in soybean hypocotyls.

Superoxide dismutase, catalase and peroxidase have collectively been

viewed as a defensive team, whose combined purpose is to protect cells from active

oxygen damage (Fridovich, 1988). Superoxide dismutase (SOD) are metallo-

enzymes that convert O2•- to H2O2 in all aerobic organisms. Therefore, SOD is

considered as a primary defense against oxygen radicals (Bannister et al., 1987).

Reports on the activity of SOD in different plant species under salinity as well as

other stress conditions reflect its important role in the defense mechanism against

ROS. The product of SOD, hydrogen peroxide requires further detoxification which

is achieved by other enzymes such as peroxidase, catalase, glutathione etc. Thus like



SOD, peroxidase and catalase also play a vital role in plant defense against oxidative

stress.

The levels of superoxide dismutase activity were lower in browning tissues

than that in non-browning tissues in culture condition in Scot pine (Laukkanen et

al., 2000). In Virginia pine, tissue browning decreases the efficiency of in vitro

regeneration through somatic embryogenesis (Tang and Newton, 2004). It was

reported that, in non-browning callus cultures, PPO activity declined while in

browning calli, PPO activity continued to increase. It is believed that the increased

PPO activity in browning calli results from wounding or oxidative damage.

Numerous experiments have recently been carried out on several plant species with

the objective of explaining the role of oxidative stress in plant morphogenesis

(Gupta and Datta, 2004; Libik et al., 2005). Higher levels of intracellular H2O2

induce and promote embryo-genesis of L. barbarum L. callus (Kairong et al., 1999).

However, the relationship between the ROS and the callus differentiation and

regeneration has not been well understood till now (Tian et al., 2003). Therefore, it

is very important to note the level of these enzymes at different stages of in vitro

propagation, particularly, when cultures are maintained for longer time under in

vitro conditions.

2.3. Evaluation of genetic fidelity of micropropagated plants

Micropropagation through axillary shoot proliferation is considered safer and

preferred method for the commercial propagation of hardwoods species, because it

maintains genetic stability better than propagation by organogenesis (McCown and

McCown, 1987).

Shenoy and Vasil (1992) reported that micropropagation through explants

containing organized meristem is generally associated with lower risk of genetic

instability, because they are generally more resistant to genetic changes that might

occur during all divisions or differentiation under in vitro condition. However,

prolong shoot multiplication under different nutrient, hormones and incubation

conditions may pose problem of genetic variation. Clonal trueness is of major concern

in commercial micropropagation and particularly, in forest trees and other woody

plants having long rotation period, the economic consequence of unrecognized

somaclonal variation could be enormous. Therefore, genetic analysis of



micropropagated plant material is essential prior to use for large scale plantation

programme (Leena and Tuiza, 2005).

Genetic fidelity is the maintenance of the genetic constitution of a particular

clone through its life span (Chaterjee and Prakash, 1996). There are various molecular

tools to assess variability. Most commonly; RAPD, RFLP, AFLP and ISSR markers

are used to assess genetic fidelity. Among the molecular markers used for such

assessment, RAPD is the simplest, cheapest and appears to be a useful tool for

analysis of genetic fidelity of in vitro propagated plants (Williams et al., 1990; Rout

and Das, 2002; Singh et al., 2002; Martins et al., 2004; Leena and Tuija, 2005).

Identification of possible somaclonal variants at an early stage of development is

considered to be very useful for quality control in plant tissue culture, transgenic plant

and in the introduction of variants.

RAPD based assessment of genetic fidelity of micropropagated plants has

been reported in many plant species; Paulownia tomentosa (Rout et al., 2001),

Curcuma longa (Salvi et al., 2002), Olive cultivar (Leva et al., 2002), Robinia

pseudoacacia (Shu et al., 2003), Quercus robur (Valladares et al., 2006), Musa spp.

(Venkatachalam et al., 2007), Mucuna pruriens (Sathyanarayana et al., 2008), Olea

europaea (Peyvandi et al., 2009) and Musa spp. (Khan et al., 2011b). Molecular and

physiological analyses of tissue culture regenerated plants in different species have

shown the presence of genetic variation between the mother and regenerated plants

(Hashmi et al., 1997; Modgil et al., 2005; Peredo et al., 2006).

Rani and Raina (1998) studied RAPD profiles generated by 20 arbitrary

primers for evaluation of genetic fidelity of micropropagated plants and their

corresponding mother plants of Eucalyptus tereticornis and E. camaldulensis.

Banding pattern produced from the amplification profiles indicated the presence of

monomorphic bands between mother plant and the progenies.

Piola et al. (1999) performed genetic fidelity studies of the microcuttings of

four clones (A1, A2, P9, and P10) of Cedrus libani by using RAPD markers. Clones

were evaluated using 18 tenmer primers (chosen out of 31 primers, which provided

amplification products). Out of eighteen markers, 11 provided heterogeneous profiles

between the four clones. Monomorphic bands were produced in each individual

microcuttings within each clone, which suggests genetic stability of the

micropropagated plants.



Leva et al. (2002) reported that no variation was observed in the

micropropagated plants of Italian olive cultivar (maurino). Similarly, Shu et al. (2003)

compared RAPD profiles of mother plants of Robinia pseudoacacia with the

micropropagated plants using 25 arbitrary primers. Presence of monomorphic bands

established that the regenerants are genetically stable.

Olmos et al. (2002) tested genetic analysis in micropropagated plants raised

through axillary shoot proliferation of M. azedarach using ten 10-base oligonucleotide

primers (series P and G OPP-01, 02, 03, 04, 06; OPG-02, 03, 04, 08 and 10). They

detected a total of 46 polymorphic bands shared in all the clones analyzed and

observed total diversity obtained by using these primers ranged from 0.62 to 0.89 with

a mean value of 0.79 indicating high level of genetic variability.

Martins et al. (2004) did not observe variation between the mother plants and

micropropagated plantlets of Prunus dulcis, when fidelity was tested using 64 RAPD

primers. The results strongly suggested that axillary branching strategy ensures

maintenance of genomic integrity in almond shoot propagation.

Ryynänen and Aronen (2005) tested genetic fidelity of the tissue culture raised

plants of five genotypes (E1987, E5201, E5382, E5387 and E5398) of Betula pendula

by comparing their RAPD profiles with the markers amplified with the original donor

trees. A polymorphic amplification pattern was observed in 18 of the tested 20

primers separating all the tested clones from each other. No reproducible intraclonal

variation could be observed in the RAPD profiles.

Valladares et al. (2006) conducted RAPD analysis on embryogenic lines

derived from 3 genotypes of Quercus robur using 40 arbitrary 10-mer primers using

Kits A and S. No evidence of genetic variation either within or between the

embryogenic lines established from three of these trees, or between these lines and

trees of origin, or between somatic embryos derived plantlets and the trees of origin

were found.

In Curcuma longa, RAPD analysis carried for in vitro multiplication cultures

at six month interval using 20 arbitatory decamer primers up to 2 years, showed

monomorphic bands indicating no variation in the micropropagated plants when

compared with control plants (Panda et al., 2007).

Mathur et al. (2008) reported strong uniformity of micropropagated plants in

relation to the parent genotype of Chlorophytum borivilianum through RAPD

analysis. Similarly, Dubey (2009) analyzed RAPD profile of the 10 randomly selected



micropropagated plants and one mother plant of J. curcus using RAPD primers and

found that micropropagated plants were genetically stable with mother plant.

Arora et al. (2010) tested RAPD profiles generated by 5 arbitrary primers for

micropropagated plants (nodal stem segment-regenerated plants) and their

corresponding mother plants of A. indica. Of the 7 primers tested, 5 (OPF-01, OPF03,

OPF-04, OPU-19 and OPU-20) produced good amplification products in terms of

quality and quantity of band patterns. The bands obtained using all 5 primers were

monomorphic across all the micropropagated plants thus confirmed the genetic

stability. Similarly, Arora et al. (2011) reported that based on the RAPD profile of

micropropagated plants (plants regenerated from root explant) all the plants were true

to type nature with their corresponding mother plants in A. indica. RAPD profiling to

test genetic stability of micropropagated plants is thus obvious.

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